- Email: [email protected]
[15] Thermodynamic parameters from hydrogen exchange measurements
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[15]
Acknowledgments Work in the author's laboratory has been supported by Grant GM-29207 from the National Institutes of Health. I am grateful to B. A. Connolly, W. J. Stec, and the members of their laboratories, who collaborated in some of the studies discussed. I thank J. M. Roscnberg for providing atomic coordinates of the EcoRl endonuclease-DNA complex and L. A. Jacobson for helpful discussions.
[15] T h e r m o d y n a m i c P a r a m e t e r s f r o m H y d r o g e n Exchange Measurements
By Y A W E N
B A I , JOAN J. E N G L A N D E R , L E L A N D M A Y N E , J O H N S. M I L N E ,
and S. WALTER ENGLANDER Introduction To understand the factors that determine the stability, interactions, and function of biomolecules, the stability and changes in stability due to temperature, solvent conditions, functional state, amino acid substitutions, and other modifications must be measured in terms of real thermodynamic parameters. At this time, the measurement of thermodynamic stability depends on carrying a protein or nucleic acid through its global unfolding transition where Ku,f, the equilibrium constant for global unfolding, can be measured. One can then try to extrapolate these measurements to obtain stability parameters at milder solution conditions. The measurement of locally resolved stability parameters depends on similarly indirect methods, such as mutating the specific interaction to be studied, and then measuring the effect on global stability as just indicated. Hydrogen exchange (HX) measurements can provide a more direct means for obtaining thermodynamic parameters. This capability depends on the fact that the HX rates of structurally slowed hydrogens are determined by an equilibrium structural unfolding event. Measured HX rates can be interpreted in terms of the stability of molecular structure against the unfolding event in real free energy terms. Because protein molecules continually cycle through their globally unfolded state even under mild solution conditions, the exchange of hydrogens dependent on this behavior can provide the free energy for global unfolding at conditions far below the melting transition. The exchange of hydrogens that depend on local unfolding events can allow direct measurements of local thermodynamic
METHODS IN ENZYMOLOGY,VOL. 259
Copyright ~¢)1995 by Academic Press, Inc. All righls of reproduction in any form reserved.
[ 15]
HYDROGEN
EXCHANGE
MEASUREMENTS
345
parameters, resolved to identifiable structural sites. Measurements of the dependence of H X rates on temperature and solution conditions can provide additional thermodynamic and extrathermodynamic parameters, both for global and local unfolding. We focus here on methods for obtaining local and global thermodynamic parameters from HX data and the background knowledge necessary to interpret HX data in these terms.
Chemical Basis of Hydrogen E x c h a n g e Structural effects on the hydrogen exchange behavior of protein and nucleic acid molecules are superimposed on the underlying HX chemical rates of the exchanging groups studied. Therefore, to interpret measured H X results in structural terms, knowledge of basic H X chemistry is essential. Most protein H X studies center on the well-distributed and slowly exchanging peptide group NH. Figure 1 shows the pH dependence of peptide group hydrogen exchange in a small-peptide model, reflecting simple catalysis by O H (above - pH 3 in polypeptides) and by H + at lower pH. The H X of a freely exposed NH can be sterically blocked by neighboring bulky side chains (Fig. la) and can also be modified by inductive effects (Fig. lb) due to neighboring polar side chains. These effects along with other influences such as temperature, sequence, and isotope effects
o
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346
[151
ENERGETICS OF BIOLOGICAL MACROMOLECULES 00-
(a) 300
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240
(b) ""H~N/H
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180
80-
120 •
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pH
Fie;. 2. The pH dependence of H X for some nucleotide N H groups. 4
have now been calibrated for all the naturally occurring amino acids. 12 Figure 2 shows a similar representation for some nucleotide bases. The behavior of the adenine NH2 hydrogens includes catalysis by OH at high pH and by H + at low pH. At intermediate pH, HX proceeds by a reaction that involves an initial protonation at the N-1 position (normally buried in the double helix) followed by a deprotonation either by OH or by a general base at sufficiently high concentration. The reaction rate, proportional to [H+][OH ], produces the pH-independent behavior seen through the midpH range. When these groups are involved in structured macromolecules, the basic chemical exchange rate can be decreased by large factors. The structural protection factor is commonly expressed as P - krc/kex,3 where k,-c [Eq. (2)] is the computed rate for the group in a structureless random coil, dependent on the principles just discussed and calculated from calibrations using model molecules. From the P factor and its dependence on temperature, the equilibrium constant for the determining structural unfolding event, its free energy, and other thermodynamic parameters can be determined (see below). For further discussion of the chemical principles see Englander and Kallenbach 3 and Eigen. 4 Peptide calibrations are in Bai e t al.l,2 Nucleotide 1 y . Bai, J. S. Milne, L. Mayne, and S. W. E n g l a n d e r , Proteins: Struct., Funct., Genet. 17, 75 (1993). 2 G. P. C o n n e l l y , Y. Bai, M.-F. Jeng, and S. W. E n g l a n d e r , Proteins: Struct., Funct., Genet. 1% 87 (1993). 3 S. W. Englander and N. R. K a l l e n b a c h , Q. Rev. Biophys. 16, 521 (1984). 4 M. E i g e m Angew. Chem., Int. Eng. Ed. 3, 1 (1964).
[ 151
HYDROGEN EXCHANGEMEASUREMENTS
347
H X chemistry is discussed in more detail in Teitelbaum and Englander 5 and Gudron and Leroy. 6
S t r u c t u r a l P h y s i c s of H y d r o g e n E x c h a n g e H y d r o g e n exchange slowing by folded structure is due to a physical blocking of the chemical steps just described. Structurally slowed hydrogens are, in the great majority of cases (but not always), involved in hydrogen bonding. The rate-limiting chemical exchange event, dependent on direct attack by solvent species, cannot occur unless the blocking structure is removed. Under native conditions, this requires a transient opening reaction that has been modeled by various authors as a breaking of individual hydrogen bonds, v a local cooperative unfolding, 6 or even a gross wholemolecule transient unfolding, s Other possible mechanisms, based on various kinds of catalyst and solvent penetrational processes, have been suggested. 4.s Opening dependent exchange can be schematized as in Eq. (1). k.p
Closed
kr,
, open
> exchange:
Kop = kop/kop
(1)
/%1
In the steady state, this reaction sequence produces H X rates given by Eq. (2) kex - kopkrc/(kop + kcl + krc)
(2)
At the so-called EX1 limit 7 where kc~ ~ krc, k~x = kop, which has hardly ever been seen. Equation (3) gives the H X rate at the usually observed EX2 limit, where kcl >> k,c. kex -
(kop/kcl)krc -
Kopkrc
(3)
These equations assume that structure is stable, so that kop <{ kd and Kop ~ 1. Kop is then essentially equal to the fraction of time the restraining structure is open. For proteins, kop can be small, so that H X rates range from the free peptide rate (Fig. 1) down to rates that are slower by 10 orders of magnitude or more. Slowing factors typically seen for nucleic acids are smaller by far. Because values for k~, the H X rate for N H groups in a random chain polypeptide, are known,l'2 the m e a s u r e m e n t of k¢~ leads to Kop [Eq. (6)], H. Teitelbaum and S. W. Englander, J. Mol. Biol. 92, 55 (1975). ~M. Gudron and J.-L. Leroy, Nucleic Acids Mol. Biol. 6, 1 (1992). 7 A. Hvidt and S. O. Nielsen, Adv. Protein Chem. 21,287 (1966). s C. K. Woodward, B. D. Hilton, and E. Tuchsen, Mol. Cell. Biochem. 48, 135 (1982).
348
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[15]
and thus to the free energy for the underlying structural opening reactions, according to Eq. (4). AGHx
=
- R T in
Kop =
-RT
ln(kex/krc)
(4)
Measurement of the dependence on temperature can then provide AH and AS of the dynamic opening reaction. Effects of mutations, functional state, and other factors on stability can similarly be studied. Protein Stability P a r a m e t e r s To obtain protein stability parameters, the global unfolding equilibrium constant, K,nf, must be measured. K..f can be measured by standard methods through the region of the unfolding transition, but this requires rather severe solution conditions, involving high temperatures or fairly high concentrations of denaturants. Denaturant and thermal melting results often suffer from sloping or even curved baselines, and proteins may exhibit some degree of irreversibility or molecular association under the extreme conditions used. To obtain stability parameters under more normal solution conditions, it is necessary to circumvent these complications by analysis, and then to perform a rather lengthy extrapolation that uses the treated data obtained only over a limited range and depends on assumptions about the shape of the extrapolated curve. The Boltzmann equation requires that global unfolding occurs continuously and often reversibly even under normal solution conditions. It now appears that the exchange of the slowest NH groups in many proteins is controlled by the global unfolding reaction itself. Equation (4) connects H X rate to the free energy of the opening reaction that controls the exchange. Thus the measurement of the slow exchange rates can access the global transition and can, in principle, reveal its thermodynamic parameters, even under conditions far below the melting transition. We briefly describe work ~ that demonstrates this capability.
Denaturant Dependence Figure 3a simulates results from hypothetical denaturation experiments. Data from denaturation experiments occupy the region of Fig. 3a marked "melting region," about iGu,¢ = 0. The solid curve represents the usual linear extrapolation of these data to obtain AG,nt at zero denaturant. Whether the extrapolant should be linear 1° or curved] 1 as shown by the ~)Y. Bai, J. S. Milne, L. Mayne, and S. W. Englander, Proteins: Struct., Funct., Gener 20, 4 (1994). m C. N. Pace, this series, Vol. 131, p. 266. it K. C. A u n e and C. Tanford, Biochemistry 8, 4586 (1969).
[ 15]
HYDROGEN EXCHANGEMEASUREMENTS
349
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[GdmCl](M) FI(~. 3. Hydrogen exhange behavior expected for global and local unfolding as a function of denaturant concentration. (a) General behavior predicted by a linear extrapolation model (solid line) and a denaturant binding model (dashed line). (b) How NH groups controlled by local unfolding are progressively swept up by global unfolding. (c and d) Real data obtained from cytochrome c. 3
dashed line, is uncertain. The slope of these curves is determined by the well-known m value defined in Eq. (5). The size of m correlates with the additional denaturant binding surface exposed in the unfolding reaction. 2xG(Den) = AG(0) - re[Den]
(5)
The dependence of 2~G(Den) in the uncertain region of low denaturant concentration (Fig. 3a) can be obtained directly by HX measurements using one-dimensional nuclear magnetic resonance (1D NMR) methods. The protein is placed into D 2 0 under any conditions one chooses to investigate. After some time, most of the N H groups will exchange to ND and become invisible to proton NMR. The small number of very slowly exchanging
350
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[15]
NHs can then be resolved and measured simply by recording 1D NMR spectra as exchange protocols. Figure 3b simulates H X behavior that one can expect for N H groups that exchange by way of global and local unfolding reactions. The slowest NH groups of some hypothetical protein, controlled by the global unfolding reaction, might trace out the upper solid curve, labeled 1. If some residual structure continues to block the H X of these N H groups in the unfolded state, a subsequent unfolding reaction of even higher energy will be required, and the slowest N H groups will indicate this higher energy level, illustrated by the upper dashed curve. The lower curves in Fig. 3b (curves 2-4) indicate the behavior expected for faster exchanging N H groups. Because these N H groups are exposed to exchange by much smaller fluctuations with low m values, their rates are insensitive to denaturant concentration. As the global unfolding reaction is promoted, it ultimately overtakes and progressively dominates the exchange of the faster and faster N H groups. Figure 3c and d plot real data for cytochrome c in this context. Hydrogen exchange rates of the most slowly exchanging N H groups, in the 90s helix, follow a curving line as shown, and smoothly join denaturation data measured by a fluorescence method. The upward curvature at low denaturant is consistent with a denaturant binding model) 2 The close agreement between the H X and melting data at high denaturant suggests that the H X analysis does correctly report the global unfolding free energy. Figure 3d focuses on behavior at low denaturant. The faster exchanging NH groups in Fig. 3d initially show small m values because the fluctuations that allow their exchange expose little new surface. As the denaturant concentration increases, the global unfolding reaction is selectively promoted and comes to dominate the exchange of even the faster N H groups.
Thermal Stability Figure 4 shows hypothetical curves for the expected dependence of unfolding reactions on temperature. Curve 1 in Fig. 4a shows the typical dependence of a global unfolding reaction on temperature, with melting temperature (Tm) at 87 ° and a strong curvature as temperature decreases, dictated by a normal value for ACp, the specific heat increment between the native and unfolded forms. Free energy calculated from the H X rate of N H groups exposed to exchange by the same global unfolding [Eq. (4a)] may be expected to trace out the same curve. Curves 2-4 in Fig. 4a suggest behavior that might be found for the exchange of N H groups controlled 12 S. W. Englander and J. J. Englander, in "Structure and Dynamics of Nucleic Acids and Proteins" (E. Clementi and R. H. Sarma, eds.), p. 421. Adenine Press, New York, 1983.
[ 15]
HYDROGEN
EXCHANGE
MEASUREMENTS
351
~E98~Y97+A96
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40
60
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100
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40
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Temperature(°C) Fit;. 4. Hypothetical curves and real data for temperature-dependent HX controlled by global and local unfolding reactions? The uppermost curving line results from an opening reaction with high ,.XCp, as for global unlk)lding. Smaller unfoldings may have high (curve 2) or low (curves 3 and 4) kS~,,f. (a) Idealized curves. (b) Hydrogen exchange and melting data for cytochrome c.
by smaller unfoldings. In these latter stimulations, ~Cp was set at zero, because only a small additional exposure of hydrophobic surface is expected. Curves 3 and 4 (Fig. 4a) are for small unfoldings with small unfolding entropy (d2XGu°j/dT= 2~Su.f). Curve 2 (Fig. 4a) simulates an unfolding with somewhat larger ~Sunf. Figure 4b shows real HX data obtained well below the thermal transition for some NH groups in cytochrome c, together with thermal melting data obtained through the region of 7".,. As before (Fig. 3c), HX data for the slowest NH groups, when processed through Eq. (4), yield values for AGu,~ that merge smoothly with the conventional melting data. Again here, as temperature is increased, the global unfolding measured by HX increases and comes to dominate the exchange of even the faster exchanging NH groups, just as expected (Fig. 4a). These results, and similar data for ribonuclease A 13 reinterpreted in these terms, appear to be in good agreement with available denaturation results measured in the usual way. Thus it appears that HX results for the slowest NH groups in both cytochrome c and ribonuclease A are in fact determined by the transient global unfolding reaction. These early results suggest that HX measured for the slowest NH groups in (some) proteins can provide values for the free energy, enthalpy, and entropy of global unfolding, for the specific heat increment, and for the m value on denaturant unfolding. It is particularly promising that the HX I.~S. L. Mayo and R. L. Baldwin, Science 262, 873 (1969).
352
E N E R G E T I C S OF B I O L O G I C A L M A C R O M O L E C U L E S
[15]
measurements necessary can be made under mild conditions in which proteins are normally stable. Only 1D NMR spectra are required. The extension of these measurements to low temperatures at which Kop [Eq. (4)] becomes small and exchange rates become very slow may be accomplished by the use of higher pH, which compensates by increasing the underlying chemical exchange rate as illustrated in Fig. 1.
Local Free Energy a n d Protein Function Let us consider hemoglobin as a paradigm for protein function. Whenhemoglobin binds its initial oxygen ligands, they are bound with reduced energy. The energy is not lost but is converted into structure change energy. Parts of the protein are raised to a higher energy level. The structure changes serve as a vehicle for containing the energy and for carrying it through the protein to remote sites not yet liganded, where it is effectively converted back into functionally useful form so that subsequently bound ligand can be bound with higher affinity. The early low affinity and the later high affinity produce the S-shaped binding curve that is the signature of the allosteric function. In short, the currency of the allosteric interaction is structural free energy. The attempt to understand allostery without measuring the flow and interchange of structural energy is like trying to understand economics without measuring the interchange of money. Most of the exchangeable hydrogens in proteins are exposed to exchange by conformational fluctuations that are much smaller than the global unfolding reaction. It now appears that these fluctuations represent local unfolding reactions. If so, then locally resolved HX measurements together with Eq. (4) can make it possible to determine structurally resolved free energies of stabilization at many points throughout a protein molecule. Equation (6), derived from Eq. (4) by simple differentiation, suggests that change in the free energy of structural stabilization should destabilize affected local unfolding units, promote their transient unfolding equilibrium, and produce an increase in the HX rate of the NH groups that are controlled by those unfoldings. Thus functionally important energy changes might be quantified and located in the protein by measuring the changes in HX rate at identifiable NH sites. AAGHx --
- R T Aln gop = - R T In
kex,2/kex, i
(6)
Locating Energetic Changes by Hydrogen Exchange Interesting molecules such as hemoglobin are too large for NMR study. To use the power of hydrogen exchange to study structure and energy
[ 15]
HYDROGEN EXCHANGEMEASUREMENTS X in
353
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200
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FIG. 5. Illustration of functional labeling and fragment separation techniques and results. (a) Exchange-out data from a fully labeled Hb in deoxy (upper curve) and oxy (lower curve) forms, and the dropout curve resulting from religanding a deoxy sample during exchangeoutJ 4 (b) lllustration of the functional labeling approach using a limited exchange-in for oxy (fast form) Hb, switch to the deoxy (slow exchanging) form, and a subsequent exchange-out chase to remove label from functionally insensitive sites. (c) The "jump class" NH groups as they exchange-out in deoxyHb after being labeled for 1 rain in oxyHb, m (d) An HPLC trace from a fragment separation experiment after functional labeling of the sensitive set of NH groups at the u-chain N terminus.
change in hemoglobin, it has been necessary to develop special lritium exchange labeling methods. These are illustrated in Fig. 5. F i g u r e 5 a t4 shows tritium exchange-out data for fully labeled oxy- and deoxyHb. In these experiments, oxyHb is first exposed to exchange-in in tritiated water for a long time (3 days) at elevated pH and temperature to promote full exchange (pH 9, 37°). The free tritium is then removed by passage through a short gel-filtration column. The bound tritium starts to exchange out, each NH site at its own rate. Samples are taken in tin]e, the 14S. W. Englander and C. Mauel, J. Biol. Chem. 247, 2387 (1972).
354
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[15]
still partially tritiated protein is separated from the newly free tritium by gel filtration, and the eluant is measured for protein concentration (absorbance) and carried tritium level (liquid scintillation). These data are calculated to obtain the exchange-out curves shown (Fig. 5a). The curves in Fig. 5a show that some hydrogens are slower in deoxyHb than in oxyHb. When ligand is added back to exchanging deoxyHb, the protein reverts to its fast form, and the allosterically sensitive N H sites that are slowed in the deoxy form become fast again. We want to locate these sensitive N H groups in the protein and measure their number and their rates in both allosteric forms. For this purpose it is necessary to separate the allosterically sensitive sites from the large background of insensitive sites, This can be done by a "functional labeling" method. 12 Figure 5b illustrates a method that causes allosterically sensitive sites to become selectively labeled. Hemoglobin in the fast exchanging oxy form is initially exchanged in for only a limited time. NH groups that exchange in this time period, including sensitive and insensitive sites, become tritium labeled. Hemoglobin is then switched to its slow exchanging deoxy form and exchanged out using gel-filtration as before. Tritium on insensitive sites exchanges out as fast as it exchanged in and is soon lost. But label on sensitive sites is now locked in to a more slowly exchanging form. Thus after some exchange-out time, one is left with a sample having tritium label just on allosterically sensitive sties. These can be measured in the slow, deoxy form, or their accelerated exchange-out in liganded Hb can be measured by adding back 02 or CO. By use of the functional labeling method, the entire multidecade time scale of Hb HX has been surveyed for allosterically sensitive N H groups. 15 It turns out that only about one-fourth of Hb peptide N H groups are sensitive to the allosteric form of the protein. These are always faster in the oxy form than in deoxyHb, by factors ranging from - 15-fold to more t h a n 10 4. An extreme example is shown in Fig. 5c. 16 Here a small set of three Nh groups that become labeled during 1 min of exchange-in in oxyHb are hugely slowed in deoxyHb, and exchange out on a 100-hr time scale. This behavior can be interpreted in terms of the destabilization of local structural elements on ligand binding, as in Eq. (6). One wants to locate these interesting sites in the protein. Fragmentation-separation methods can accomplish this] 5-19 Here a sample of protein 15 E. L. Malin and S. W. Englander, J. Biol. Chem. 255, 10695 (1980). tt~ R. K. H. Liem, D. B. Calhoun, J. J. Englander, and S. W. Englander, J. Biol. Chem. 255,
10687 (1980). 17 j. j. Rosa and F. M. Richards, J. Mol. Biol. 133, 399 (1979). isj. R. Rogero, J. J. Englander, and S. W. Englander, this series, Vol. 131L, p. 508. 19 N. M. Allewell, J. Biochem. Biophys. Methods 7, 345 (1983).
[ 15]
HYDROGEN EXCHANGE MEASUREMENTS
355
TABLE I LGSS OF COOPERATIVE FREE ENERGY IN HEMOBEOGIN MEASURED BY HYDROGEN EXCHANGE AND GLOBAL METHODS a
Allosteric cooperativity Hb
Hydrogen exchange (structure stability)
No PPi Bunbury NES DesHis Kariya NES-desArg Oxygenation
0.75 1.1 3.0 3.0 4.5 6.2 8.1
Subunit dissociation 0.5 3.0 2.7 5.7
0.2 3.6 2.7 5.7
8.2 8.3
02 binding 0.4 0.6 2.5 3.9 6.3 6.9 8.3
"The HX values are the sum of the free energy losses found at the a-chain N terminus and the/~-chain C terminus. The subunit dissociation values either were obtained from the change in subunit assembly equilibrium constant for unliganded Hb or include also the small correction for changes in R-state Hb. The O2-binding values were obtained from the change in median partial pressure of liganding. Conditions: pH 7.4, 0 to 5°, no phosphate effector. (From Englander et al.21)
s e l e c t i v e l y l a b e l e d as just d e s c r i b e d is p l u n g e d into slow e x c h a n g e conditions, p H - 3 at 0 ° (see Fig. 1). T h e p r o t e i n d e n a t u r e s , b u t e x c h a n g e halft i m e h e r e is o v e r 1 hr. This allows s o m e t i m e to f r a g m e n t t h e p r o t e i n with acid p r o t e a s e ( p e p s i n , 5 min), a n d s e p a r a t e the f r a g m e n t s b y highp e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) run at p H 3 a n d 0 °. A trace o f this k i n d is s h o w n in Fig. 5d. F r a c t i o n s of the e l u a n t c o n t a i n i n g s e p a r a t e d f r a g m e n t s can be a n a l y z e d for f r a g m e n t c o n c e n t r a t i o n a n d carried t r i t i u m level. In this w a y the a l l o s t e r i c a l l y sensitive sites can b e l o c a t e d at m o d e r a t e r e s o l u t i o n . F u r t h e r s u b f r a g m e n t a t i o n e x p e r i m e n t s m a y m o r e closely i d e n tify t h e i r p r e c i s e locations. D a t a such as this f r o m s a m p l e s t a k e n as a f u n c t i o n of e x c h a n g e - o u t t i m e can t h e n b e u s e d to m e a s u r e the e x c h a n g e r a t e s o f t h e s e sites in b o t h a l l o s t e r i c f o r m s a n d thus i n d i c a t e the free e n e r g y c h a n g e to be a t t r i b u t e d to the p o s i t i o n s identified.
Allosteric Energy Equal to Structural Free Energy Change C r y s t a l l o g r a p h i c results i n d i c a t e t h a t t h e b i n d i n g of h e m o g l o b i n ligands causes s o m e s t r u c t u r a l salt links to b r e a k . 2° T h e s e b o n d c h a n g e s m a y f o r m p a r t of t h e chain of e n e r g e t i c e v e n t s that i n t e r c o n v e r t s l i g a n d - b i n d i n g ene r g y a n d s t r u c t u r a l c h a n g e e n e r g y . T h e ability o f the m e t h o d s just d e s c r i b e d to m e a s u r e locally r e s o l v e d e n e r g i e s has b e e n t e s t e d in a series of h e m o g l o 2oM. F. Perutz, Nature (London) 228, 726 (1970).
356
E N E R G E T I C S OF B I O L O G I C A L M A C R O M O L E C U L E S
[15]
bin molecules in which particular candidate linkages have been broken by mutation, chemical modification, or the withdrawal of allosteric effector. In these variants, the loss of cooperative, allosteric energy was measured at two positions thought to be centrally important for hemoglobin allostery, namely at the wchain N terminus and the/3-chain C terminus. The same molecules were tested by established methods that can measure cooperative energy at a global level, namely by ligand binding and by subunit dissociation experiments. Results available are summarized in Table I. 2~ When Hb is fully liganded and switched to the R state, global methods register a loss of allosteric energy of 8.3 kcal/mol (at 0°, pH 7.4). The H X measurements, summed at the two positions noted, indicate 8.1 kcal/mol. Modifications that still leave deoxyHb in the T state impose lesser losses of cooperative energy. Table I shows that the structural energy loss obtained by HX methods tracks the energies obtained by the global methods rather well. Summary Just as exchangeable hydrogens that are controlled by global unfolding can be used to measure thermodynamic parameters at a global level, hydrogens that are exposed to exchange by local unfolding reactions may be used to obtain locally resolved energy parameters. Results with the hemoglobin system demonstrate the ability of H X methods to locate functionally important changes in a protein and to measure the energetic contribution of each. These results offer the promise that H X measurements may be used to delineate, in terms of definable bonds and their energies and interactions, the network of interactions that Hb and other proteins use to produce their various functions. Acknowledgment This work was supported by NIH Grants DKll295 and GM31847.
21S. W. Englander,J. J. Englander, R. E. McKinnie,G. K. Ackers, G. J. Turner, J. A. Westrick, and S. J. Gill, Science 256, 1684 (1992).
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[15]
Acknowledgments Work in the author's laboratory has been supported by Grant GM-29207 from the National Institutes of Health. I am grateful to B. A. Connolly, W. J. Stec, and the members of their laboratories, who collaborated in some of the studies discussed. I thank J. M. Roscnberg for providing atomic coordinates of the EcoRl endonuclease-DNA complex and L. A. Jacobson for helpful discussions.
[15] T h e r m o d y n a m i c P a r a m e t e r s f r o m H y d r o g e n Exchange Measurements
By Y A W E N
B A I , JOAN J. E N G L A N D E R , L E L A N D M A Y N E , J O H N S. M I L N E ,
and S. WALTER ENGLANDER Introduction To understand the factors that determine the stability, interactions, and function of biomolecules, the stability and changes in stability due to temperature, solvent conditions, functional state, amino acid substitutions, and other modifications must be measured in terms of real thermodynamic parameters. At this time, the measurement of thermodynamic stability depends on carrying a protein or nucleic acid through its global unfolding transition where Ku,f, the equilibrium constant for global unfolding, can be measured. One can then try to extrapolate these measurements to obtain stability parameters at milder solution conditions. The measurement of locally resolved stability parameters depends on similarly indirect methods, such as mutating the specific interaction to be studied, and then measuring the effect on global stability as just indicated. Hydrogen exchange (HX) measurements can provide a more direct means for obtaining thermodynamic parameters. This capability depends on the fact that the HX rates of structurally slowed hydrogens are determined by an equilibrium structural unfolding event. Measured HX rates can be interpreted in terms of the stability of molecular structure against the unfolding event in real free energy terms. Because protein molecules continually cycle through their globally unfolded state even under mild solution conditions, the exchange of hydrogens dependent on this behavior can provide the free energy for global unfolding at conditions far below the melting transition. The exchange of hydrogens that depend on local unfolding events can allow direct measurements of local thermodynamic
METHODS IN ENZYMOLOGY,VOL. 259
Copyright ~¢)1995 by Academic Press, Inc. All righls of reproduction in any form reserved.
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MEASUREMENTS
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parameters, resolved to identifiable structural sites. Measurements of the dependence of H X rates on temperature and solution conditions can provide additional thermodynamic and extrathermodynamic parameters, both for global and local unfolding. We focus here on methods for obtaining local and global thermodynamic parameters from HX data and the background knowledge necessary to interpret HX data in these terms.
Chemical Basis of Hydrogen E x c h a n g e Structural effects on the hydrogen exchange behavior of protein and nucleic acid molecules are superimposed on the underlying HX chemical rates of the exchanging groups studied. Therefore, to interpret measured H X results in structural terms, knowledge of basic H X chemistry is essential. Most protein H X studies center on the well-distributed and slowly exchanging peptide group NH. Figure 1 shows the pH dependence of peptide group hydrogen exchange in a small-peptide model, reflecting simple catalysis by O H (above - pH 3 in polypeptides) and by H + at lower pH. The H X of a freely exposed NH can be sterically blocked by neighboring bulky side chains (Fig. la) and can also be modified by inductive effects (Fig. lb) due to neighboring polar side chains. These effects along with other influences such as temperature, sequence, and isotope effects
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pD FK;. 1. Hydrogen exhange behavior of Dee peptide NH in small-molecule models. Stcric blocking effects (a) and inductive effects (b) of neighboring amino acid side chains are illustrated. I Polypeptides show pH minima between pH 2.5 and 3.
346
[151
ENERGETICS OF BIOLOGICAL MACROMOLECULES 00-
(a) 300
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240
(b) ""H~N/H
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180
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120 •
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have now been calibrated for all the naturally occurring amino acids. 12 Figure 2 shows a similar representation for some nucleotide bases. The behavior of the adenine NH2 hydrogens includes catalysis by OH at high pH and by H + at low pH. At intermediate pH, HX proceeds by a reaction that involves an initial protonation at the N-1 position (normally buried in the double helix) followed by a deprotonation either by OH or by a general base at sufficiently high concentration. The reaction rate, proportional to [H+][OH ], produces the pH-independent behavior seen through the midpH range. When these groups are involved in structured macromolecules, the basic chemical exchange rate can be decreased by large factors. The structural protection factor is commonly expressed as P - krc/kex,3 where k,-c [Eq. (2)] is the computed rate for the group in a structureless random coil, dependent on the principles just discussed and calculated from calibrations using model molecules. From the P factor and its dependence on temperature, the equilibrium constant for the determining structural unfolding event, its free energy, and other thermodynamic parameters can be determined (see below). For further discussion of the chemical principles see Englander and Kallenbach 3 and Eigen. 4 Peptide calibrations are in Bai e t al.l,2 Nucleotide 1 y . Bai, J. S. Milne, L. Mayne, and S. W. E n g l a n d e r , Proteins: Struct., Funct., Genet. 17, 75 (1993). 2 G. P. C o n n e l l y , Y. Bai, M.-F. Jeng, and S. W. E n g l a n d e r , Proteins: Struct., Funct., Genet. 1% 87 (1993). 3 S. W. Englander and N. R. K a l l e n b a c h , Q. Rev. Biophys. 16, 521 (1984). 4 M. E i g e m Angew. Chem., Int. Eng. Ed. 3, 1 (1964).
[ 151
HYDROGEN EXCHANGEMEASUREMENTS
347
H X chemistry is discussed in more detail in Teitelbaum and Englander 5 and Gudron and Leroy. 6
S t r u c t u r a l P h y s i c s of H y d r o g e n E x c h a n g e H y d r o g e n exchange slowing by folded structure is due to a physical blocking of the chemical steps just described. Structurally slowed hydrogens are, in the great majority of cases (but not always), involved in hydrogen bonding. The rate-limiting chemical exchange event, dependent on direct attack by solvent species, cannot occur unless the blocking structure is removed. Under native conditions, this requires a transient opening reaction that has been modeled by various authors as a breaking of individual hydrogen bonds, v a local cooperative unfolding, 6 or even a gross wholemolecule transient unfolding, s Other possible mechanisms, based on various kinds of catalyst and solvent penetrational processes, have been suggested. 4.s Opening dependent exchange can be schematized as in Eq. (1). k.p
Closed
kr,
, open
> exchange:
Kop = kop/kop
(1)
/%1
In the steady state, this reaction sequence produces H X rates given by Eq. (2) kex - kopkrc/(kop + kcl + krc)
(2)
At the so-called EX1 limit 7 where kc~ ~ krc, k~x = kop, which has hardly ever been seen. Equation (3) gives the H X rate at the usually observed EX2 limit, where kcl >> k,c. kex -
(kop/kcl)krc -
Kopkrc
(3)
These equations assume that structure is stable, so that kop <{ kd and Kop ~ 1. Kop is then essentially equal to the fraction of time the restraining structure is open. For proteins, kop can be small, so that H X rates range from the free peptide rate (Fig. 1) down to rates that are slower by 10 orders of magnitude or more. Slowing factors typically seen for nucleic acids are smaller by far. Because values for k~, the H X rate for N H groups in a random chain polypeptide, are known,l'2 the m e a s u r e m e n t of k¢~ leads to Kop [Eq. (6)], H. Teitelbaum and S. W. Englander, J. Mol. Biol. 92, 55 (1975). ~M. Gudron and J.-L. Leroy, Nucleic Acids Mol. Biol. 6, 1 (1992). 7 A. Hvidt and S. O. Nielsen, Adv. Protein Chem. 21,287 (1966). s C. K. Woodward, B. D. Hilton, and E. Tuchsen, Mol. Cell. Biochem. 48, 135 (1982).
348
ENERGETICS OF BIOLOGICAL MACROMOLECULES
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and thus to the free energy for the underlying structural opening reactions, according to Eq. (4). AGHx
=
- R T in
Kop =
-RT
ln(kex/krc)
(4)
Measurement of the dependence on temperature can then provide AH and AS of the dynamic opening reaction. Effects of mutations, functional state, and other factors on stability can similarly be studied. Protein Stability P a r a m e t e r s To obtain protein stability parameters, the global unfolding equilibrium constant, K,nf, must be measured. K..f can be measured by standard methods through the region of the unfolding transition, but this requires rather severe solution conditions, involving high temperatures or fairly high concentrations of denaturants. Denaturant and thermal melting results often suffer from sloping or even curved baselines, and proteins may exhibit some degree of irreversibility or molecular association under the extreme conditions used. To obtain stability parameters under more normal solution conditions, it is necessary to circumvent these complications by analysis, and then to perform a rather lengthy extrapolation that uses the treated data obtained only over a limited range and depends on assumptions about the shape of the extrapolated curve. The Boltzmann equation requires that global unfolding occurs continuously and often reversibly even under normal solution conditions. It now appears that the exchange of the slowest NH groups in many proteins is controlled by the global unfolding reaction itself. Equation (4) connects H X rate to the free energy of the opening reaction that controls the exchange. Thus the measurement of the slow exchange rates can access the global transition and can, in principle, reveal its thermodynamic parameters, even under conditions far below the melting transition. We briefly describe work ~ that demonstrates this capability.
Denaturant Dependence Figure 3a simulates results from hypothetical denaturation experiments. Data from denaturation experiments occupy the region of Fig. 3a marked "melting region," about iGu,¢ = 0. The solid curve represents the usual linear extrapolation of these data to obtain AG,nt at zero denaturant. Whether the extrapolant should be linear 1° or curved] 1 as shown by the ~)Y. Bai, J. S. Milne, L. Mayne, and S. W. Englander, Proteins: Struct., Funct., Gener 20, 4 (1994). m C. N. Pace, this series, Vol. 131, p. 266. it K. C. A u n e and C. Tanford, Biochemistry 8, 4586 (1969).
[ 15]
HYDROGEN EXCHANGEMEASUREMENTS
349
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[GdmCl](M) FI(~. 3. Hydrogen exhange behavior expected for global and local unfolding as a function of denaturant concentration. (a) General behavior predicted by a linear extrapolation model (solid line) and a denaturant binding model (dashed line). (b) How NH groups controlled by local unfolding are progressively swept up by global unfolding. (c and d) Real data obtained from cytochrome c. 3
dashed line, is uncertain. The slope of these curves is determined by the well-known m value defined in Eq. (5). The size of m correlates with the additional denaturant binding surface exposed in the unfolding reaction. 2xG(Den) = AG(0) - re[Den]
(5)
The dependence of 2~G(Den) in the uncertain region of low denaturant concentration (Fig. 3a) can be obtained directly by HX measurements using one-dimensional nuclear magnetic resonance (1D NMR) methods. The protein is placed into D 2 0 under any conditions one chooses to investigate. After some time, most of the N H groups will exchange to ND and become invisible to proton NMR. The small number of very slowly exchanging
350
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[15]
NHs can then be resolved and measured simply by recording 1D NMR spectra as exchange protocols. Figure 3b simulates H X behavior that one can expect for N H groups that exchange by way of global and local unfolding reactions. The slowest NH groups of some hypothetical protein, controlled by the global unfolding reaction, might trace out the upper solid curve, labeled 1. If some residual structure continues to block the H X of these N H groups in the unfolded state, a subsequent unfolding reaction of even higher energy will be required, and the slowest N H groups will indicate this higher energy level, illustrated by the upper dashed curve. The lower curves in Fig. 3b (curves 2-4) indicate the behavior expected for faster exchanging N H groups. Because these N H groups are exposed to exchange by much smaller fluctuations with low m values, their rates are insensitive to denaturant concentration. As the global unfolding reaction is promoted, it ultimately overtakes and progressively dominates the exchange of the faster and faster N H groups. Figure 3c and d plot real data for cytochrome c in this context. Hydrogen exchange rates of the most slowly exchanging N H groups, in the 90s helix, follow a curving line as shown, and smoothly join denaturation data measured by a fluorescence method. The upward curvature at low denaturant is consistent with a denaturant binding model) 2 The close agreement between the H X and melting data at high denaturant suggests that the H X analysis does correctly report the global unfolding free energy. Figure 3d focuses on behavior at low denaturant. The faster exchanging NH groups in Fig. 3d initially show small m values because the fluctuations that allow their exchange expose little new surface. As the denaturant concentration increases, the global unfolding reaction is selectively promoted and comes to dominate the exchange of even the faster N H groups.
Thermal Stability Figure 4 shows hypothetical curves for the expected dependence of unfolding reactions on temperature. Curve 1 in Fig. 4a shows the typical dependence of a global unfolding reaction on temperature, with melting temperature (Tm) at 87 ° and a strong curvature as temperature decreases, dictated by a normal value for ACp, the specific heat increment between the native and unfolded forms. Free energy calculated from the H X rate of N H groups exposed to exchange by the same global unfolding [Eq. (4a)] may be expected to trace out the same curve. Curves 2-4 in Fig. 4a suggest behavior that might be found for the exchange of N H groups controlled 12 S. W. Englander and J. J. Englander, in "Structure and Dynamics of Nucleic Acids and Proteins" (E. Clementi and R. H. Sarma, eds.), p. 421. Adenine Press, New York, 1983.
[ 15]
HYDROGEN
EXCHANGE
MEASUREMENTS
351
~E98~Y97+A96
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<] 20
40
60
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100
20
40
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80
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Temperature(°C) Fit;. 4. Hypothetical curves and real data for temperature-dependent HX controlled by global and local unfolding reactions? The uppermost curving line results from an opening reaction with high ,.XCp, as for global unlk)lding. Smaller unfoldings may have high (curve 2) or low (curves 3 and 4) kS~,,f. (a) Idealized curves. (b) Hydrogen exchange and melting data for cytochrome c.
by smaller unfoldings. In these latter stimulations, ~Cp was set at zero, because only a small additional exposure of hydrophobic surface is expected. Curves 3 and 4 (Fig. 4a) are for small unfoldings with small unfolding entropy (d2XGu°j/dT= 2~Su.f). Curve 2 (Fig. 4a) simulates an unfolding with somewhat larger ~Sunf. Figure 4b shows real HX data obtained well below the thermal transition for some NH groups in cytochrome c, together with thermal melting data obtained through the region of 7".,. As before (Fig. 3c), HX data for the slowest NH groups, when processed through Eq. (4), yield values for AGu,~ that merge smoothly with the conventional melting data. Again here, as temperature is increased, the global unfolding measured by HX increases and comes to dominate the exchange of even the faster exchanging NH groups, just as expected (Fig. 4a). These results, and similar data for ribonuclease A 13 reinterpreted in these terms, appear to be in good agreement with available denaturation results measured in the usual way. Thus it appears that HX results for the slowest NH groups in both cytochrome c and ribonuclease A are in fact determined by the transient global unfolding reaction. These early results suggest that HX measured for the slowest NH groups in (some) proteins can provide values for the free energy, enthalpy, and entropy of global unfolding, for the specific heat increment, and for the m value on denaturant unfolding. It is particularly promising that the HX I.~S. L. Mayo and R. L. Baldwin, Science 262, 873 (1969).
352
E N E R G E T I C S OF B I O L O G I C A L M A C R O M O L E C U L E S
[15]
measurements necessary can be made under mild conditions in which proteins are normally stable. Only 1D NMR spectra are required. The extension of these measurements to low temperatures at which Kop [Eq. (4)] becomes small and exchange rates become very slow may be accomplished by the use of higher pH, which compensates by increasing the underlying chemical exchange rate as illustrated in Fig. 1.
Local Free Energy a n d Protein Function Let us consider hemoglobin as a paradigm for protein function. Whenhemoglobin binds its initial oxygen ligands, they are bound with reduced energy. The energy is not lost but is converted into structure change energy. Parts of the protein are raised to a higher energy level. The structure changes serve as a vehicle for containing the energy and for carrying it through the protein to remote sites not yet liganded, where it is effectively converted back into functionally useful form so that subsequently bound ligand can be bound with higher affinity. The early low affinity and the later high affinity produce the S-shaped binding curve that is the signature of the allosteric function. In short, the currency of the allosteric interaction is structural free energy. The attempt to understand allostery without measuring the flow and interchange of structural energy is like trying to understand economics without measuring the interchange of money. Most of the exchangeable hydrogens in proteins are exposed to exchange by conformational fluctuations that are much smaller than the global unfolding reaction. It now appears that these fluctuations represent local unfolding reactions. If so, then locally resolved HX measurements together with Eq. (4) can make it possible to determine structurally resolved free energies of stabilization at many points throughout a protein molecule. Equation (6), derived from Eq. (4) by simple differentiation, suggests that change in the free energy of structural stabilization should destabilize affected local unfolding units, promote their transient unfolding equilibrium, and produce an increase in the HX rate of the NH groups that are controlled by those unfoldings. Thus functionally important energy changes might be quantified and located in the protein by measuring the changes in HX rate at identifiable NH sites. AAGHx --
- R T Aln gop = - R T In
kex,2/kex, i
(6)
Locating Energetic Changes by Hydrogen Exchange Interesting molecules such as hemoglobin are too large for NMR study. To use the power of hydrogen exchange to study structure and energy
[ 15]
HYDROGEN EXCHANGEMEASUREMENTS X in
353
x out
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sensiti,,'o NHs
insensitive NHs
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,
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,
.
,
2
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.
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.
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t
i
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Ol
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150
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200
o
60
30
90
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FIG. 5. Illustration of functional labeling and fragment separation techniques and results. (a) Exchange-out data from a fully labeled Hb in deoxy (upper curve) and oxy (lower curve) forms, and the dropout curve resulting from religanding a deoxy sample during exchangeoutJ 4 (b) lllustration of the functional labeling approach using a limited exchange-in for oxy (fast form) Hb, switch to the deoxy (slow exchanging) form, and a subsequent exchange-out chase to remove label from functionally insensitive sites. (c) The "jump class" NH groups as they exchange-out in deoxyHb after being labeled for 1 rain in oxyHb, m (d) An HPLC trace from a fragment separation experiment after functional labeling of the sensitive set of NH groups at the u-chain N terminus.
change in hemoglobin, it has been necessary to develop special lritium exchange labeling methods. These are illustrated in Fig. 5. F i g u r e 5 a t4 shows tritium exchange-out data for fully labeled oxy- and deoxyHb. In these experiments, oxyHb is first exposed to exchange-in in tritiated water for a long time (3 days) at elevated pH and temperature to promote full exchange (pH 9, 37°). The free tritium is then removed by passage through a short gel-filtration column. The bound tritium starts to exchange out, each NH site at its own rate. Samples are taken in tin]e, the 14S. W. Englander and C. Mauel, J. Biol. Chem. 247, 2387 (1972).
354
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[15]
still partially tritiated protein is separated from the newly free tritium by gel filtration, and the eluant is measured for protein concentration (absorbance) and carried tritium level (liquid scintillation). These data are calculated to obtain the exchange-out curves shown (Fig. 5a). The curves in Fig. 5a show that some hydrogens are slower in deoxyHb than in oxyHb. When ligand is added back to exchanging deoxyHb, the protein reverts to its fast form, and the allosterically sensitive N H sites that are slowed in the deoxy form become fast again. We want to locate these sensitive N H groups in the protein and measure their number and their rates in both allosteric forms. For this purpose it is necessary to separate the allosterically sensitive sites from the large background of insensitive sites, This can be done by a "functional labeling" method. 12 Figure 5b illustrates a method that causes allosterically sensitive sites to become selectively labeled. Hemoglobin in the fast exchanging oxy form is initially exchanged in for only a limited time. NH groups that exchange in this time period, including sensitive and insensitive sites, become tritium labeled. Hemoglobin is then switched to its slow exchanging deoxy form and exchanged out using gel-filtration as before. Tritium on insensitive sites exchanges out as fast as it exchanged in and is soon lost. But label on sensitive sites is now locked in to a more slowly exchanging form. Thus after some exchange-out time, one is left with a sample having tritium label just on allosterically sensitive sties. These can be measured in the slow, deoxy form, or their accelerated exchange-out in liganded Hb can be measured by adding back 02 or CO. By use of the functional labeling method, the entire multidecade time scale of Hb HX has been surveyed for allosterically sensitive N H groups. 15 It turns out that only about one-fourth of Hb peptide N H groups are sensitive to the allosteric form of the protein. These are always faster in the oxy form than in deoxyHb, by factors ranging from - 15-fold to more t h a n 10 4. An extreme example is shown in Fig. 5c. 16 Here a small set of three Nh groups that become labeled during 1 min of exchange-in in oxyHb are hugely slowed in deoxyHb, and exchange out on a 100-hr time scale. This behavior can be interpreted in terms of the destabilization of local structural elements on ligand binding, as in Eq. (6). One wants to locate these interesting sites in the protein. Fragmentation-separation methods can accomplish this] 5-19 Here a sample of protein 15 E. L. Malin and S. W. Englander, J. Biol. Chem. 255, 10695 (1980). tt~ R. K. H. Liem, D. B. Calhoun, J. J. Englander, and S. W. Englander, J. Biol. Chem. 255,
10687 (1980). 17 j. j. Rosa and F. M. Richards, J. Mol. Biol. 133, 399 (1979). isj. R. Rogero, J. J. Englander, and S. W. Englander, this series, Vol. 131L, p. 508. 19 N. M. Allewell, J. Biochem. Biophys. Methods 7, 345 (1983).
[ 15]
HYDROGEN EXCHANGE MEASUREMENTS
355
TABLE I LGSS OF COOPERATIVE FREE ENERGY IN HEMOBEOGIN MEASURED BY HYDROGEN EXCHANGE AND GLOBAL METHODS a
Allosteric cooperativity Hb
Hydrogen exchange (structure stability)
No PPi Bunbury NES DesHis Kariya NES-desArg Oxygenation
0.75 1.1 3.0 3.0 4.5 6.2 8.1
Subunit dissociation 0.5 3.0 2.7 5.7
0.2 3.6 2.7 5.7
8.2 8.3
02 binding 0.4 0.6 2.5 3.9 6.3 6.9 8.3
"The HX values are the sum of the free energy losses found at the a-chain N terminus and the/~-chain C terminus. The subunit dissociation values either were obtained from the change in subunit assembly equilibrium constant for unliganded Hb or include also the small correction for changes in R-state Hb. The O2-binding values were obtained from the change in median partial pressure of liganding. Conditions: pH 7.4, 0 to 5°, no phosphate effector. (From Englander et al.21)
s e l e c t i v e l y l a b e l e d as just d e s c r i b e d is p l u n g e d into slow e x c h a n g e conditions, p H - 3 at 0 ° (see Fig. 1). T h e p r o t e i n d e n a t u r e s , b u t e x c h a n g e halft i m e h e r e is o v e r 1 hr. This allows s o m e t i m e to f r a g m e n t t h e p r o t e i n with acid p r o t e a s e ( p e p s i n , 5 min), a n d s e p a r a t e the f r a g m e n t s b y highp e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) run at p H 3 a n d 0 °. A trace o f this k i n d is s h o w n in Fig. 5d. F r a c t i o n s of the e l u a n t c o n t a i n i n g s e p a r a t e d f r a g m e n t s can be a n a l y z e d for f r a g m e n t c o n c e n t r a t i o n a n d carried t r i t i u m level. In this w a y the a l l o s t e r i c a l l y sensitive sites can b e l o c a t e d at m o d e r a t e r e s o l u t i o n . F u r t h e r s u b f r a g m e n t a t i o n e x p e r i m e n t s m a y m o r e closely i d e n tify t h e i r p r e c i s e locations. D a t a such as this f r o m s a m p l e s t a k e n as a f u n c t i o n of e x c h a n g e - o u t t i m e can t h e n b e u s e d to m e a s u r e the e x c h a n g e r a t e s o f t h e s e sites in b o t h a l l o s t e r i c f o r m s a n d thus i n d i c a t e the free e n e r g y c h a n g e to be a t t r i b u t e d to the p o s i t i o n s identified.
Allosteric Energy Equal to Structural Free Energy Change C r y s t a l l o g r a p h i c results i n d i c a t e t h a t t h e b i n d i n g of h e m o g l o b i n ligands causes s o m e s t r u c t u r a l salt links to b r e a k . 2° T h e s e b o n d c h a n g e s m a y f o r m p a r t of t h e chain of e n e r g e t i c e v e n t s that i n t e r c o n v e r t s l i g a n d - b i n d i n g ene r g y a n d s t r u c t u r a l c h a n g e e n e r g y . T h e ability o f the m e t h o d s just d e s c r i b e d to m e a s u r e locally r e s o l v e d e n e r g i e s has b e e n t e s t e d in a series of h e m o g l o 2oM. F. Perutz, Nature (London) 228, 726 (1970).
356
E N E R G E T I C S OF B I O L O G I C A L M A C R O M O L E C U L E S
[15]
bin molecules in which particular candidate linkages have been broken by mutation, chemical modification, or the withdrawal of allosteric effector. In these variants, the loss of cooperative, allosteric energy was measured at two positions thought to be centrally important for hemoglobin allostery, namely at the wchain N terminus and the/3-chain C terminus. The same molecules were tested by established methods that can measure cooperative energy at a global level, namely by ligand binding and by subunit dissociation experiments. Results available are summarized in Table I. 2~ When Hb is fully liganded and switched to the R state, global methods register a loss of allosteric energy of 8.3 kcal/mol (at 0°, pH 7.4). The H X measurements, summed at the two positions noted, indicate 8.1 kcal/mol. Modifications that still leave deoxyHb in the T state impose lesser losses of cooperative energy. Table I shows that the structural energy loss obtained by HX methods tracks the energies obtained by the global methods rather well. Summary Just as exchangeable hydrogens that are controlled by global unfolding can be used to measure thermodynamic parameters at a global level, hydrogens that are exposed to exchange by local unfolding reactions may be used to obtain locally resolved energy parameters. Results with the hemoglobin system demonstrate the ability of H X methods to locate functionally important changes in a protein and to measure the energetic contribution of each. These results offer the promise that H X measurements may be used to delineate, in terms of definable bonds and their energies and interactions, the network of interactions that Hb and other proteins use to produce their various functions. Acknowledgment This work was supported by NIH Grants DKll295 and GM31847.
21S. W. Englander,J. J. Englander, R. E. McKinnie,G. K. Ackers, G. J. Turner, J. A. Westrick, and S. J. Gill, Science 256, 1684 (1992).