Backbone–Backbone H‐Bonds Make Context‐Dependent Contributions to Protein Folding Kinetics and Thermodynamics: Lessons from Amide‐to‐Ester Mutations

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS TO PROTEIN FOLDING KINETICS AND THERMODYNAMICS: LESSONS FROM AMIDE‐TO‐ESTER MUTATIONS By EVAN T. POWERS, SONGPON DEECHONGKIT, AND JEFFERY W. KELLY Department of Chemistry and The Skaggs Institute for Chemical Biology The Scripps Research Institute, La Jolla, California 92037

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Nomenclature and Synthesis of Amide‐to‐Ester Mutants . . . . . . . . . . . . . . . . . III. Esters as Amide Replacements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Geometry and Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Structural Effects of Amide‐to‐Ester Mutations . . . . . . . . . . . . . . . . . . . . . . IV. Interpretation of Energetic Data from Amide‐to‐Ester Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. H‐Bond Energies and the Thermodynamic Analysis of Amide ‐to ‐Ester Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Kinetic Analysis of Amide‐to‐Ester Mutants . . . . . . . . . . . . . . . . . . . . . . . . . V. Amide‐to‐Ester Mutations in Studies of Protein Function . . . . . . . . . . . . . . . . VI. Amide‐to‐Ester Mutations in Studies of Protein Folding Thermodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Analysis of

Gb and

Gf Values from Amide‐to‐Ester Mutants . . . . . . . . . A. General Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Quantitative Analysis of

Gf/b Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Amide‐to‐Ester Mutations in Studies of Protein Folding Kinetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40 41 44 44 46 48 48 55 56 58 61 61 64 68 69 70

Abstract The contribution of backbone‐backbone hydrogen bonds (H‐bonds) to protein folding energetics has been controversial. This is due, at least in part, to the inability to perturb backbone‐backbone H‐bonds by traditional methods of protein mutagenesis. Recently, however, protein backbone mutagenesis has become possible with the development of chemical and biological methods to replace individual amides in the protein backbone with esters. Here, we review the use of amide‐to‐ester mutation as a tool to evaluate the contribution of backbone‐backbone H‐bonds to protein folding kinetics and thermodynamics.

ADVANCES IN PROTEIN CHEMISTRY, Vol. 72 DOI: 10.1016/S0065-3233(05)72002-7

39

Copyright 2006, Elsevier Inc. All rights reserved. 0065-3233/06 $35.00

40

POWERS ET AL.

I. Introduction Hydrogen bonds between backbone amides are a prominent feature of protein structures. A survey (Baker and Hubbard, 1984) of protein crystal structures revealed that backbone amide NH groups are the donors in 46% of all H‐bonds to backbone amide carbonyls and that backbone amide carbonyls are the acceptors in 68% of all H‐bonds to backbone amide NHs (the apparent discrepancy is due to the tendency of amide carbonyls to accept H‐bonds from more than one donor). It is perhaps surprising, then, that the energetic contributions of backbone–backbone H-bonding to the stability of folded proteins has been a long‐running debate in the protein folding field (Baldwin, 2003). The earliest theories of protein denaturation proposed that ‘‘the force of attraction between polar groups’’ (Wu, 1931) or, in other words, H‐bonds (Mirsky and Pauling, 1936) held the protein molecule in a defined native conformation. This view was reinforced by the proposal that low energy conformations of polypeptides would be stabilized primarily by backbone–backbone H‐bonds and led to the structures of the a‐helix as well as parallel and antiparallel b-sheets (Pauling and Corey, 1951; Pauling et al., 1951). Soon after, however, Kauzmann (1959) proposed that protein native states could also be stabilized by the hydrophobic effect. That the hydrophobic effect is an important driving force for protein folding became more widely accepted when studies on N‐methylacetamide, a model compound mimic of backbone amides, suggested that the formation of backbone–backbone H‐bonds in aqueous solution had an enthalpy close to 0 kcal/mol (Klotz and Franzen, 1962). This was consistent with the view that the potential stabilization of the native state by backbone–backbone H‐bonds was offset by the stabilization of the denatured state by H‐bonds between water and amide carbonyl and NH groups, making the net contribution of backbone–backbone H‐bonding to protein folding thermodynamics negligible (Klotz and Franzen, 1962). By the early 1990s, many believed that the hydrophobic effect was the primary source of protein stability and that hydrogen bonding contributed to the specificity of protein native states (i.e., the existence of a group of closely related low energy structures), but not to their stabilities (Dill, 1990; Honig and Yang, 1995). Dissent from this view increased, however, as site‐directed mutagenesis experiments consistently showed that eliminating side chain–backbone or side chain–side chain H‐bonds tended to destabilize the native state (Myers and Pace, 1996). The disagreement over the role of backbone– backbone H‐bonds in protein folding thermodynamics also extended to disagreement over their contributions to protein folding kinetics (Baldwin, 1989). The importance of backbone–backbone H‐bonds in the transition state was emphasized by proponents of the framework model of protein folding, which states that formation of a framework of secondary structures is the key step in protein folding. In contrast, the importance of the

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

41

hydrophobic effect in protein folding kinetics was emphasized by proponents of the hydrophobic collapse model, which states that the formation of a condensed intermediate (in which hydrophobic side chains are buried, but the formation of native contacts is incomplete) is the key step in protein folding. A plausible explanation for the difference of opinion over the contribution of H‐bonding to protein folding thermodynamics and kinetics is that the energy of H‐bond formation is likely context dependent. Because H‐bonding is largely an electrostatic interaction (Umeyama and Morokuma, 1977), it is easy to imagine that the strength of a given H‐bond would be influenced by the microenvironment enveloping it, especially by the local dielectric constant. Evidence for side chain H‐bonding energetics being context dependent has been obtained from traditional site‐directed mutagenesis experiments (Myers and Pace, 1996). The strengths of backbone–backbone H‐bonds are also expected to be context sensitive. For example, it has been suggested that the strength of backbone–backbone H‐bonds that occur in linear arrays should increase with the length of the array (Dannenberg, 2002; Guo and Karplus, 1992; Kobko and Dannenberg, 2003). Thus, backbone–backbone H‐bond strengths in a‐helices and b‐sheets should increase with the length of the helix or the number of strands in the sheet (Wu and Zhao, 2001; Zhao and Wu, 2002). Miller and co‐workers (2002) have provided experimental evidence for this phenomenon in peptide a‐helices. These observations notwithstanding, experimental evidence for the context dependence of H‐bond energies has not been obtained until relatively recently for backbone–backbone H‐bonds, largely because the protein backbone cannot be structurally altered using traditional site‐directed mutagenesis. A convenient approach to mutate and thus perturb the H‐bonding capability of the protein backbone is to replace one or more of the amide bonds with ester bonds (Yang et al., 2004). Amide‐to‐ester mutations, unlike traditional side chain mutations, do not significantly alter the conformational preferences of the backbone (see Section III.A), but the ester oxygen cannot serve as an H‐bond donor, and the ester carbonyl is a weaker H‐bond acceptor than the amide carbonyl (Abraham and Platts, 2001). Advances in biological and chemical methods of protein synthesis have enabled the preparation of proteins with amide‐to‐ester mutations (see Section II), which has in turn enabled studies of the contributions of specific backbone–backbone H‐bonds to protein folding thermodynamics and kinetics. The justification for using amide‐to‐ester mutants to study backbone–backbone H‐bonding and the results obtained from such studies are discussed later. These results are then interpreted in terms of the contribution of backbone–backbone H‐bonds to protein folding thermodynamics and kinetics.

42

POWERS ET AL.

II. Nomenclature and Synthesis of Amide‐to‐Ester Mutants Peptides and proteins containing ester linkages are often referred to as depsipeptides. The synthetic methodology for the preparation of depsipeptides has been established because many biologically active depsipeptides are known (Ballard et al., 2002). The nomenclature indicating the identity and position of a‐hydroxy acids in peptide or protein sequences uses lowercase Greek letters corresponding to the one‐letter code of the analogous a‐amino acids (Deechongkit et al., 2004a,b). Thus, a represents lactic acid, the a‐hydroxy acid equivalent of alanine, A. The one‐letter codes of the other a‐hydroxy acids are shown in Fig. 1. The preparation of amide‐to‐ester mutants of proteins (which are formally depsipeptides) by solid‐phase peptide synthesis is conceptually straightforward. The chemical synthesis methodology (Scheme 1) is essentially the same as that employed for normal polypeptides, except that one or more a‐hydroxy acid residues are incorporated into the sequence at desired positions instead of an a‐amino acid residue (Baca and Kent, 2000; Beligere and Dawson, 2000; Blankenship et al., 2002; Deechongkit et al., 2004a; Low and Hill, 2000; Lu et al., 1997, 1999; Nakhle et al., 2000; Wales and Fitzgerald, 2001; Zheng et al., 2003; Zhou et al., 1998). The Boc/benzyl protecting group strategy for solid‐phase synthesis is used instead of the

Fig. 1.

One‐letter codes of a‐hydroxy acids.

Scheme 1. Reagents: (a) Standard solid‐phase peptide synthesis with a Boc/benzyl protecting group strategy. Coupling step: Boc protected a‐amino acid, HBTU, DIEA; deprotection step: trifluoroacetic acid. (b) a‐Hydroxy acid (1.1 equiv), DIC (1 equiv), HOBt (1.2 equiv), NEM (0.4 equiv). (c) Boc protected a‐amino acid (1.1 equiv), DIC (1 equiv), NEM (0.4 equiv), catalytic DMAP. (d) Cleavage and deprotection: HF, 4% p‐cresol.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

43

Fmoc/tBu approach because repeated removal of the Fmoc a‐amino protecting groups by treatment with piperidine can lead to aminolysis of the ester bonds. The ester linkages are stable to the deprotection conditions required by the Boc/benzyl strategy (treatment with trifluoroacetic acid to remove the Boc groups and treatment with HF to remove side chain protecting groups and cleave the peptide from the resin). a‐Hydroxy acids can be coupled to growing peptide chains by amide bond formation without protecting the a‐hydroxyl group (reactive side chains, however, must be protected). Typically, the carboxyl group of the a‐hydroxy acid is activated with diisopropyl carbodiimide (DIC) in the presence of 1‐hydroxybenzotraizole (HOBt) in a mixed solvent (dichloromethane/dimethylformamide), followed by coupling to the amino terminus of the growing peptide chain in the same mixed solvent in the presence of a substoichiometric amount of N‐ ethyl morpholine (NEM). Hexafluoroacetone acetals of a‐hydroxy acids (see Scheme 1) are also convenient activated building blocks for solid‐ phase depsipeptide synthesis (Albericio et al., 2005). Coupling of the next a‐amino acid residue through ester bond formation is more difficult than typical amide couplings and therefore requires the use of catalytic amounts of dimethylaminopyridine (DMAP, a powerful acylation catalyst) and longer coupling times (60 min). Couplings to hindered a‐hydroxy acids can require even more forcing conditions. Peggion and co‐workers (2002) found that coupling Boc‐Ala‐OH to a‐methyl hydroxyvaline‐OBzl (the benzyl ester of the a‐hydroxy acid corresponding to Val with an additional methyl on the a carbon) required the use of the combined catalysts scandium triflate and DMAP. Care must be taken to avoid cleaving the ester bond during purification of the crude depsipeptide. In our experience, depsipeptides can be purified by high‐pressure liquid chromatography using mobile phases containing
0.1% of trifluoroacetic acid without ester hydrolysis. In contrast, using neutral or basic aqueous buffers increases the risk of ester hydrolysis, especially if the amide‐to‐ester replacement is in a solvent exposed part of the protein and/or is close to Ser or His residues (Deechongkit et al., 2004a). The only commercially available a‐hydroxy acids are those bearing unfunctionalized side chains: glycolic acid (g), l‐lactic acid (a), l‐phenyllactic acid (f), l‐leucic acid (l), l‐isoleucic acid (i), and l‐hydroxyvaline (ϖ). Deechongkit et al. (2004c) reported convenient syntheses of the remaining l‐a‐hydroxy acids with side chain protecting groups that are appropriate for the Boc/benzyl strategy of solid‐phase depsipeptide synthesis. It should be noted that straightforward methods for the solution phase synthesis of depsipeptides have also been reported (Katakai et al., 2004). The methodology described earlier can be used to synthesize amide‐ to‐ester mutants of proteins up to about 50 residues in length. Longer

44

POWERS ET AL.

depsipeptides can be prepared using native chemical ligation (Dawson and Kent, 2000). Amide‐to‐ester mutants of turkey ovomucoid third domain (50 residues) (Lu et al., 1997), eglin C (70 residues) (Lu et al., 1999), and a single chain version of the HIV protease dimer (202 residues) (Baca and Kent, 2000) have been synthesized by native chemical ligation. A biological approach for the synthesis of amide‐to‐ester mutants using the nonsense suppression technique to incorporate a‐hydroxy acids into proteins has been reported elsewhere (Chapman et al., 1997; Ellman et al., 1992; Koh et al., 1997; Shin et al., 1997). This complementary method for incorporating a‐hydroxy acid residues into proteins has been reviewed by Thorson et al. (1998).

III.

Esters as Amide Replacements

A. Geometry and Conformation The bond lengths and angles of esters and amides are generally very similar (Fig. 2). In addition, both amides and esters are stabilized by resonance (Wiberg and Laidig, 1987). The partial double bond character in C0 –N and C0 –Oe bonds results in Ca–C0 –N/OeCa atoms having a strong

Fig. 2. Bond lengths, bond angles, and resonance forms of peptide amides (Engh and Huber, 1991) and esters (Ramakrishnan and Mitra, 1978). Side chains are omitted for clarity. The dipole moment of peptide amides (Matthew, 1985) and esters (Brant et al., 1969) are shown as well.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

45

preference to lie in the same plane (Chakrabarti and Dunitz, 1982; Schweizer and Dunitz, 1982).1 Importantly, both amides and esters prefer a trans geometry about the C0 –N or C0 –Oe bonds (Chakrabarti and Dunitz, 1982; Schweizer and Dunitz, 1982). Calculations have yielded enthalpy differences of 4.4 and 2.9 kcal/mol between the cis and the trans isomers of methyl acetate and N‐methylacetamide in aqueous solution, respectively (using a polarized continuum model for the solvent) (Kallies and Mitzner, 1996b). Experimental data on the thermodynamics of the cis–trans interconversion of esters in aqueous solution are scarce, but the calculated enthalpy of cis–trans interconversion of N‐methylacetamide (2.9 kcal/ mol) is comparable to the free energy measured by nuclear magnetic resonance (NMR) in aqueous solution (2.0 kcal/mol) (Barker and Boudreaux, 1967). In both N‐methylacetamide and methyl acetate, the cis form is destabilized by steric repulsions between the CH3–C0 and the N/ OeCH3 methyl groups. This cis form of methyl acetate is further destabilized by dipole–dipole interactions between the C¼O and the OeCH3 bonds; the calculated dipole moment of trans‐methyl acetate (2.0 D) is much lower than that of cis‐methyl acetate (4.8 D) (Kallies and Mitzner, 1996b). The calculated difference between the dipole moments of trans (4.1 D) and cis (4.4 D) N‐methylacetamide is much smaller (Kallies and Mitzner, 1996b). The most important difference between amides and esters, aside from the inability of esters to donate H‐bonds, is that the resonance stabilization of amides is greater than that of esters. This phenomenon manifests itself in several ways. In infrared spectra, the C¼O stretch of amides occurs at lower wavenumbers (1670 to 1700 cm1) (Challis and Challis, 1979) than that of esters (1735 to 1750 cm1) (Sutherland, 1979), indicating that the amide carbonyl has more single bond character than the ester carbonyl. The activation barrier for cis–trans interconversion (rotation about the C0 – N/O bond) has been calculated to be substantially higher for N‐methylacetamide (20 kcal/mol) than for methyl acetate (11 kcal/mol; Fig. 2b) (Kallies and Mitzner, 1996a,b). Furthermore, the C0 OeCa bond angle (117 ) is smaller than the C0 NCa bond angle (123 ), indicating that the hybridization of the ester oxygen is closer to sp3 than that of the amide nitrogen, which is closer to sp2 hybridization. The geometrical differences between amides and esters outlined in the preceding paragraph are, for the most part, outweighed by their similarities. In fact, the differences between the two only become apparent in We follow the naming convention in which C0 refers to carbonyl carbons and Ca refers to a‐ carbons. In ester groups, the oxygen atom that is singly bonded to C0 will be denoted Oe. The amide nitrogen and carbonyl oxygen will be denoted N and O, respectively. 1

46

POWERS ET AL.

homopolymers. For example, random coils of poly(l‐lactic acid) are more compact than random coils of poly(l‐alanine), largely because of the relatively small C0 OeCa bond angle of esters and larger dipole–dipole interactions in poly(l‐alanine) (Brant and Flory, 1965a,b,c; Brant et al., 1969; Tonelli and Flory, 1969). However, calculated Ramachandran plots of lactic acid residues in poly(l‐alanine‐l‐lactic acid) are very similar to those of alanine residues (Ingwall and Goodman, 1974), including conformational energy minima at f and c dihedral angles close to those required for a and 310 helices, b‐sheets, and polyproline II helices. As long as the side chains are not altered, a‐hydroxy acids can be substituted for a‐ amino acids without creating significant conformational disturbances. Thermodynamic and kinetic effects caused by amide‐to‐ester substitutions therefore can be interpreted largely in terms of changes in H‐bonding. This conclusion is supported by experimental data (see Section III.B), and in particular by the pioneering work of Goodman and co‐workers (Arad and Goodman, 1990a,b; Becktel et al., 1981, 1985; Goodman, 1978; Goodman et al., 1972, 1974, 1981; Ingwall and Goodman, 1974; Ingwall et al., 1976, 1978; Katakai and Goodman, 1982; Mammi and Goodman, 1986; Mathias et al., 1978; Nissen et al., 1975; Wouters et al., 1982).

B.

Structural Effects of Amide‐to‐Ester Mutations

Several crystal structures of peptides and one crystal structure of a protein with one or more amide‐to‐ester mutations have been reported. In most of these structures, the amide‐to‐ester mutation is found in a helix (Aravinda et al., 2002; Karle et al., 2001; Ohyama et al., 2000, 2001; Oku et al., 2004a). These crystal structures have, in general, shown that the a‐hydroxy acid residue can reside comfortably in a helical conformation. The ester group(s) in these helical depsipeptides tends to occur at boundaries between the 310 and the a‐helical structure, which allows the peptide to minimize the loss of H‐bonds (Aravinda et al., 2002; Karle et al., 2001; Ohyama et al., 2000, 2001; Oku et al., 2004a). In an a‐helical peptide, there is an H‐bond between the amide NH of residue i and the amide carbonyl of residue i‐4. If an amide‐to‐ester mutation were made at residue i (i.e., if residue i were replaced by an a‐hydroxy acid), the amide carbonyl at position i‐4 would lose its H‐bond donor. However, if the helix geometry shifted from a to 310 at the site of the amide‐to‐ester mutation, the amide carbonyl of residue i‐4 would be able to H‐bond to the amide NH of residue i‐1. Similar slight alterations in helix geometry have been observed in computational studies of the influence of amide‐to‐ester mutations on peptide helices (Cieplak and Surmeli, 2004). Larger structural

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

47

perturbations can also occur; an example in which a depsipeptide helix was kinked by 40 has been reported (Oku et al., 2004a), but this is likely because the peptide in question, Boc‐LLA(LLa)3LL‐OEt (recall that a represents lactic acid), had three amide‐to‐ester substitutions. In addition, substantial conformational changes were observed by NMR in an amide‐to‐ ester mutant of insulin (Kurapkat et al., 1997) and were proposed to occur in an amide‐to‐ester mutant of eglin C (Lu et al., 1999). In general, however, spectroscopic and functional assays have shown that amide‐to‐ ester mutations cause negligible changes in protein structures, as required if the energetic perturbations caused by amide‐to‐ester mutation are to be interpreted in terms of changes in backbone–backbone H‐bonding (Yang et al., 2004). Distances between the ester Oe and the amide carbonyls at the i‐3 or i‐4 positions in the crystal structures of helical depsipeptides described earlier tend to be longer (3.1 to 4.0 A˚) (Aravinda et al., 2002; Karle et al., 2001; Ohyama et al., 2000, 2001; Oku et al., 2004a) than distances between the amide N and amide carbonyl in a typical backbone–backbone H‐bond (3.0 A˚) (Jeffrey and Saenger, 1991). This observation and a similar observation in the crystal structure of dipeptide (Oku et al., 2003) suggest that the amide‐to‐ester mutation introduces an electrostatic repulsion between the ester Oe and nearby amide carbonyl oxygens. This repulsion does not usually cause significant structural changes, but it must be considered in analyses of the thermodynamic effects of amide‐to‐ester mutations (see Section IV). The ester carbonyl in amide‐to‐ester mutants (the carbonyl of residue i‐1 for an amide‐to‐ester mutant at residue (i) appears to form relatively long (and therefore weak) H‐bonds with amide NH groups (Aravinda et al., 2002; Karle et al., 2001), as expected based on scales of H‐bond acceptor strengths (Abraham and Platts, 2001). Studies of the structural effects of amide‐to‐ester mutations in other protein secondary structures are not abundant, but existing data are consistent with the expectation that a‐hydroxy acid residues in proteins are able to adopt conformations analogous to those of a‐amino acid residues. The crystal structure of the L18l amide‐to‐ester mutant of turkey ovomucoid third domain in complex with Streptomyces griseus proteinase B has been solved. The l18 residue in the amide‐to‐ester mutant is in a conformation that is almost identical to L18 in the wild type (Bateman et al., 2001). A crystal structure of Ac‐l‐Pro‐l‐Lac‐NHMe shows that a lactic acid residue can occupy the conformation required for the iþ2 residue in a type 1 b turn (Lecomte et al., 1974). Finally, the f and c angles of the lactic acid residue in the crystal structure of Boc‐l‐Ala‐l‐Lac‐OBzl (f ¼ 69 , c ¼ 163 ) are close to those expected for a polyproline II conformation (f ¼ 78 , c ¼ 149 ) (Oku et al., 2004b).

48

POWERS ET AL.

IV. Interpretation of Energetic Data from Amide‐to‐Ester Mutants A. H‐Bond Energies and the Thermodynamic Analysis of Amide ‐to ‐Ester Mutants The effect of an amide‐to‐ester mutation on protein folding is usually judged from the value of

Gf; for example, the difference between the free energy of folding of the mutant protein (
Gf,mut) and that of the wild‐ type protein (
Gf,wt), where

Gf ¼
Gf,mut –
Gf,wt. The value of

Gf can be interpreted at either a qualitative or a quantitative level. At the qualitative level, the value of

Gf is used as an indicator of the importance of the perturbed backbone–backbone H‐bond to the stability of the native state; large, positive

Gf values indicate important H‐bonds, whereas small

Gf values indicate unimportant H‐bonds. Qualitative interpretation of

Gf values has proven useful in understanding the role of individual backbone–backbone H‐bonds in the folding of the three‐stranded b‐sheet of the Pin WW domain, identifying a group of H‐bonds that are energetically important for folding and another group of H-bonds that are not (Deechongkit et al., 2004b). At the quantitative level, an attempt is made to extract the intrinsic energies of backbone–backbone H‐bonds from

Gf values. Amide‐to‐ ester mutations are nearly ideal for quantitative interpretations of

Gf. As discussed previously, the primary result of an amide‐to‐ester mutation is to eliminate the H‐bond donor and weaken the H‐bond acceptor of the mutated amide; amide‐to‐ester mutations do not generally introduce complicating conformational or steric effects. However, the effects of mutations, even very conservative ones, on protein folding thermodynamics are often not straightforward (Fersht et al., 1992). Eliminating amide NH groups by amide‐to‐ester mutations can leave amide carbonyls without H‐bonding partners and, as noted in Section III.B, it can introduce electrostatic repulsions between the ester Oe and nearby amide carbonyls. These effects must be accounted for in order to extract the energies of backbone–backbone H‐bonds from

Gf values (Beligere and Dawson, 2000; Blankenship et al., 2002; Deechongkit et al., 2004a; Koh et al., 1997; Lu et al., 1997, 1999; Yang et al., 2004). Two quantities of fundamental importance for understanding the role of backbone–backbone H‐bonding in protein folding thermodynamics can be obtained by quantitatively analyzing

Gf values from amide‐to‐ester mutants. The first is the intrinsic H‐bond energy (
Ghb), and the second is the net contribution of an H‐bond to the stability of the native state (
Gnethb). We define
Ghb as the energy required to break a

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

49

backbone–backbone H‐bond in the native state of a protein. Thus,
Ghb is the free energy difference between having an H‐bonded donor/acceptor pair in a natively folded protein and having the donor and acceptor not H‐bonded within an otherwise natively folded protein. Large, positive values of
Ghb indicate strong H‐bonds. This definition ensures that backbone–backbone H‐bonds are always considered to stabilize the native state. We believe that this point of view is intuitively satisfying; because H‐bonds are observed in natively folded proteins, they should be said to stabilize the native state (in other words, they would not exist in the native state if they did not stabilize the native state). Note that this definition largely excludes entropic contributions to
Ghb so that
Ghb 
Hhb. The quantity
Gnethb is the difference between the extent to which a backbone–backbone H‐bond stabilizes the native state and the extent to which solvated amides stabilize the denatured state. The former quantity is simply
Ghb. The latter quantity is the sum of the free energies of transfer of the H‐bond acceptor, an amide carbonyl (
Gt,amCO), and the H‐bond donor, an amide NH (
Gt,amNH), from water into their environment in the natively folded protein. Thus,
Gnethb can be expressed as
Gnethb ¼
Ghb  ð
Gt;amCO þ
Gt;amNH Þ

ð1Þ

Large, positive values of
Gnethb indicate that formation of a given H‐bond strongly favors the native state over the denatured state. It should be noted that the controversy over H‐bonding described in the Section I surrounds the value of
Gnethb, so the use of thermodynamic data from amide‐to‐ ester mutants to determine this value is of particular interest.

1. Extraction of DGhb from DDGf Values of Amide ‐to ‐Ester Mutants Amide‐to‐ester mutations can be divided into three types (Fig. 3), each of which has a different relationship between
Ghb and

Gf. In a type 1 mutant, the mutated amide both donates and accepts an H‐bond. Type 1 mutants result when an amide‐to‐ester mutation is made in the central strand of a b‐sheet or at an interior position of an a‐helix. In a type 2 mutant, the mutated amide only accepts an H‐bond. Type 2 mutants result when an amide‐to‐ester mutation is made in an outer strand of a b sheet or at the N terminus of an a‐helix. In a type 3 mutant, the mutated amide only donates an H‐bond. Type 3 mutants result when an amide‐to‐ester mutation is made in an outer strand of a b‐sheet or at the C terminus of an a helix. In most analyses of

Gf values from amide‐to‐ester mutants (and from traditional side chain mutants), the overall free energy of folding of mutant and wild‐type proteins is divided into contributions from processes

50

POWERS ET AL.

Fig. 3. Types of amide‐to‐ester mutations. Dashed black lines indicate amide–amide H‐bonds, dashed gray lines indicate weakened H‐bonds between an ester carbonyl and an amide NH, and solid black lines indicate repulsions between an ester Oe and an amide carbonyl.

that directly involve the mutated residue (local processes) and contributions from processes that do not (nonlocal processes) (Fersht et al., 1992). Figure 4 compares the local processes in a protein folding reaction for a wild‐type protein and a type 1 amide‐to‐ester mutant. For the wild‐type protein, the local processes are transfer of the amide to be mutated and its H‐bonding partners from aqueous solution into a protein‐like environment, followed by formation of interactions (two backbone–backbone H‐bonds). The transfer process contributes
Gt,wt to
Gf,wt, while the interactions contribute
Gi,wt to
Gf,wt. Thus,
Gf,wt can be written as
Gf;wt ¼
Gt;wt þ
Gi;wt þ
Gnl;wt

ð2Þ

where
Gnl,wt is the contribution to
Gf,wt of all of the nonlocal processes (
Gnl,wt accounts for desolvation of, and interactions formed by, the rest of the wild‐type protein, as well as configurational entropy loss). The H‐bonds accepted and donated by the amide to be mutated are denoted A and D, respectively, as shown in Fig. 4. The term
Gi,wt can be separated into individual contributions from each of these H‐bonds,
GAhb;wt and
GD hb;wt , which will not necessarily be equal. Thus,
Gi;wt ¼ ð
GAhb;wt þ
GD hb;wt Þ

ð3Þ

The term
Gt,wt can be separated into contributions from the amide carbonyl and NH groups, which participate in H‐bonds A and D, respectively.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

51

52

POWERS ET AL.

D
Gt;wt ¼ ð
GAt;amCO þ
GAt;amNH Þ þ ð
GD t;amCO þ
Gt;amNH Þ

ð4Þ

Because transfer free energies, like H‐bond energies, are likely to be context dependent, the values of
GAt;amCO and
GD t;amCO will not necessarily be equal to each other. The same is true of the values of
GAt;amNH and
GD t;amNH . By substituting Eqs. (3) and (4) into Eq. (2),
Gf,wt can be written as D
Gf;wt ¼ ð
GAt;amCO þ
GAt;amNH Þ þ ð
GD t;amCO þ
Gt;amNH Þ A D ð
Ghb;wt þ
Ghb;wt Þ þ
Gnl;wt

ð5Þ

A similar expression can be written for
Gf,mut, the free energy of folding of the amide‐to‐ester mutant, with the following changes. (1) The term
GAt;amCO is replaced by
GAt;esCO (the transfer free energy of an ester carbonyl) in the first set of parentheses, as the amide carbonyl from which this term arises is replaced by an ester carbonyl. (2) The term D
GD t;amNH in the second set of parentheses is replaced by
Gt;esO (the transfer free energy of an ester Oe), as the amide NH from which this term arises is replaced by an ester Oe. (3) The term
GAhb;wt in the third set of parentheses is replaced by
GAhb;mut , as H‐bond A is weakened by the amide‐to‐ester mutation. (4) The term
GD hb;wt in the third set of parentheses is replaced by the term
GOOrep, as H‐bond D is replaced by a repulsion between the ester Oe and the amide carbonyl. The value of
Gf,mut is then given by D
Gf;mut ¼ ð
GAt;esCO þ
GAt;amNH Þ þ ð
GD t;amCO þ
Gt;esO Þ A 
Ghb;mut þ
GOOrep þ
Gnl;mut

ð6Þ

Using Eqs. (5) and (6),

Gf can be written as

Gf ¼
Gf;mut 
Gf;wt ¼ ð
GAt;esCO 
GAt;amCO Þ D A A þð
GD t;esO 
Gt;amNH Þ þ ð
Ghb;wt 
Ghb;mut Þ D þ
Ghb;wt þ
GOOrep þ ð
Gnl;mut 
Gnl;wt Þ

ð7Þ

Fig. 4. Local processes in a protein folding reaction for a wild‐type protein and an amide‐to‐ester mutant divided into two stages. The first stage involves transfer of the amide to be mutated and its H‐bonding partners to a native‐like environment, but without H‐bond formation. The second stage involves the formation of two backbone– backbone H-bonds, labeled A (in which the mutated amide is the acceptor) and D (in which the mutated amide is the donor). Gray areas in the boxes represent solvent.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

53

The term ð
GAhb;wt 
GAhb;mut Þ represents the effect of weakening H‐bond A by exchanging the amide carbonyl for an ester carbonyl. For brevity, this is written as

GAhb . The term ð
Gnl;mut 
Gnl;wt Þ represents the difference in the nonlocal contributions to the free energy of folding between the amide and ester mutants; that is, it represents effects due to reorganization of the protein as a result of the amide‐to‐ester mutation. This is written as
Greorg. As noted by Fersht et al. (1992), it is difficult to interpret

Gf values if reorganization cannot be neglected. The value of
Greorg is thus usually taken to be 0 kcal/mol if the mutant protein can be shown by a technique that assesses structure (such as circular dichroism spectroscopy, NMR spectroscopy, or X‐ray crystallography) or function (such as binding or activity assays) to have native structure (Yang et al., 2004). Neglecting
Greorg and rearranging Eq. (7) to collect the terms corresponding to H‐bond strengths one side of the equation gives

GAhb þ
GD hb;wt ¼



Gf  ð
GAt;esCO 
GAt;amCO Þ D ð
GD t;esO 
Gt;amNH Þ 
GOOrep

ð8Þ

The effects of H‐bonding on the folding of type 2 and 3 amide‐to‐ester mutants can be derived easily from the preceding analysis, as only the effects relating to H‐bond A are relevant to type 2 mutants, and only the effects relating to H‐bond D are relevant to type 3 mutants. The value of

GAhb can therefore be determined from the

Gf of a type 2 amide‐to‐ester mutant as

GAhb ¼

Gf  ð
GAt;esCO 
GAt;amCO Þ

ð9Þ

is similarly related to the value of

Gf of a type 3 The value of amide‐to‐ester mutant:
GD hb;wt

D D
GD hb;wt ¼

Gf  ð
Gt;esO 
Gt;amNH Þ 
GOOrep

ð10Þ

A D Equations (8)–(10) show that

GAhb þ
GD hb ,

Ghb , and
Ghb can be determined from type 1, 2, and 3 amide‐to‐ester mutants, respectively, if
GOOrep and the transfer free energies of amide carbonyl, amide NH, ester carbonyl, and ester Oe groups can be estimated. The availability of such estimates is discussed later. It should be noted, however, that

GAhb is a quantity of secondary interest because it is not an intrinsic H‐bond energy (it is a difference between intrinsic H‐bond energies). Type 1 and 2 amide‐to‐ester mutants are therefore not as useful as type 3 amide‐to‐ester mutants in the analysis presented earlier; only

Gf values from type 3 mutants can be exclusively related to an intrinsic H‐bond energy (
GD hb ).

54 2.

POWERS ET AL.

Extraction of DGnethb from DDGf Values of Amide‐to‐Ester Mutants

The expression for
Gf,wt for a type 1 amide‐to‐ester mutant in Eq. (5) can be rearranged and then combined with Eq. (1) to yield
Gf;wt

¼ ð
GAt;amCO þ
GAt;amNH Þ 
GAhb;wt þ D D ð
GD t;amCO þ
Gt;amNH Þ 
Ghb;wt þ
Gnl;wt A D ¼ 
Gnethb;wt 
Gnethb;wt þ
Gnl;wt

ð11Þ

Similarly, the expression for
Gf,mut can be rewritten as
Gf;mut

¼ ð
GAt;esCO þ
GAt;amNH Þ 
GAhb;mut þ D ð
GD t;amCO þ
Gt;esO Þ þ
GOOrep þ
Gnl;mut D ¼ 
GAnethb;mut þ ð
GD t;amCO þ
Gt;esO Þþ
GOOrep þ
Gnl;mut

ð12Þ

The value of

Gf for a type 1 amide‐to‐ester mutation is then

Gf

¼
Gf;mut 
Gf;wt D D ¼

GAnethb þ
GD nethb;wt þ ð
Gt;amCO þ
Gt;esO Þ þ
GOOrep þ
Greorg

ð13Þ

where

GAnethb ¼ ð
GAnethb;wt 
GAnethb;mut Þ. As with Eq. (8), neglecting
Greorg and collecting the H‐bonding terms together yields D D

GAnethb þ
GD nethb;wt ¼

Gf  ð
Gt;amCO þ
Gt;esO Þ 
GOOrep

ð14Þ

The corresponding equations for type 2 and 3 mutants are

GAnethb ¼

Gf

ð15Þ

D D
GD nethb;wt ¼

Gf  ð
Gt;amCO þ
Gt;esO Þ 
GOOrep

ð16Þ

and

respectively. Again,

GAnethb is not the net contribution of an H‐bond to native state stability; it is a difference between the net contributions of two different types of H‐bonds. Type 1 and 2 mutants are therefore not as useful as type 3 mutants in addressing the net contribution of H‐bonds to protein folding thermodynamics. Only the

Gf values of type 3 amide‐to‐ ester mutants can be related exclusively to the net contribution of an H‐bond to native state stability.

3.

Comments on the Quantitative Analysis of DDGf Values

The strengths of individual backbone–backbone H‐bonds are characterD A A ized by
GD hb and
Gnethb and, to a lesser extent, by

Ghb and

Gnethb .

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

55

The preceding sections showed that determining these quantities requires one parameter that can be measured experimentally (

Gf) and several correction terms that must be estimated computationally or from model A=D A=D compound data ð
Gt;amCO ;
Gt;amNH ;
GAt;esCO ;
GD t, esO ; and
GOOrep Þ. As a result, the reliability of conclusions made about the role of H‐bonding in protein folding thermodynamics based on

Gf values depends critically on the reliability of the estimates of the correction terms. Many sources of error will contribute to the uncertainty in the correction terms. For example, many models are available for estimating the transfer free energies of functional groups in proteins, from water–octanol partition coefficients (Eisenberg and McLachlan, 1986; Fauchere and Pliska, 1983; Kellogg and Abraham, 2000; Wimley et al., 1996) to electrostatic salvation‐free energies (Avbelj, 2000; Avbelj and Baldwin, 2003; Baldwin, 2002) to interatomic potentials based on protein engineering data (Lomize et al., 2002), and it is not necessarily clear which of these is most appropriate for use in Eqs. (8)–(10) and (14)–(16). It is also not clear how the transfer‐free energies of amides and esters should be partitioned between their carbonyl and NH or Oe groups. The uncertainty just described is especially problematic for determining
Gnethb, as, in general,
Gnethb <
Ghb. Equation (16) shows that
Gnethb is a sum of four terms; if the error in each term were on the order of 0.5 kcal/mol, the error in the calculated value of
Gnethb would be on the order of 1 kcal/mol. Accordingly,
GD nethb would have to be greater than 1.4 kcal/mol (or less than 1.4 kcal.mol) to allow the conclusion that the net contribution of backbone–backbone H‐bonds to the native state was stabilizing (or destabilizing) to be made with more than 90% confidence. This uncertainty suggests that caution must be exercised when quantitatively interpreting

Gf values from amide‐to‐ester mutants, especially when

Gf is small. Nevertheless, creating amide‐to‐ester mutations is a strategy by which this question can be addressed and is perhaps the only approach whereby backbone–backbone H‐bonds can be studied individually. The analysis presented in the preceding sections can still be instructive, despite the caveats noted earlier.

B.

Kinetic Analysis of Amide‐to‐Ester Mutants

Amide‐to‐ester mutations enable the contributions of backbone–backbone H‐bonds to protein folding kinetics as well as thermodynamics to be evaluated. In fact, the use of M values renders the analysis of kinetic data from amide‐to‐ester mutants much simpler than the analysis of thermodynamic data. The M value for a given mutation is defined as the influence

56

POWERS ET AL.

of a mutation on the free energy of the transition state divided by its influence on the free energy of a native state (Fersht et al., 1992). The latter quantity is simply

Gf, which was defined earlier. The former quantity, referred to as

Gy, can be obtained from the rate constants for the folding of the amide‐to‐ester and wild‐type proteins:

Gy ¼
Gymut 
Gywt ¼ RT lnðkwt =kmut Þ

ð17Þ

The M value for a given mutation is therefore M ¼



Gy

Gf

ð18Þ

The theory of M values has been described in detail elsewhere and is beyond the scope of this review (Fersht et al., 1992; Nymeyer et al., 2000). We simply note the following. A M value of 1 for an amide‐to‐ester mutation indicates that the perturbed H‐bond(s) exists in both the native state and the folding transition state. A M value of 0 indicates that the perturbed H‐bond(s) exists in the native state but not in the folding transition state. Values of M between 0 and 1 are more difficult to interpret, but can be taken to indicate a situation intermediate between the two extremes described earlier (Nymeyer et al., 2000). It should be noted that amide‐to‐ester mutations, unlike side chain mutations, are direct probes of secondary structure, as secondary structures can be defined in terms of their backbone–backbone H‐bonding patterns. For this reason, M values obtained from amide‐to‐ester mutants reflect the degree of secondary structure that exists in the folding transition state.

V.

Amide‐to‐Ester Mutations in Studies of Protein Function

The prevalence of backbone–backbone H‐bonds in protein structures suggests that they are likely to be important not only for folding, but also for function. For example, backbone–backbone H‐bonds play an important role in substrate and inhibitor binding by proteinases (Bode and Huber, 1992). Amide‐to‐ester mutations have been an effective tool for studying the contribution of backbone–backbone H‐bonds to protein function. In many such studies, the amide‐to‐ester mutations were introduced into peptide analogs of protein–protein interaction sites, as peptides are shorter and more accessible by chemical synthesis than proteins. Amide‐to‐ester mutants of peptides have been used to study the role of backbone–backbone H‐bonds in the stereospecificity of a‐chymotrypsin (Ingles and Knowles, 1968); the activity and degradation of the peptide

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

57

hormone bradykinin (Ravdel et al., 1967; Shchukina et al., 1965; Shemyaki et al., 1966); the binding of hormones to neurophysin (Carlson and Breslow, 1981); the association and conductance of gramicidin A channels (Jude et al., 2001); and the binding and conformation of substrates for cAMP‐dependent protein kinase (Bramson et al., 1985; Thomas et al., 1987), thermolysin (Morgan et al., 1991), papain (Berti et al., 1991; Liu and Hanzlik, 1993), and chymotrypsin (Coombs et al., 1999). Amide‐ to‐ester mutations have also been used to develop inhibitors of the aggregation of peptides associated with Alzheimer’s disease (Gordon and Meredith, 2003) and type II diabetes (Rijkers et al., 2002). Perhaps the most striking example of the effect of an amide‐to‐ester mutation on peptide binding comes from the evolution of antibiotic resistance (Williams and Bardsley, 1999). Vancomycin (a member of the glycopeptide family of antibiotics) functions by binding the sequence Lys‐d‐Ala‐d‐Ala at C termini of bacterial cell wall precursors, thereby interfering with cell wall biosynthesis. This mode of action was remarkably robust; vancomycin resistance was unknown for roughly 30 years after its introduction into clinical use. Unfortunately, vancomycin‐resistant strains of Enterococcus appeared in the late 1980s. Their vancomycin resistance stemmed from a set of five genes that enabled them to synthesize cell wall precursors in which the C‐terminal d‐Ala residue was mutated to d‐Lac (d‐a). This amide‐to‐ester mutation prevents formation of a critical backbone–backbone H‐bond in complexes of vancomycin with cell wall precursors. The importance of this H‐bond is illustrated by the 1000‐fold decrease in the affinity of vancomycin for the amide‐to‐ester mutant AcLys(Ac)‐d‐Ala‐d‐Lac relative to its affinity for AcLys(Ac)‐d‐Ala‐d‐Ala (Bugg et al., 1991). Improved chemical and biological techniques for protein synthesis (see Section II) have enabled the preparation of amide‐to‐ester mutants of proteins to probe the role of specific backbone–backbone H‐bonds in protein function and protein–protein interactions. For example, the contribution of backbone–backbone H‐bonding to the binding of proteinases by protein proteinase inhibitors has been studied using amide‐to‐ester mutants of bovine pancreatic trypsin inhibitor (BPTI) (Groeger et al., 1994), turkey ovomucoid third domain (Bateman et al., 2001; Lu et al., 1997), and eglin C (Lu et al., 1999). Similarly, an amide‐to‐ester mutant of the homodimeric enzyme HIV protease has been used to show that backbone–backbone H‐bonds from only one of the two subunits are required for substrate binding and catalytic activity (Baca and Kent, 2000). Amide‐ to‐ester mutants have also been used to study the contribution of backbone H‐bonds to the enzyme‐catalyzed conversion of b,g‐unsaturated ketones to a,b‐unsaturated ketones (Cisneros et al., 2004), to the reduction

58

POWERS ET AL.

potential of Fe4S4 clusters in high‐potential iron proteins (Low and Hill, 2000), and to electron transfer in a designed four‐helix bundle (Zheng et al., 2003; Zhou et al., 1998) or a b turn (Williamson and Bowler, 1996, 1998, 2000). Finally, the nonsense suppression technique was used to study amide‐to‐ester mutants of a ligand‐gated ion channel (nicotinic acetylcholine receptor) in Xenopus oocytes (England et al., 1999). These mutants were used in vivo to probe the structural changes that occur during ligand gating. The difference between the free energy of binding of the amide‐to‐ester mutant (
Gb,mut) and the wild‐type peptide or protein (
Gb,wt) to their ligands was measured in many of the studies cited previously. This quantity,

Gb, is directly analogous to

Gf and depends on H‐bond energies in the same way (see Section IV). The values of

Gb are listed in Table I for each case in which this quantity was determined. Data in Table I are discussed in Section VII.

VI.

Amide‐to‐Ester Mutations in Studies of Protein Folding Thermodynamics

Amide‐to‐ester mutations have been used to study the role of backbone– backbone H‐bonds in peptide structure acquisition and in peptide models of protein folding. For example, they were used in conformational studies of the cyclic peptide gramicidin S (Krit et al., 1975; Zhuze et al., 1974); in studies of b‐turn formation by short peptides (Gallo and Gellman, 1993, 1994; Haque et al., 1996; Liang et al., 1992) and elastin repeating sequences (Arad and Goodman, 1990a,b); in studies of normal vs bifurcated H‐bonds (Yang and Gellman, 1998); in studies of templated helix formation (Kemp et al., 1995); in studies of the conformational stability of the collagen triple helix (Jenkins et al., 2005; Mammi and Goodman, 1986); and even in studies of the folding of b‐peptides (i.e., peptides composed of b‐amino acids) (Seebach et al., 1996, 2002). Such studies have been extended in the past decade to proteins. Examples include insulin (Kurapkat et al., 1997; Wollmer et al., 1994), Staphylococcal nuclease (Chapman et al., 1997; Shin et al., 1997), T4 lysozyme (Koh et al., 1997), a designed metal‐assembled three‐helix bundle (Zheng et al., 2003; Zhou et al., 1998), chymotrypsin inhibitor 2 (Beligere and Dawson, 2000), 4‐oxalocrotonate tautomerase (Nakhle et al., 2000; Silinski and Fitzgerald, 2003), eglin C (Lu et al., 2000), a hyperstable mutant of P22 arc repressor (Wales and Fitzgerald, 2001), the GCN4 coiled coil domain of the bZIP repressor (Blankenship et al., 2002), and the WW domain from the human PIN1 protein (Deechongkit et al., 2004a,b). Values of

Gf were determined for the amide‐to‐ester

Peptide or protein with amide‐to‐ester mutation Eglin C

Z‐Gly‐c(PO2NH)‐Leu‐NH2 Z‐Gly‐c(PO2NH)‐Gly‐Leu Z‐Gly‐c(PO2NH)‐Phe‐Leu Z‐Gly‐c(PO2NH)‐Ala‐Leu Z‐Gly‐c(PO2NH)‐Leu‐Leu Ac‐Phe‐Gly‐OMe Moc‐Phe‐Gly‐OMe Ac‐l‐Lys‐d‐Ala‐d‐Ala YYGAKIYRPDKM

Entry 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 2a 2b 2c 2d 2e 3a 3b 4 5a 5b

Binding partner Chymotrypsin Subtilisin Elastase Chymotrypsin Subtilisin Elastase Chymotrypsin Subtilisin Elastase Chymotrypsin Subtilisin Elastase Thermolysin Thermolysin Thermolysin Thermolysin Thermolysin Papain Papain Vancomycin Trypsin Trypsin

Mutated residue b

(V)L43l (V)L43lb (V)L43lb (T)L44lc (T)L44lc (T)L44lc L45l L45l L45l L47l L47l L47l NH2 to OMe G2g F2f A2a L2l F1f G2g dA3da G3g A4a

Type



Gb (kcal/mol)

3 3 3 2 1 2 3 3 3 3 3 3 1 1 1 1 1 3 1 3 2 3

1.2 0 2.9 –3.6d –0.7d –0.4d 3.7e 4.5e 2.1e 2.0 1.8 1.4 2.8e 2.3e 2.8e 2.6e 2.8e 2.7f 2.6f 4.1 1.2 1.3

Reference Lu et al. (1999)

Morgan et al. (1991)

Berti et al. (1991) Bugg et al. (1991) Coombs et al. (1999)

59

(continued)

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

Table I

Gb Values (in kcal/mol) of Amide‐to‐Ester Mutants from Studies of Protein Binding Thermodynamicsa

60

Table I Peptide or protein with amide‐to‐ester mutation Turkey ovomucoid third domain

Ac‐Leu‐Gly‐CN Ac‐Met‐Gly‐CN BPTI

Binding partner

6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 8

Chymotrypsin Pancreatic elastase Subtilisin Proteinase A Proteinase B Leukocyte elastase Papain Papain Papain Papain Trypsin

Mutated residue L18l L18l L18l L18l L18l L18l F1f G2g G2g G2g (R)G17gg

Type 3 3 3 3 3 3 3 1 1 1 3



Gb (kcal/mol) 1.6e 2.0e 1.3e 1.5e 1.7e 0.8e 2.1 0.8 1.0 1.2 1.9

Reference Lu et al. (1997)

Liu and Hanzlik (1993)

Groeger et al. (1994)

Also listed are the peptide or protein mutated, the binding partner with which

Gb values were measured, the mutated residue, and the type of mutation made (according to the categories in Fig. 2). b The wild‐type residue (V) was mutated to L, and the amide‐to‐ester mutant, L43l, was compared to the V43L mutant. c The wild‐type residue (T) was mutated to L, and the amide‐to‐ester mutant, L44l, was compared to the T44L mutant. d Negative

Gb values attributed to a conformational change. e Backbone‐side chain or bifurcated H‐bonds perturbed. f

Gb values derived from kcat/Km instead of true binding constants. g The wild‐type residue (R) was mutated to G, and the amide‐to‐ester mutant, G17g, was compared to the R17G mutant. a

POWERS ET AL.

Ac‐Phe‐Gly‐CN

Entry

(continued )

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

61

mutants of most of the proteins listed previously. They are listed in Table II and are discussed in Section VII.

VII.

Analysis of

Gb and

Gf Values from Amide‐to‐Ester Mutants

A. General Observations The vast majority (69 of 72) of the

Gb and

Gf values in Tables I and II are positive, demonstrating that amide‐to‐ester mutations almost always destabilize protein native states or protein–protein complexes. However, we are particularly interested in the effects of amide‐to‐ester mutations when the mutated amide is involved in backbone–backbone H‐ bonding. We will exclude from further consideration cases in which the mutated amide is involved in backbone‐side chain or bifurcated H‐bonds (entries 1g‐1i, 2a‐2e, and 6a‐6f from Table I and case 8q from Table II). In addition, Lu and co‐workers (1999) speculated that the negative

Gb values observed for amide‐to‐ester mutants at the P2 position of eglin C (entries 1d‐1f of Table I) were due to a conformational change (i.e.,
Greorg 6¼ 0) and the

Gb values from the amide‐to‐ester mutants of a papain substrate (entries 3a and 3b of Table I) were derived from kcat/Km instead of binding constants (Berti et al., 1991). These cases are also excluded from further analysis. This leaves 51

Gf/b values from amide‐to‐ester mutations in which only backbone–backbone H‐bonds are perturbed: 18 type 1 mutants, 12 type 2 mutants, and 21 type 3 mutants.2 The distribution of the remaining

Gf/b values in Tables I and II is displayed in Fig. 5. All of the

Gf/b values are positive. The distribution has a maximum between 1.0 and 1.5 kcal/mol and then tapers off slowly as

Gf/b increases. The average value of

Gf/b for all types of amide‐to‐ ester mutants is 1.7 kcal/mol (SD ¼ 1.2 kcal/mol). The average value of

Gf/b is 2.3 kcal/mol (SD ¼ 1.4 kcal/mol) for type 1 mutants, 1.0 kcal/ mol (SD ¼ 0.6 kcal/mol) for type 2 mutants, and 1.7 kcal/mol (SD ¼ 1.1 kcal/mol) for type 3 mutants. The sum of the average values of

Gf/b for the type 2 and 3 mutants is within error of the average

Gf/b value of type 1 mutants, indicating that the effects of eliminating an H‐bond donor and weakening an H‐bond 2 It is worth noting that a control experiment in which an amide‐to‐ester mutation was made at a solvent exposed position of the Pin WW domain (L7l) yielded a

Gf ¼ 0 (Deechongkit et al., 2004a). This observation justifies the expectation that the destabilization that accompanies amide‐to‐ester mutation is caused by H‐bond perturbation.

62

Table II

Gf Values (in kcal/mol) of Amide‐to‐Ester Mutants from Studies of Protein Folding Thermodynamics Protein Metal‐assembled helix bundle

Staphylococcal nuclease

T4 lysozyme

4‐Oxalocrotonate tautomerase (hexamer)

P22 Arc repressor (dimer)

Mutation

Type

Secondary structure



Gf (kcal/mol)

1a 1b 1c 2

1 1 1 3

Helix Helix Helix Helix

3a 3b 3c 3d 3e 3f 4a 4b 4c 5a 5b 5c 6a

L7l Q8y H9
V13a/A16a/ V19a/A22a L6l V10ϖ L13l L20l V24ϖ L27l L14l (I)L72lb (K)L84lc L39l (S)L44ld (I)L50le I2i

1 1 1 1 1 1 2 2 2 2 1 3 1

Helix Helix Helix Helix Helix Helix Sheet Sheet Turn Helix Helix Helix Sheet

6b

I7i

1

Sheet

7

L8l

3

Sheet

0.7 0.7 1.1 1.0 (2.9 for three H-bonds 0.7 3.2 3.1 2.4 3.6 3.2 2.5 1.5 1.6 0.9 1.7 0.7 3.7 (21.9 for six subunits) 4.1 (24.6 for six subunits) 1.2 (2.4 for two subunits)

Reference Zheng et al. (2003); Zhou et al. (1998) Beligere and Dawson (2000) Blankenship et al. (2002)

Chapman et al. (1997) Shin et al. (1997) Koh et al. (1997)

Nakhle et al. (2000); Silinski and Fitzgerald (2003)

Wales and Fitzgerald (2001)

POWERS ET AL.

Chymotrypsin inhibitor (CI2) GCN4

Entry

Pin WW domain

a

W11o E12E K13k R14r M15m S16s R17r S19s V22ϖ Y23c Y24c F25f N26n H27
N30n A31a S32s Q33y W34o G70g

3 3 2 3 2 3 2 3 2 3 1 1 1 2 3 3 2 3 2 3

Turn Sheet Sheet Sheet Sheet Sheet Turn Turn Sheet Sheet Sheet Sheet Sheet Turn Turn Sheet Sheet Sheet Sheet Sheet

1.1 1.5 0.7 3.9f 0.4 1.1 0.3 0.6 0.6 2.2 1.1 4.2f 4.8f 0.8 1.8 0.8g 1.0 3.1f 0.5 0.4

Deechongkit et al. (2004a,b)

Lu et al. (2000)

Also listed are the protein mutated, the mutated residue, the type of mutation made (according to the categories in Fig. 2), and the secondary structure in which the mutation was made. b The wild‐type residue (I) was mutated to L, and the amide‐to‐ester mutant, L72l, was compared to the I72L mutant. c The wild‐type residue (K) was mutated to L, and the amide‐to‐ester mutant, L84l, was compared to the K84L mutant. d The wild‐type residue (S) was mutated to L, and the amide‐to‐ester mutant, L44l, was compared to the S44L mutant. e The wild‐type residue (I) was mutated to L, and the amide‐to‐ester mutant, L50l, was compared to the I50L mutant. f Trimethylamine N‐oxide was required to measure

Gf of very unstable Pin WW domain variants. g Backbone‐side chain H‐bond perturbed.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

Eglin C

8a 8b 8c 8d 8e 8f 8g 8i 8j 8k 8l 8m 8n 8o 8p 8q 8r 8s 8t 9

63

64

POWERS ET AL.

Fig. 5. Distribution of

Gf/b values from amide‐to‐ester mutants in which only backbone–backbone H‐bonds were perturbed.

acceptor are approximately additive. More importantly, the high standard deviations of all of the average

Gf/b values are consistent with the expectation that the energetics of backbone–backbone H‐bond formation should be context dependent. This context dependence may involve the type of secondary structure in which the mutated amide resides; for example, the average

Gf values for type 1 mutations is 2.0 kcal/mol (SD ¼ 1.2 kcal/mol, n ¼ 10) in a‐helices and 3.6 kcal/mol (SD ¼ 1.4 kcal/mol, n ¼ 5) in b‐sheets. However, there are not yet enough data on each type of mutation in each type of secondary structure to reach a statistically sound conclusion. It has also been observed in amide‐to‐ester mutants of GCN4 (Blankenship et al., 2002) and the Pin WW domain (Deechongkit et al., 2004a,b) that backbone–backbone H‐bonds enveloped by a hydrophobic core appear to contribute more to native state stability than solvent exposed H‐bonds.

B.

Quantitative Analysis of DDGf/b Values

Quantitatively analyzing

Gf/b values in order to estimate intrinsic backbone–backbone H‐bond free energies, or the net contribution of backbone–backbone H‐bonds to native state stabilities, requires estimates of the correction factors described in Section IV.A, namely A=D A=D A=D
GA;D t;amCO ;
Gt;amNH ;
Gt;esCO ;
Gt;esO , and
GOOrep. To avoid the complexities of tailoring the correction terms to the specific environment of each amide‐to‐ester variant in Tables I and II, we analyzed only the average values of

Gf/b for type 2 and 3 amide‐to‐ester mutants and used values

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

65

for the correction terms that should be valid for an ‘‘average’’ protein environment. The superscripts A and D are therefore dropped from the correction terms. The average value of

Gf/b for type 1 amide‐to‐ester mutants are not analyzed because it is, to a first approximation, simply the sum of the average

Gf/b values for the type 2 and 3 amide‐to‐ester mutants.

1. Values for the Correction Terms (DGt,amCO, DGt,amNH, DGt,esCO, DGt,esO, and DGOOrep) Accurate estimates of free energies of transfer from aqueous solution to the protein interior have long been sought because of their importance to protein folding and binding. As mentioned earlier, one of the methods for estimating these transfer‐free energies is to use water–octanol partition coefficients (Eisenberg and McLachlan, 1986; Fauchere and Pliska, 1983; Kellogg and Abraham, 2000; Wimley et al., 1996). Calculations of functional group contributions to water–octanol partition coefficients yield a transfer free energy of 2.0 kcal/mol for a protein backbone amide (Wimley et al., 1996).3 Similar calculations suggest that the difference in the water–octanol transfer free energies of an amide –C(O)NH– fragment and an ester –C (O)O– fragment is 1.4 kcal/mol (Meylan and Howard, 2000), so 0.6 kcal/ mol will be used for the transfer free energy of an ester –C(O)O– fragment. These transfer free energies must still be partitioned between the amide and ester carbonyls and their NH or Oe fragments. The simplest way to do this is to assign half to each fragment; we have used this approach in the past (Deechongkit et al., 2004a). It seems likely, though, that the carbonyl fragments contribute more than the NH or Oe fragments to amide and ester solvation in water. Evidence for this assertion comes from the following observations. (1) N‐Methylacetamide accepts two H‐bonds from water to its carbonyl, but only donates one H‐bond from its NH to water (Eaton et al., 1989). (2) The H‐bonds between water and the amide carbonyl have been calculated to be stronger than those between the amide NH and water (Rablen et al., 1998). (3) Acetamide, N‐methylacetamide, and N,N‐dimethylacetamide have similar vapor/water distribution coefficients, suggesting that replacing donatable hydrogen atoms with methyl groups has a small effect on amide solvation in water (Wolfenden, 1978). The ester carbonyl likely dominates ester solvation to an even greater extent. Data for ester 3

This is lower than the value of 2.7 kcal/mol that has been suggested for amides from data on small molecules (Meylan and Howard, 2000; Roseman, 1988). At least part of the difference can be attributed to side chain shielding of the backbone amide from the solvent (Wimley et al., 1996). We also note that the transfer energies quoted earlier may not be valid for amides close in sequence to N and C termini (R. Baldwin, personal communication).

66

POWERS ET AL.

solvation in water are not as abundant as they are for amide solvation, but it has been shown computationally that H‐bonds between the water O–H group and the ester carbonyl are much stronger than hydrogen bonds between the water O–H and the ester Oe (Rablen et al., 1998). We will therefore assign two‐thirds of the transfer free energy of amides to the amide carbonyl and one‐third to the amide NH, yielding
Gt,amCO ¼ 1.3 kcal/mol and
Gt,amNH ¼ 0.7 kcal/mol. All of the transfer free energy of esters will be assigned to the ester carbonyl, yielding
Gt,esCO ¼ 0.6 kcal/mol and
Gt,esO ¼ 0 kcal/mol. The uncertainty in these values is difficult to assess, but it seems likely that the standard deviation will be somewhat smaller than the values themselves. We will assign a standard deviation of 0.5 kcal/mol to each of the transfer free energies described earlier for the purposes of argument, but this should be considered a lower limit. The value of
GOOrep was estimated to be between 1.5 and 2.4 kcal/ mol in a computational study of amide‐to‐ester mutations by Cieplak and Surmeli (2004). This value is consistent with an experimental value for
GOOrep of 2.6 kcal/mol derived by comparing the affinity of vancomycin for AcLys(Ac)‐d‐Ala‐d‐Ala to its affinity for two analogs: the amide‐to‐ester mutant AcLys(Ac)‐d‐Ala‐d‐Lac and an analog in which the d‐Ala‐d‐Ala linkage was replaced by a ketomethylene group (McComas et al., 2003). However, it has also been found that mutants of BPTI in which an amide in the binding loop was replaced with an ester or a ketomethylene group bind to trypsin with equal affinities, suggesting a value of
GOOrep close to 0 kcal/mol (Groeger et al., 1994). Similarly, papain substrates with ester or ketomethylene groups at the P20 position yielded comparable kcat/Km values, suggesting a small value for
GOOrep (Berti et al., 1991). We will use
GOOrep ¼ 1 kcal/mol as a compromise value. The variability in the reported values of
GOOrep suggests that the standard deviation of this quantity should be substantial. We will use a standard deviation of 1 kcal/ mol for the analysis in the following section.

2.

Backbone–Backbone H‐Bond Energies

The average values of

GAhb and
GD hb can be estimated by inserting the average

Gf value for type 2 and 3 amide‐to‐ester mutants and the correction terms quoted earlier into Eqs. (9) and (10), respectively. This calculation yields

GAhb ¼ 1:7 kcal=mol (SD ¼ 0.9 kcal/mol) and A
GD hb;wt ¼ 1:4 kcal=mol (SD ¼ 1.6 kcal/mol). The average value of

Ghb agrees well with the difference determined by Gallo and co‐workers (1993) between the enthalpies of amide–amide and amide– ester H‐bond formation in CH2Cl2 (1.6 kcal/mol). It is also noteworthy that the values of A A A

GAhb and
GD hb;wt are similar. Because

Ghb ¼
Ghb;wt 
Ghb;mut

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

67

and the average values of
GAhb;wt and
GD hb;wt should be the same,
GAhb;mut should be close to 0 kcal/mol. This implies that the H‐bond between an ester carbonyl and an amide NH is quite weak, consistent with the observation made in Section III.B that these H‐bonds are relatively long. A calculated average value of 1.4 kcal/mol for
GD hb;wt indicates that the intrinsic free energy of backbone–backbone H‐bonds in proteins, which is roughly the same as their enthalpy (see Section IV.A), is substantial. Measurements of amide–amide H‐bond enthalpies are abundant, but the conditions under which they were measured vary widely. It is therefore difficult to make a useful comparison between the average value of
GD hb;wt and literature data. We note, however, that the average value of
GD hb;wt is comparable to the enthalpy of H‐bond formation in peptide helices (Chou and Scheraga, 1971; Hermans, 1966; Lopez et al., 2002; Rialdi and Hermans, 1966; Scholtz et al., 1991) and to the enthalpy of dimerization of N‐methylacetamide in a variety of moderately polar solvents: 1.5 kcal/mol in chloroform (Tsuboi, 1955), 1.6 kcal/mol in cis‐dichloroethylene (Franzen and Stephens, 1963), and 0.8 kcal/mol in dioxane (Klotz and Franzen, 1962). The large standard deviation of
GD hb;wt is partly due to the uncertainties in assigning the values of the correction terms, but it also reflects the context dependence of backbone–backbone H‐bond strengths, which has already been mentioned. The average values of

GAnethb and
GD nethb;wt can be estimated by inserting the average

Gf values for type 2 and 3 amide‐to‐ester mutants and the correction terms quoted earlier into Eqs. (15) and (16), respectively. This calculation yields

GAnethb ¼ 1:0 kcal=mol (SD ¼ 0.6 kcal/ mol) and
GD nethb;wt ¼ 0:6 kcal=mol (SD ¼ 1.6 kcal/mol). The former quantity requires little comment; it simply shows that weakening the H‐ bond acceptor in a backbone–backbone H‐bond decreases the net contribution of that backbone–backbone H‐bond to native state stability by 1.0 kcal/mol. The value of

GAnethb is lower than that of

GAhb because transfer of the ester group into a protein environment is less energetically costly than transfer of an amide. The average value of
GD nethb;wt is more interesting. Although the uncertainties in the correction factors used to derive
GD nethb;wt are too large to allow assertions to be made with great confidence, it seems likely that the average value of
GD nethb;wt is close to 0 kcal/mol. This finding suggests that the net contribution of backbone–backbone H‐bonding to protein folding thermodynamics on average neither stabilizes nor destabilizes protein native states. Another way of phrasing the preceding statement is perhaps more illuminating: the net contribution of many backbone–backbone H‐bonds to protein folding thermodynamics is to destabilize the native

68

POWERS ET AL.

state, but the net contribution of just as many others is to stabilize the native state, particularly those formed in a low dielectric microenvironment. The likely role of backbone–backbone H‐bonds that fall into the destabilizing category is to ensure the existence of a unique lowest energy native state, that is, they contribute to the specificity of protein folding (Dill, 1990; Honig and Yang, 1995). In contrast, the likely role of backbone–backbone H‐bonds that fall into the stabilizing category is to supplement the free energy gains from hydrophobic side chain burial. The category into which a specific backbone–backbone H‐bond falls can be determined from the

Gf value of the relevant amide‐to‐ester mutants. Among the type 3 mutants of the Pin WW domain (Deechongkit et al., 2004a,b), the H‐bonds donated by R14 and Q33 likely belong in the net stabilizing category, as

Gf >3.0 kcal/mol for both R14r and Q33y (a full standard deviation above the average for type 3 mutants). In contrast, the H‐bond donated by S19 likely belongs in the net destabilizing category, as

Gf ¼ 0.6 kcal/mol for S19s (almost a full standard deviation below the average).

VIII. Amide‐to‐Ester Mutations in Studies of Protein Folding Kinetics The effect of backbone perturbation on protein folding kinetics can be analyzed using M values, as discussed in Section IV.B. The use of M values from amide‐to‐ester mutants, or from other backbone modifications, to study the role of backbone–backbone H‐bonding in protein folding kinetics is still in its infancy, but we expect that this technique will soon prove itself extremely valuable. We have used M values from amide‐ to‐ester mutants to map secondary structure formation during the folding of the Pin WW domain (Deechongkit et al., 2004b). The Pin WW domain is a 34‐residue, three‐stranded b‐sheet protein with two loops. It folds very quickly; the folding half‐life is approximately 100 ms at 50 C. Consequently, this process can only be studied using the laser temperature jump technique or the equivalent (Gruebele et al., 1998). Traditional side chain mutagenesis revealed that the residues in loop 1 have the highest M values, suggesting that native‐like contacts are formed in the folding transition state by the side chains in this segment of the Pin WW domain (Jager et al., 2001). The M values for residues in the b‐strands and loop 2 are smaller, suggesting that these segments are less structured in the folding transition state. The M values of side chain mutants are informative, but they are only indirect probes of secondary structure formation in the folding transition state. In contrast, M values of amide‐to‐ester mutants are direct probes of

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

69

Fig. 6. Structure of the Pin WW domain, with side chains omitted for clarity. The light gray atoms are carbon, the dark gray atoms are nitrogen, and the intermediate gray atoms are oxygen. Dashed black lines represent H‐bonds. Values of M for amide‐ to‐ester mutants were measured at the indicated positions.

secondary structure formation in the folding transition state (see Section IV.B). The M values were measured for eight of the Pin WW domain amide‐to‐ester mutants in Table II (all of the mutants with thermal unfolding midpoints above 35 C) to determine which elements of secondary structure exist in its folding transition state. These M values, which are shown in Fig. 6, fall into three categories (Deechongkit et al., 2004b). Those for the amide‐to‐ester mutants in loop 1 are the closest to 1, indicating that the H‐bonds formed by these amides are almost fully engaged in the folding transition state and that loop 1 has a native‐like secondary structure in the folding transition state. The M values for the amide‐to‐ester mutants in the b‐strands and loop 2 are smaller, but still greater than 0, indicating that the b‐strands and loop 2 are partly structured in the folding transition state. Finally, the M value of W34o is close to 0, showing that the ends of the strands are unstructured in the folding transition state. These M values, in conjunction with the M values from traditional side chain mutants, have yielded an unusually detailed picture of the folding of the Pin WW domain.

IX. Conclusions and Future Directions Creating amide‐to‐ester protein variants represents a powerful tool for studying protein folding thermodynamics and kinetics. Amide‐to‐ester

70

POWERS ET AL.

mutants allow individual backbone–backbone H‐bonds to be perturbed and examined in the extremely complicated environment of a folded, or folding, protein. The studies discussed earlier have already yielded striking results, which future work in the field will further scrutinize and extend. For example, it was suggested in Section VII.B.2 that a given backbone– backbone H‐bond is just as likely to make a net stabilizing contribution to the native state as to make a net destabilizing contribution. This was based on an analysis of average

Gf and

Gb values using correction factors that were meant to be appropriate for a generalized protein environment. This analysis would be vastly improved if, for example, the values of the correction terms (the transfer‐free energies and
GO–Orep) were calculated for the specific context of each amide‐to‐ester mutant. The substantial advances realized in computational biology and chemistry should enable more sophisticated corrections. Similarly, the role of secondary structure formation in transition state energetics will become clearer as M value analyses of amide‐to‐ester mutants in more proteins become available. Continued work with amide‐to‐ester mutants (and other backbone–backbone H‐bond perturbing mutations) will surely occur, producing more high‐quality data and strategies for extracting H‐bond strengths that will have a great impact on our understanding of the role of backbone–backbone H‐bonding in protein folding.

References Abraham, M. H., and Platts, J. A. (2001). Hydrogen bond structural group constants. J. Org. Chem. 66, 3484–3491. Albericio, F., Burger, K., Ruiz‐Rodriguez, J., and Spengler, J. (2005). A new strategy for solid‐phase depsipeptide synthesis using recoverable building blocks. Org. Lett. 7, 597–600. Arad, O., and Goodman, M. (1990a). Depsipeptide analogs of elastin repeating sequences: Conformational analysis. Biopolymers 29, 1651–1668. Arad, O., and Goodman, M. (1990b). Depsipeptide analogs of elastin repeating sequences: Synthesis. Biopolymers 29, 1633–1649. Aravinda, S., Shamala, N., Das, C., and Balaram, P. (2002). Structural analysis of peptide helices containing centrally positioned lactic acid residues. Biopolymers 64, 255–267. Avbelj, F. (2000). Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins. J. Mol. Biol. 300, 1335–1359. Avbelj, F., and Baldwin, R. L. (2003). Role of backbone solvation and electrostatics in generating preferred peptide backbone conformations: Distributions of phi. Proc. Natl. Acad. Sci. USA 100, 5742–5747. Baca, M., and Kent, S. B. H. (2000). Protein backbone engineering through total chemical synthesis: New insight into the mechanism of HIV‐1 protease catalysis. Tetrahedron 56, 9503–9513. Baker, E. N., and Hubbard, R. E. (1984). Hydrogen bonding in globular proteins. Prog. Biophys. Mol. Biol. 44, 97–179.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

71

Baldwin, R. L. (1989). How does protein folding get started? Trends. Biochem. Sci. 14, 291–294. Baldwin, R. L. (2002). Relation between peptide backbone solvation and the energetics of peptide hydrogen bonds. Biophys. Chem. 101, 203–210. Baldwin, R. L. (2003). In search of the energetic role of peptide hydrogen bonds. J. Biol. Chem. 278, 17581–17588. Ballard, C. E., Yu, H., and Wang, B. (2002). Recent developments in depsipeptide research. Curr. Med. Chem. 9, 471–498. Barker, R. H., and Boudreaux, G. J. (1967). NMR evidence for cis‐trans isomerism in N‐methyl acetamide. Spectrochim. Acta A 23, 727. Bateman, K. S., Huang, K., Anderson, S., Lu, W. Y., Qasim, M. A., Laskowski, M., and James, M. N. G. (2001). Contribution of peptide bonds to inhibitor‐protease binding: Crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3‐Pro18l and OMTKY3‐C[COO]‐Leu18l in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY3‐C [CH2 NHþ 2 ]‐Asp19l. J. Mol. Biol. 305, 839–849. Becktel, W. J., Mathias, L. J., and Goodman, M. (1981). Polydepsipeptides. 8. Configurational contributions to the conformation of polydepsipeptides and analogous polypeptides. Macromolecules 14, 203–207. Becktel, W. J., Wouters, G., Simmons, D. M., and Goodman, M. (1985). Polydepsipeptides. 11. Conformational analysis of polydepsipeptides containing methyl, isopropyl, and isobutyl side chains. Macromolecules 18, 630–634. Beligere, G. S., and Dawson, P. E. (2000). Design, synthesis, and characterization of 4‐ester CI2, a model for backbone hydrogen bonding in protein a‐helices. J. Am. Chem. Soc. 122, 12079–12082. Berti, P. J., Faerman, C. H., and Storer, A. C. (1991). Cooperativity of papain substrate interaction energies in the S2 to S20 subsites. Biochemistry 30, 1394–1402. Blankenship, J. W., Balambika, R., and Dawson, P. E. (2002). Probing backbone hydrogen bonds in the hydrophobic core of GCN4. Biochemistry 41, 15676–15684. Bode, W., and Huber, R. (1992). Natural protein proteinase inhibitors and their interaction with proteinases. Eur. J. Biochem. 204, 433–451. Bramson, H. N., Thomas, N. E., and Kaiser, E. T. (1985). The use of N‐methylated peptides and depsipeptides to probe the binding of heptapeptide substrates to cAMP‐dependent protein‐kinase. J. Biol. Chem. 260, 5452–5457. Brant, D. A., and Flory, P. J. (1965a). The configuration of random polypeptide chains. I. Experimental results. J. Am. Chem. Soc. 87, 2788–2791. Brant, D. A., and Flory, P. J. (1965b). The configuration of random polypeptide chains. II. Theory. J. Am. Chem. Soc. 87, 2791–2800. Brant, D. A., and Flory, P. J. (1965c). The role of dipole interactions in determining polypeptide configurations. J. Am. Chem. Soc. 87, 663–664. Brant, D. A., Tonelli, A. E., and Flory, P. J. (1969). Configurational statistics of random poly(lactic acid) chains. II. Theory. Macromolecules 2, 228–235. Bugg, T. D. H., Wright, G. D., Dutkamalen, S., Arthur, M., Courvalin, P., and Walsh, C. T. (1991). Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: Biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry 30, 10408–10415. Carlson, J., and Breslow, E. (1981). Contribution of the peptide backbone to the binding of peptides and hormones to neurophysin. Biochem. Biophys. Res. Commun. 100, 455–462.

72

POWERS ET AL.

Chakrabarti, P., and Dunitz, J. D. (1982). Structural characteristics of the carboxylic amide group. Helv. Chim. Acta 65, 1555–1562. Challis, B. A., and Challis, J. A. (1979). Amides and related compounds. In ‘‘Comprehensive Organic Chemistry’’ (D. H. R. Barton and W. D. Ollis, Eds.), p. 990. Pergamon, Oxford. Chapman, E., Thorson, J. S., and Schultz, P. G. (1997). Mutational analysis of backbone hydrogen bonds in Staphylococcal nuclease. J. Am. Chem. Soc. 119, 7151–7152. Chou, P. Y., and Scheraga, H. A. (1971). Calorimetric measurement of enthalpy change in the isothermal helix‐coil transition of ploy‐l‐lysine in aqueous solution. Biopolymers 10, 657–680. Cieplak, A. S., and Surmeli, N. B. (2004). Single‐site mutation and secondary structure stability: An isodesmic reaction approach. The case of unnatural amino acid mutagenesis Ala to Lac. J. Org. Chem. 69, 3250–3261. Cisneros, G. A., Wang, M., Silinski, P., Fitzgerald, M. C., and Yang, W. T. (2004). The protein backbone makes important contributions to 4‐oxalocrotonate tautomerase enzyme catalysis: Understanding from theory and experiment. Biochemistry 43, 6885–6892. Coombs, G. S., Rao, M. S., Olson, A. J., Dawson, P. E., and Madison, E. L. (1999). Revisiting catalysis by chymotrypsin family serine proteases using peptide substrates and inhibitors with unnatural main chains. J. Biol. Chem. 274, 24074–24079. Dannenberg, J. J. (2002). Cooperativity in hydrogen bonded aggregates: Models for crystals and peptides. J. Mol. Struct. 615, 219–226. Dawson, P. E., and Kent, S. B. (2000). Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69, 923–960. Deechongkit, S., Dawson, P. E., and Kelly, J. W. (2004a). Toward assessing the position‐ dependent contributions of backbone hydrogen bonding to b‐sheet folding thermodynamics employing amide‐to‐ester perturbations. J. Am. Chem. Soc. 126, 16762–16771. Deechongkit, S., Nguyen, H., Powers, E. T., Dawson, P. E., Gruebele, M., and Kelly, J. W. (2004b). Context‐dependent contributions of backbone hydrogen bonding to b‐sheet folding energetics. Nature 430, 101–105. Deechongkit, S., You, S. L., and Kelly, J. W. (2004c). Synthesis of all nineteen appropriately protected chiral a‐hydroxy acid equivalents of the a‐amino acids for Boc solid‐phase depsi‐peptide synthesis. Org. Lett. 6, 497–500. Dill, K. A. (1990). Dominant forces in protein folding. Biochemistry 29, 7133–7155. Eaton, G., Symons, M. C. R., and Rastogi, P. P. (1989). Spectroscopic studies of the solvation of amides with N‐H Groups. 1. The carbonyl group. J. Chem. Soc. Faraday Trans. I 85, 3257–3271. Eisenberg, D., and McLachlan, A. D. (1986). Solvation energy in protein folding and binding. Nature 319, 199–203. Ellman, J. A., Mendel, D., and Schultz, P. G. (1992). Site‐specific incorporation of novel backbone structures into proteins. Science 255, 197–200. Engh, R. A., and Huber, R. (1991). Accurate bond and angle parameters for X‐ray protein structure refinement. Acta Crystallogr. Sect. A 47, 392–400. England, P. M., Zhang, Y. N., Dougherty, D. A., and Lester, H. A. (1999). Backbone mutations in transmembrane domains of a ligand‐gated ion channel: Implications for the mechanism of gating. Cell 96, 89–98. Fauchere, J. L., and Pliska, V. (1983). Hydrophobic parameters p of amino acid side chains from the partitioning of N‐acetyl amino acid amides. Eur. J. Med. Chem. 18, 369–375.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

73

Fersht, A. R., Matouschek, A., and Serrano, L. (1992). The folding of an enzyme. 1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771–782. Franzen, J. S., and Stephens, R. E. (1963). Effect of a dipolar solvent system on interamide hydrogen bonds. Biochemistry 2, 1321–1327. Gallo, E. A., and Gellman, S. H. (1993). Hydrogen‐bond‐mediated folding in depsipeptide models of b‐turns and a‐helical turns. J. Am. Chem. Soc. 115, 9774–9788. Gallo, E. A., and Gellman, S. H. (1994). Effect of a C‐terminal cationic group on the competition between a‐helical turn and b‐turn in a model depsipeptide. J. Am. Chem. Soc. 116, 11560–11561. Goodman, M. (1978). Matrix‐controlled synthesis and conformational studies of polydepsipeptides. J. Polymer Sci. Polymer Symp. 62, 173–188. Goodman, M., Becktel, W., Katakai, R., and Wouters, G. (1981). Synthesis and conformational analysis of polydepsipeptides. Makromol. Chem. Suppl. 4, 100–115. Goodman, M., Gilon, C., and Knobler, Y. (1972). Polydepsipeptides. 1. Synthesis and characterization of copolymers of a‐amino and a‐hydroxy acids. Isr. J. Chem. 10, 867–879. Goodman, M., Gilon, C., Palumbo, M., and Ingwall, R. T. (1974). Polydepsipeptides. 2. Synthesis and preliminary conformational studies of an alternating a‐amino and a‐hydroxy acid polymer. Isr. J. Chem. 12, 67–77. Gordon, D. J., and Meredith, S. C. (2003). Probing the role of backbone hydrogen bonding in beta‐amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions. Biochemistry 42, 475–485. Groeger, C., Wenzel, H. R., and Tschesche, H. (1994). BPTI backbone variants and implications for inhibitory activity. Int. J. Pept. Protein Res. 44, 166–172. Gruebele, M., Sabelko, J., Ballew, R., and Ervin, J. (1998). Laser temperature jump induced protein refolding. Acc. Chem. Res. 31, 699–707. Guo, H., and Karplus, M. (1992). Ab initio studies of hydrogen bonding of N‐methylacetamide: Structure, cooperativity, and internal rotational barriers. J. Phys. Chem. 96, 7273–7287. Haque, T. S., Little, J. C., and Gellman, S. H. (1996). Stereochemical requirements for b‐hairpin formation: Model studies with four‐residue peptides and depsipeptides. J. Am. Chem. Soc. 118, 6975–6985. Hermans, J. J. (1966). Experimental free energy and enthalpy of formation of the a‐helix. J. Phys. Chem. 70, 510–515. Honig, B., and Yang, A. S. (1995). Free energy balance in protein folding. Adv. Prot. Chem. 46, 27–58. Ingles, D. W., and Knowles, J. R. (1968). Stereospecificity of a‐chymotrypsin. Biochem. J. 108, 561. Ingwall, R. T., Gilon, C., Becktel, W. J., and Goodman, M. (1978). Polydepsipeptides. 7. Conformational analysis of poly(l‐alanyl‐l‐alanyl‐l‐lactic acid). Macromolecules 11, 540–545. Ingwall, R. T., Gilon, C., and Goodman, M. (1976). Polydepsipeptides. 5. Experimental conformational analysis of poly(l‐alanyl‐l‐lactic acid) and related model compounds. Macromolecules 9, 802–808. Ingwall, R. T., and Goodman, M. (1974). Polydepsipeptides. 3. Theoretical conformational analysis of randomly coiling and ordered depsipeptide chains. Macromolecules 7, 598–605. Jager, M., Nguyen, H., Crane, J. C., Kelly, J. W., and Gruebele, M. (2001). The folding mechanism of a b‐sheet: The WW domain. J. Mol. Biol. 311, 373–393.

74

POWERS ET AL.

Jeffrey, G. A., and Saenger, W. (1991). Hydrogen bonding in biological structures. Springer‐Verlag, Berlin. Jenkins, C. L., Vasbinder, M. M., Miller, S. J., and Raines, R. T. (2005). Peptide bond isosteres: Ester or (E)‐alkene in the backbone of the collagen triple helix. Org. Lett. 7, 2619–2622. Jude, A. R., Providence, L. L., Schmutzer, S. E., Shobana, S., Greathouse, D. V., Andersen, O. S., and Koeppe, R. E. (2001). Peptide backbone chemistry and membrane channel function: Effects of a single amide‐to‐ester replacement on gramicidin channel structure and function. Biochemistry 40, 1460–1472. Kallies, B., and Mitzner, R. (1996a). Study of electron densities of methyl acetate, N‐methylacetamide and N,N’‐dimethylurea by quantum mechanical investigations. 1. Gas phase. J. Chem. Soc. Perkin Trans. 2, 1397–1401. Kallies, B., and Mitzner, R. (1996b). Study of electron densities of methyl acetate, N‐methylacetamide and N,N’‐dimethylurea by quantum mechanical investigations. 2. Solvent models. J. Chem. Soc. Perkin Trans. 2, 1403–1408. Karle, I. L., Das, C., and Balaram, P. (2001). Effects of hydrogen bond deletion on peptide helices: Structural characterization of depsipeptides containing lactic acid. Biopolymers 59, 276–289. Katakai, R., and Goodman, M. (1982). Polydepsipeptides. 9. Synthesis of sequential polymers containing some amino acids having polar side chains and (S)‐lactic acid. Macromolecules 15, 25–30. Katakai, R., Kobayashi, K., Yamada, K., Oku, H., and Emori, N. (2004). Synthesis of sequential polydepsipeptides utilizing a new approach for the synthesis of depsipeptides. Biopolymers 73, 641–644. Kauzmann, W. (1959). Some factors in the interpretation of protein denaturation. Adv. Prot. Chem. 14, 1–63. Kellogg, G. E., and Abraham, D. J. (2000). Hydrophobicity: Is log Po/w more than the sum of its parts? Eur. J. Med. Chem. 35, 651–661. Kemp, D. S., Allen, T. J., and Oslick, S. L. (1995). The energetics of helix formation by short templated peptides in aqueous solution. 1. Characterization of the reporting helical template Ac‐Hel1. J. Am. Chem. Soc. 117, 6641–6657. Klotz, I. M., and Franzen, J. S. (1962). Hydrogen bonds between model peptide groups in solution. J. Am. Chem. Soc. 84, 3461–3466. Kobko, N., and Dannenberg, J. J. (2003). Cooperativity in amide hydrogen bonding chains: Relation between energy, position, and H‐bond chain length in peptide and protein folding models. J. Phys. Chem. A 107, 10389–10395. Koh, J. T., Cornish, V. W., and Schultz, P. G. (1997). An experimental approach to evaluating the role of backbone interactions in proteins using unnatural amino acid mutagenesis. Biochemistry 36, 11314–11322. Krit, N. A., Zhuze, A. L., Andronova, T. M., Filatova, M. P., Ravdel, G. A., and Shchukina, L. A. (1975). Synthesis of gramicidin S depsipeptide analogs. Bioorg. Khim. 1, 247–256. Kurapkat, G., De Wolf, E., Grotzinger, J., and Wollmer, A. (1997). Inactive conformation of an insulin despite its wild‐type sequence. Protein Sci. 6, 580–587. Lecomte, C., Aubry, A., Protas, J., Boussard, G., and Marraud, M. (1974). Study of molecular conformation of solid state of N‐acetyl‐l‐prolyl‐l‐lactylmethylamide. Acta Crystallogr. Sect. B B 30, 1992–1996. Liang, G. B., Rito, C. J., and Gellman, S. H. (1992). Thermodynamic analysis of b‐turn formation in Pro‐Ala, Pro‐Gly, and Pro‐Val model peptides in methylene chloride. J. Am. Chem. Soc. 114, 4440–4442.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

75

Liu, S. M., and Hanzlik, R. P. (1993). The contribution of intermolecular hydrogen bonding to the kinetic specificity of papain. Biochim. Biophys. Acta 1158, 264–272. Lomize, A. L., Riebarkh, M. Y., and Pogozheva, I. D. (2002). Interatomic potentials and solvation parameters from protein engineering data for buried residues. Protein Sci. 11, 1984–2000. Lopez, M. M., Chin, D. H., Baldwin, R. L., and Makhatadze, G. I. (2002). The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal‐ binding to induce helix formation. Proc. Natl. Acad. Sci. USA 99, 1298–1302. Low, D. W., and Hill, M. G. (2000). Backbone‐engineered high‐potential iron proteins: Effects of active‐site hydrogen bonding on reduction potential. J. Am. Chem. Soc. 122, 11039–11040. Lu, W. Y., Qasim, M. A., Laskowski, M., and Kent, S. B. H. (1997). Probing intermolecular main chain hydrogen bonding in serine proteinase‐protein inhibitor complexes: Chemical synthesis of backbone‐engineered turkey ovomucoid third domain. Biochemistry 36, 673–679. Lu, W. Y., Randal, M., Kossiakoff, A., and Kent, S. B. H. (1999). Probing intermolecular backbone H‐bonding in serine proteinase‐protein inhibitor complexes. Chem. Biol. 6, 419–427. Lu, W. Y., Starovasnik, M. A., Dwyer, J. J., Kossiakoff, A. A., and Kent, S. B. H. (2000). Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis. Biochemistry 39, 3575–3584. Mammi, S., and Goodman, M. (1986). Polydepsipeptides. 13. Synthesis and 1H‐NMR analysis of collagen model structures. Int. J. Pept. Protein Res. 28, 29–44. Mathias, L. J., Fuller, W. D., Nissen, D., and Goodman, M. (1978). Polydepsipeptides. 6. Synthesis of sequential polymers containing varying ratios of l‐alanine and l‐lactic acid. Macromolecules 11, 534–539. Matthew, J. B. (1985). Electrostatic effects in proteins. Annu. Rev. Biophys. Biophys. Chem. 14, 387–417. McComas, C. C., Crowley, B. M., and Boger, D. L. (2003). Partitioning the loss in vancomycin binding affinity for d‐Ala‐d‐Lac into lost H‐bond and repulsive lone pair contributions. J. Am. Chem. Soc. 125, 9314–9315. Meylan, W. M., and Howard, P. H. (2000). Estimating log P with atom/fragments and water solubility with log P. Perspect. Drug Discov. Design 19, 67–84. Miller, J. S., Kennedy, R. J., and Kemp, D. S. (2002). Solubilized, spaced polyalanines: A context‐free system for determining amino acid a‐helix propensities. J. Am. Chem. Soc. 124, 945–962. Mirsky, A. E., and Pauling, L. (1936). On the structure of native, denatured, and coagulated proteins. Proc. Natl. Acad. Sci. USA 22, 439–447. Morgan, B. P., Scholtz, J. M., Ballinger, M. D., Zipkin, I. D., and Bartlett, P. A. (1991). Differential binding energy: A detailed evaluation of the influence of hydrogen‐ bonding and hydrophobic groups on the inhibition of thermolysin by phosphorus‐ containing inhibitors. J. Am. Chem. Soc. 113, 297–307. Myers, J. K., and Pace, C. N. (1996). Hydrogen bonding stabilizes globular proteins. Biophys. J. 71, 2033–2039. Nakhle, B. M., Silinski, P., and Fitzgerald, M. C. (2000). Identification of an essential backbone amide bond in the folding and stability of a multimeric enzyme. J. Am. Chem. Soc. 122, 8105–8111. Nissen, D., Gilon, C., and Goodman, M. (1975). Polydepsipeptides. 4. Synthesis of the alternating polydepsipeptides poly (Ala‐Lac) and poly (Val‐Lac). Makromol. Chem. 1 (Suppl), 23–53.

76

POWERS ET AL.

Nymeyer, H., Socci, N. D., and Onuchic, J. N. (2000). Landscape approaches for determining the ensemble of folding transition states: Success and failure hinge on the degree of frustration. Proc. Natl. Acad. Sci. USA 97, 634–639. Ohyama, T., Oku, H., Hiroki, A., Maekawa, Y., Yoshida, M., and Katakai, R. (2000). The crystal structure for a depsipeptide Boc‐(Leu‐Leu‐Ala)2‐(Leu‐Leu‐Lac)3‐OEt with a 310‐helical segment. Biopolymers 54, 375–378. Ohyama, T., Oku, H., Yoshida, M., and Katakai, R. (2001). Crystal structure of a depsipeptide, Boc‐(Leu‐Leu‐Lac)3‐Leu‐Leu‐OEt. Biopolymers 58, 636–642. Oku, H., Ohyama, T., Hiroki, A., Yamada, K., Fukuyama, K., Kawaguchi, H., and Katakai, R. (2004a). Addition of a peptide fragment on an a‐helical depsipeptide induces a/310‐conjugated helix: Synthesis, crystal structure, and CD spectra of Boc‐Leu‐ Leu‐Ala‐(Leu‐Leu‐Lac)3‐Leu‐Leu‐OEt. Biopolymers 75, 242–254. Oku, H., Suda, T., Yamada, K., and Katakai, R. (2004b). A short depsipeptide: Tert‐ butoxycarbonyl‐l‐alanyl‐l‐lactic acid benzyl ester (Boc‐l‐Ala‐l‐Lac‐OBzl). Acta Crystallogr. Sect. E Struct. Rep. Online 60, 720–721. Oku, H., Yamada, K., and Katakai, R. (2003). N‐H...O¼C hydrogen bonding and O...O¼C repulsive interactions in tert‐butoxycarbonyl‐l‐leucyl‐l‐alanine ethyl ester (Boc‐l‐Leu‐l‐Ala‐OEt). Acta Crystallogr. Sect. E Struct. Rep. Online 59, 1581–1583. Pauling, L., and Corey, R. B. (1951). Configurations of polypeptide chains with favored orientations around single bonds: Two new pleated sheets. Proc. Natl. Acad. Sci. USA 37, 729–740. Pauling, L., Corey, R. B., and Branson, H. R. (1951). The structure of proteins: Two hydrogen‐bonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. USA 37, 205–211. Peggion, C., Barazza, A., Formaggio, F., Crisma, M., Toniolo, C., Villa, M., Tomasini, C., Mayrhofer, H., Pochlauer, P., Kaptein, B., and Broxterman, Q. B. (2002). (aMe)Hyv: Chemo‐enzymatic synthesis, and preparation and preferred conformation of model depsipeptides. J. Chem. Soc. Perkin Trans. 2, 644–651. Rablen, P. R., Lockman, J. W., and Jorgensen, W. L. (1998). Ab initio study of hydrogen‐ bonded complexes of small organic molecules with water. J. Phys. Chem. A 102, 3782–3797. Ramakrishnan, C., and Mitra, J. (1978). Dimensions of the ester unit. Proc. Indian Acad. Sci. Sect. A 87, 13–21. Ravdel, G. A., Filatova, M. P., Shchukina, L. A., Paskhina, T. S., Surovikina, M. S., Trapeznikova, S. S., and Egorova, T. P. (1967). 6‐Glycine‐8‐phenyllactic acid bradykinin: Its synthesis, biological activity, and splitting by kininase (carboxypeptidase N). J. Med. Chem. 10, 242–246. Rialdi, G., and Hermans, J. J. (1966). Calorimetric heat of the helix‐coil transition of poly‐l‐glutamic acid. J. Am. Chem. Soc. 88, 5719–5720. Rijkers, D. T. S., Hoppener, J. W. M., Posthuma, G., Lips, C. J. M., and Liskamp, R. M. J. (2002). Inhibition of amyloid fibril formation of human amylin by N‐alkylated amino acid and a‐hydroxy acid residue containing peptides. Chem.‐Eur. J. 8, 4285–4291. Roseman, M. A. (1988). Hydrophilicity of polar amino acid side chains is markedly reduced by flanking peptide bonds. J. Mol. Biol. 200, 513–522. Scholtz, J. M., Marqusee, S., Baldwin, R. L., York, E. J., Stewart, J. M., Santoro, M., and Bolen, D. W. (1991). Calorimetric determination of the enthalpy change for the a‐helix to coil transition of an alanine peptide in water. Proc. Natl. Acad. Sci. USA 88, 2854–2858.

BACKBONE–BACKBONE H‐BONDS MAKE CONTEXT‐DEPENDENT CONTRIBUTIONS

77

Schweizer, W. B., and Dunitz, J. D. (1982). Structural characteristics of the carboxylic ester group. Helv. Chim. Acta 65, 1547–1554. Seebach, D., Ciceri, P. E., Overhand, M., Jaun, B., Rigo, D., Oberer, L., Hommel, U., Amstutz, R., and Widmer, H. (1996). Probing the helical secondary structure of short‐chain b‐peptides. Helv. Chim. Acta 79, 2043–2066. Seebach, D., Mahajan, Y. R., Senthilkumar, R., Rueping, M., and Jaun, B. (2002). b‐Depsipeptides: The effect of a missing and a weakened hydrogen bond on the stability of the beta‐peptidic 314‐helix. Chem. Commun. 159, 8–1599. Shchukina, L. A., Ravdel, G. A., Filatova, M. P., and Zhuze, A. L. (1965). Depsipeptide analogues of biologically active peptides. 2. Synthesis of bradykinin and its depsipeptide analogues. Acta Chim. Acad. Sci. Hung. 44, 205. Shemyakin, M. M., Shchukina, L. A., Vinogradova, E. I., Ravdel, G. A., and Ovchinnikow, Y. A. (1966). Mutual replaceability of amide and ester groups in biologically active peptides and depsipeptides. Experientia 22, 535–536. Shin, I. J., Ting, A. Y., and Schultz, P. G. (1997). Analysis of backbone hydrogen bonding in a b‐turn of Staphylococcal nuclease. J. Am. Chem. Soc. 119, 12667–12668. Silinski, P., and Fitzgerald, M. C. (2003). Comparative analysis of two different amide‐to‐ ester bond mutations in the b‐sheet of 4‐oxalocrotonate tautomerase. Biochemistry 42, 6620–6630. Sutherland, I. O. (1979). Esters. In ‘‘Comprehensive Organic Chemistry’’ (D. H. R. Barton and W. D. Ollis, Eds.), p. 911. Pergamon Press, Oxford. Thomas, N. E., Bramson, H. N., Miller, W. T., and Kaiser, E. T. (1987). Role of enzyme‐ peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities. Biochemistry 26, 4461–4466. Thorson, J. S., Cornish, V. W., Barrett, J. E., Cload, S. T., Yano, T., and Schultz, P. G. (1998). A biosynthetic approach for the incorporation of unnatural amino acids into proteins. Methods. Mol. Biol. 77, 43–73. Tonelli, A. E., and Flory, P. J. (1969). Configuration statistics of random poly(lactic acid) chains. I. Experimental results. Macromolecules 2, 225–227. Tsuboi, M. (1955). Energy of the molecular association of N‐methyl acetamide. J. Chem. Soc. Japan 76, 376–379. Umeyama, H., and Morokuma, K. (1977). The origin of hydrogen‐bonding: An energy decomposition study. J. Am. Chem. Soc. 99, 1316–1332. Wales, T. E., and Fitzgerald, M. C. (2001). The energetic contribution of backbone‐ backbone hydrogen bonds to the thermodynamic stability of a hyperstable P22 arc repressor mutant. J. Am. Chem. Soc. 123, 7709–7710. Wiberg, K. B., and Laidig, K. E. (1987). Barriers to rotation adjacent to double bonds. 3. The C‐O barrier in formic acid, methyl formate, acetic acid, and methyl acetate. The origin of ester and amide resonance. J. Am. Chem. Soc. 109, 5935–5943. Williams, D. H., and Bardsley, B. (1999). The vancomycin group of antibiotics and the fight against resistant bacteria. Angew. Chem. Int. Edit. 38, 1173–1193. Williamson, D. A., and Bowler, B. E. (1996). Synthesis of a b‐turn forming depsipeptide for hydrogen bond mediated electron transfer studies. Tetrahedron 52, 12357–12372. Williamson, D. A., and Bowler, B. E. (1998). Electron transfer through the hydrogen‐ bonded interface of a b‐turn‐forming depsipeptide. J. Am. Chem. Soc. 120, 10902–10911. Williamson, D. A., and Bowler, B. E. (2000). Porphyrin to quinone electron transfer across a depsipeptide which forms an a‐helical turn. Inorg. Chim. Acta 297, 47–55.

78

POWERS ET AL.

Wimley, W. C., Creamer, T. P., and White, S. H. (1996). Solvation energies of amino acid side chains and backbone in a family of host‐guest pentapeptides. Biochemistry 35, 5109–5124. Wolfenden, R. (1978). Interaction of the peptide bond with solvent water: A vapor phase analysis. Biochemistry 17, 201–204. Wollmer, A., Gilge, G., Brandenburg, D., and Gattner, H. G. (1994). An insulin with the native sequence but virtually no activity. Biol. Chem. Hoppe‐Seyler 375, 219–222. Wouters, G., Katakai, R., Becktel, W. J., and Goodman, M. (1982). Polydepsipeptides. 10. Helix‐coil transitions of sequential polydepsipeptides having protected polar side chains. Macromolecules 15, 31–35. Wu, H. (1931). Studies on denaturation of proteins XIII: A theory of denaturation. Chin. J. Physiol. 5, 321–344. Wu, Y. D., and Zhao, Y. L. (2001). A theoretical study on the origin of cooperativity in the formation of 310‐ and a‐helices. J. Am. Chem. Soc. 123, 5313–5319. Yang, J. H., and Gellman, S. H. (1998). Energetic superiority of two‐center hydrogen bonding relative to three‐center hydrogen bonding in a model system. J. Am. Chem. Soc. 120, 9090–9091. Yang, X. Y., Wang, M., and Fitzgerald, M. C. (2004). Analysis of protein folding and function using backbone modified proteins. Bioorganic Chem. 32, 438–449. Zhao, Y. L., and Wu, Y. D. (2002). A theoretical study of b‐sheet models: Is the formation of hydrogen‐bond networks cooperative? J. Am. Chem. Soc. 124, 1570–1571. Zheng, Y. J., Case, M. A., Wishart, J. F., and McLendon, G. L. (2003). Do main chain hydrogen bonds create dominant electron transfer pathways? An investigation in designed proteins. J. Phys. Chem. B 107, 7288–7292. Zhou, J., Case, M. A., Wishart, J. F., and McLendon, G. L. (1998). Thermodynamic and structural effects of a single backbone hydrogen bond deletion in a metal‐assembled helical bundle protein. J. Phys. Chem. B 102, 9975–9980. Zhuze, A. L., Kogan, G. A., Krit, N. A., Andronova, T. M., Filatova, M. P., Senyavina, L. B., Meshcheryakova, E. A., Ryabova, I. D., Ravdel, G. A., and Shchukina, L. A. (1974). Depsipeptide modification as an approach to elucidation of intramolecular hydrogen bond contribution to conformational stability of peptides (gramicidin S). Mol. Biol. 8, 69–74.