Peptide structures

Current Opinion in Solid State and Materials Science 6 (2002) 109–116

Peptide structures ¨ Carl Henrik Gorbitz Department of Chemistry, University of Oslo, N-0315 Oslo, Norway

Abstract Prediction of solid state peptide structures from the amino acid sequence is difficult not only because of the inherent flexibility of the molecules, but also due to the vast number of possible interactions between hydrogen bond donating and accepting groups. This review focuses on recent advances into rationalizing and controlling peptide conformations and hydrogen bond networks by utilization of special amino acid residues, cyclization, addition of terminal blocking groups and more.  2002 Published by Elsevier Science Ltd. Keywords: Helices; Turns; Molecular conformations; Hydrogen bonds; Nanotubes; Crystal engineering

1. Introduction Controlling and predicting the aggregation of molecules in the solid state is an ultimate goal in the field of research now known as crystal engineering [1–3]. The development of new materials relies most of all on the accumulated knowledge obtained from recurring packing patterns in crystal structures [**4]. Several families of compounds, such as ureas, guanidines, dicarboxylic acids and aminopyridines, have served as models to decipher the underlying mechanisms responsible for the observed interactions between various functional groups, in particular hydrogen bonds as well as metal coordination [**5]. Design strategies for supramolecular synthesis have been particularly successful for planar molecules with self-complementary (homomeric) or complimentary (heteromeric) hydrogen bonding groups that are involved in two-dimensional interaction patterns [3,**5] (although, due to the crystal packing arrangement, the over-all hydrogen bond network may actually be three-dimensional). Endeavors into the third dimension are dominated by studies of coordination compounds, reviewed in this journal last year [6]. Studies of peptides constitute an alternative approach in this respect. Systematic investigations of intermolecular interactions have been few, however [*7,*8,9], since many peptide chemists are concerned primarily with molecular conformations. In these cases hydrogen bonds are used primarily as tools to reach a target molecular structure by

¨ E-mail address: [email protected] (C.H. Gorbitz).

formation of specific intramolecular interactions. This review presents important progress that has been made in the study of peptide structures from late 2000 to early 2002.

2. Hydrogen bonding patterns in peptides and proteins The basic molecular structure of a peptide includes hydrogen bond donors (.N–H) and acceptors (.C5O) in the peptide bonds in addition to the C-terminal –COO 2 carboxylate group and the N-terminal –NH 1 3 amino group. The first three groups could easily take part in twodimensional hydrogen bond network, but the amino group seeks three distinctly unplanar hydrogen bond acceptors. The hydrogen bond patterns of free, unblocked peptides are therefore inherently three-dimensional. The total number of hydrogen bonds is n12 for a peptide with n residues (minimum value counting only interactions with N–H donors), which may nevertheless appear as a manageable number for small peptides as far as predicting the structure is concerned. However, several complicating factors are present. First of all, retention of a particular hydrogen bond motif (synthon) in a number of different crystal structures requires that hydrophobic groups, if present, can be efficiently segregated into independent regions in the crystal. Moreover, such regions must be flexible with respect to their ability to encompass hydrophobic groups of various sizes. For peptides, with ubiquitous hydrophobic

1359-0286 / 02 / $ – see front matter  2002 Published by Elsevier Science Ltd. PII: S1359-0286( 02 )00044-X

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entities of variable sizes in the side chains, finding robust modes of hydrophobic aggregation is a particular challenge. Examples of isostructural peptides are few, while examples of complete change of crystal packing and molecular interactions upon addition of a single methyl or methylene group are abundant, e.g., Arg-Asp /Arg-Glu [10]. Secondly, hydrogen bond donors and acceptors may not only be present in the peptide main chain, but also in the side chains and in cocrystallized solvent molecules. The number of possible hydrogen bond types then increase dramatically and renders any structure prediction futile. The above discussion suggests that hydrogen bonding patterns in peptide structures can be simplified in a number of ways:

• avoid residues with hydrogen bond donating and accepting groups in the side chain; • if possible avoid cocrystallization of solvent molecules; • remove multi-donating and multi-accepting terminal groups by adding N- and C-terminal blocking groups or by making a cyclic peptide. It may finally be added that the carbonyl groups of the peptide bonds are, when not accepting H atoms from N–H or O–H donors, invariably involved in weaker interactions with C a –H donors [11]. To the authors knowledge no systematic studies of the impact of C a –H???O interactions in peptides have been carried out, although several recent investigations indicate a previously unrecognized importance in protein structures [*12].

Fig. 1. Helix–loop–helix structure of a 21-residue apolar peptide. H atoms that are not involved in hydrogen bonds have been omitted for clarity. Side chains are shown as thin, black lines [**26].

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3. Linear N- and C-blocked peptides Most of the peptide structures currently being published have blocking groups at both termini and contain noncoded amino acids. Popular modifications include a,bdehydro and a,a-disubstituted amino acids, such as DZ Phe and aminoisobutyric acid (Aib), respectively. These residues are strong inducers of b-bends and 3 10 -helices, and are used in small model peptides (often without intramolecular hydrogen bonds) to study conformational preferences for selected sequences of amino acids residues [13–21]. Studies of b-sheets in somewhat larger peptides rely on formation of tight b-hairpins. – L-Pro-L-Xaa– (for type I turns) or – L-Pro-Gly– (for type I or type II turns) sequences are still in use for this purpose, but application of – D-Pro-D-Xaa / Gly– sequences are becoming more common since they promote the mirror image type I9 and type II9 turns that are stereochemically favorable for bsheet formation [*22,*23]. An elegant covalent assembly of two prefabricated units of peptide secondary structure into a single 17-residue synthetic peptide has been accomplished by Karle et al. [**24]. In the ‘Meccano set approach’ stereochemical control over peptide folding in one hairpin domain and one helical domain was achieved by incorporating residues like Aib and D-Pro. This methodology clearly departs from the ‘hydrophobic-in / hydrophilic-out’ algorithm for the amino acid residues that is usually followed in de novo protein design [25]. A second impressive example of a designer peptide is provided by Ramagopal et al. in presenting the structure of a 21-residue peptide in a helix–loop–helix motif (Fig. 1) [**26]. The strategy for reaching the desired folding pattern relied extensively on the conformation-directing control provided by a large number of DZ Phe-residues. The design furthermore involved weak interactions between the two helices in a ‘wedge into a groove’ arrangement. While most b-sheets in synthetic peptides involve antiparallel strands, there are also examples of parallel sheets. The associated model compounds must contain a non-peptide link for chain reversal. Recent examples include peptide-related systems like the D-Pro-(1,1-dimethyl)-1,2-diaminoethyl unit [*27] and diketopiperazine [*28] as well as the more exotic ferrocene structure [29]. The conformational restrictions of an a-amino acid chain constitute a driving force towards helix formation, and older textbooks on peptide structures accordingly dismissed the use of more flexible b- or g-amino acids as components in helices. Nevertheless, researchers are now becoming increasingly aware of the properties and potential usefulness of such residues, not only in structures with designed turns [*23,*27], but also in helices. An example is shown in Fig. 2, which depicts the first helix formed by a peptide constructed entirely from g-amino acid residues [*30]. Formation of an amyloid-like parallel

Fig. 2. 2.6 14 helical structure of a g-tetrapeptide. Side chains and blocking groups are shown as thin, black lines [*30].

b-sheet by the Boc-b-Ala-Aib-b-Ala-Ome tripeptide represents another interesting application for b-amino acids [*31].

4. Cyclic systems Apart from natural products (and their analogues and derivatives) [32–36], cyclic peptides are studied for conformational insight, and may include the above-mentioned non-coded residues [37]. A neat example is shown in Fig. 3 where two D-Pro-Gly type II9 turns have been introduced in a cyclodecapeptide to produce a b-sheet with all functional groups positioned on the same side of the ring. These types of scaffold are developed as tools in de novo protein design and peptide mimicry [*38]. Cyclic a-peptides with an even number of alternating Dand L-residues possess unique structural features in that they, under conditions that favor hydrogen bonding, can stack to form hollow, b-sheet-like tubular structures. These stacks can be incorporated into biological membranes, and were recently reported to possess intriguing antibacterial properties [**39]. Attention is now also focused on careful

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Fig. 3. Structural prototype of a regioselectively addressable functionalized template (RAFT). (a) b-sheet and two type II9 b-turns induced by D-Pro–Gly sequences. The side chains have been removed for clarity. (b) The side chains of the four para-nitrophenylalanine residues protrude from the same side of the cyclopeptide ring [*38].

selection of hydrophobic side chains in order to induce higher order molecular organization in the solid state through aromatic edge-to-face interactions [*7]. In the cyclic D,L-a-peptides adjacent carbonyl groups point in opposite directions, giving apolar nanotubes by stacking. By utilizing instead unsaturated d-amino acids, Gauthier et al. succeeded in aligning all carbonyl groups in the same direction, thus producing highly polarized tubes (Fig. 4) [**40]. Since all stacks in the crystal were oriented in the same direction, the resulting material was very anisotropic.

5. Free peptides The crystal structures of unblocked peptides usually 2 include one to three independent –NH 1 3 ??? OOC– hydrogen bonds, each generating a so-called head-to-tail chain. A frequent motif involves two hydrogen bonds of this type in rather planar sheets found in structures that are divided into distinct hydrophobic and hydrophilic layers. With this

type of packing arrangement an acceptor for the third amino H atom must be present in an amino acid side chain or in a cocrystallized molecule (water, other solvent or guest), since it cannot be accepted by a main chain carboxylate group. A good example is seen for (R)phenylglycyl-(R)-phenylglycine, for which the inherent demand for a third acceptor has been utilized to form an inclusion complex with methyl phenyl sulfoxide (Fig. 5) with chiral recognition of the guest molecule [*41]. A very different type of structure was observed for the first time last year for the four dipeptides Leu / Phe-Leu / Phe, which have one-dimensional hydrogen bond networks in the shape of tubes [*42]. The crystal structures result from closepacking of such tubes, mediated by hydrophobic interactions as illustrated for Phe-Phe in Fig. 6. The hydrophilic water-filled channels that run along the hexa˚ A related gonal axes have a diameter of about 10 A. structure is adopted by Trp-Gly [43], which has subsequently been shown to exhibit very rare negative thermal expansion along the helical axis [*44]. Other hydrophobic dipeptides devoid of conventional hydrogen bond acceptors (and donors) in the side chains, may form either

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hydrophobic layers or columns [45] or, when both side chains are small (not Leu or Phe), a second type of tubular structure with hydrophobic channels (Fig. 7) [46].

6. Collagen model peptides Detailed information about the structure of the fibrous protein collagen (which actually exists in at least 15 modifications) has been derived from high quality, high resolution crystallographic studies of synthetic peptides like [(Pro-Pro-Gly) 10 ] 3 [*47] and (Pro-Hyp-Gly) 3 -Ile-ThrGly-Ala-Arg-Gly-Leu-Ala-Gly-(Pro-Hyp-Gly) 4 [*48]. The work on these collagen-mimics is based on the methodology developed for solving and refining protein structures.

7. Experimental aspects Synchrotron radiation is now used routinely for collection of data sets on peptide crystals [**26,32,34,*44,49]. The ab initio crystal structure determination of the peptide Piv-Pro-Gly-NHMe (forming a Type-II b-turn) from powder diffraction data [*50] highlights the existing opportunities for structure determination when single crystals cannot be prepared.

8. Conclusions The establishment of conformational properties of regular and non-coded amino acids will continue to give important contributions to the understanding of peptide structures, and interesting applications of designed peptides become apparent, such as artificial receptors [*28]. As the ability to design molecules and domains with predetermined folding reaches maturity, more attention will probably be focused on de novo protein design, but also on intermolecular interactions and the generation of supramolecular structures, such as sheets [*31,*41] and tubular structures by cyclic molecules [*7,**39,**40]. For unblocked peptides the prediction of crystal structures will continue to be a tremendous challenge, but the unprecedented observation of nanotube formation by supramolecular aggregation of hydrophobic dipeptides illustrates nicely that taking measures to reduce the number of donors and acceptors in the molecules can yield interesting results [*42]. The left-handed dipeptide double helix of Val-Ala (Fig. 7) [46], has thus been a recurring motif in the structures of six additional dipeptides, all sharing the ¨ same hydrogen bonding pattern (C.H. Gorbitz, unpublished data). Furthermore, tripeptides with sequence Gly-Xaa1-

Fig. 4. Alignment of dipoles by stacking of cyclic tripeptides built from d-amino acids [**40].

Xaa2, where Xaa is a hydrophobic residue, display limited structural diversity. These observations indicate that reasonably stable molecular scaffolds can indeed be found for the notoriously erratic short, unblocked peptides, and that structure prediction and manipulation (crystal engineering) could be feasible. It may finally be added that for predictable construction of layered structures (such as Ref. [*41]) it would be most interesting to reduce the –NH 1 3 group from a 3-fold to a 2-fold donor (but not to a single donor by addition of blocking groups like Boc). Possible alternatives could be application of –NH 2 Me 1 (or other alkyl) or =NH 21 as the peptide N-terminal.

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Fig. 5. (a) Hydrogen bonds in a hydrophilic layer of (R)-phenylglycyl-(R)-phenylglycine. Molecules connected by hydrogen bonds are related by 2-fold screw axes. (b) Layers seen edge-on. The third amino H atom is accepted by a cocrystallized methyl phenyl sulfoxide molecule that is completely embedded in the hydrophobic layer [*41].

Fig. 6. Channels with hexagonal symmetry formed in the structure of Phe-Phe. Two amino H atoms are donated to peptide acceptors in the hydrophilic tubes generated by the peptide main chains, the third amino H-atom points straight into the channel where it is accepted by a disordered water molecule [*42].

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Fig. 7. (a) Val-Ala left-handed double helix with 3-fold screw symmetry in ball-and-stick (left) and space-fill representation (right). Side chains have been reduced to H atoms for clarity; one peptide strand is shown in a darker tone. (b) View along the channels with a double helix identified by a triangle. Side chains appear as thin, black lines [46].

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