15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide ¹⁵N is used as a probe of solvent exchange. The experiment is performed in 50% H₂O–50% D₂O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, ¹⁵N^D, into protonated species, ¹⁵N^H, as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of ¹⁵N^H/D-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5–2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.     

Detection and characterization of an early folding intermediate of T4 lysozyme using pulsed hydrogen exchange and two-dimensional NMR.

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.     

Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO). The structure of CspA has five strands of beta-sheet. Strands beta1-beta4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all-or-none global unfolding mechanism. By contrast, the protection of amide protons from strand beta5 is too weak to measure in water. Strand beta5 is hydrogen bonded to strands beta3 and beta4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands beta1-beta4 but fail to account for the weak protection in strand beta5. The most conspicuous feature of strand beta5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands beta1-beta4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand beta5 and the loops connecting the beta-strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.     

Protein hydrogen exchange mechanism: local fluctuations

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This "local fluctuation" mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix.     

The pH dependence of hydrogen-deuterium exchange in trp repressor: the exchange rate of amide protons in proteins reflects tertiary interactions,not only secondary structure.

The pH dependence of amide proton exchange rates have been measured for trp-repressor. One class of protons exchanges too fast to be measured in these experiments. Among the protons that have measurable hydrogen-deuterium exchange rates, two additional classes may be distinguished. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly with increasing base concentration (log kex versus pH). The third class of amide protons is characterized by much higher protection against exchange at higher pH. These protons are located in the core of the protein, in helices B and C. The exchange rate in the core region does not increase linearly with pH, but rather goes through a minimum around pH 6. The mechanism of exchange for the slowly exchanging core protons is interpreted in terms of the two-process model of Hilton and Woodward (1979, Biochemistry 18:5834-5841), i.e., exchange through both a local mechanism that does not require unfolding of the protein, and a mechanism involving global unfolding of the protein. The increase in exchange rates at low pH is attributed to a partial unfolding of the repressor. It is concluded that the formation of secondary structure alone is insufficient to account for the high protection factors seen in the core of native proteins at higher pH, and that tertiary interactions are essential to stabilize the structure.     

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature,pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10³-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.     

The solvent dependence of hydrogen exchange kinetics of folded proteins.

The effects of ethanol, ethylene glycol, dioxane, and other organic co-solvents upon the hydrogen exchange rates of randomly coiled oxidized RNase, native RNase, and native trypsin have been measured. The exchange rate of oxidized RNase, the model compound for the proton transfer step in hydrogen exchange, is decreased by all of the co-solvents studied at temperatures in the range 3-20 degrees. This has been ascribed to the combined effects of the disruption of peptide bond solvation due to a reduction in the concentration of water, and of changes in [OH-] ion concentration due to changes in the acid dissociation constant of water, Kw. The solvent dependence for both native RNase and native trypsin is similar in all of the solvents studied. At a low temperature (3-20 degrees), the exchange rates go through a minimum as the solvent concentration is increased. At higher temperatures (20-35 degrees) the exchange rates are increased at all concentrations of the co-solvent. The apparent rate minimum at lower temperatures is due to two opposing effects. Co-solvents decrease the rate of exchange that occurs directly from the folded molecule. At higher concentrations and higer temperature. The decrease in rates for exchange directly from folded protein is primarily due to the effects on the proton transfer step, and not to binding or the solvent effects on protein structure. The solvents used in this study have no apparent effect on conformational processes contributing to the hydrogen exchange process in folded proteins.     

Solvent accessibility in folded proteins. Studies of hydrogen exchange in trypsin.

In a native protein, the exchange of a peptide amide proton with solvent occurs by one of two pathways, either directly from the folded protein, or via unfolding, exchange taking place from the unfolded protein. From the thermal unfolding rate constants, the contribution of unfolding to the over-all kinetics as a function of solvent and temperature has been determined. Exchange involving unfolding of the protein is characterized by a high activation energy, in the range of 50 to 60 Cal per mol. The activiation energy (Eapp) of the rates of exchange directly from the folded protein is approximately 20 to 25 Cal per mol. Because for the proton transfer step, Eapp approximately equal to 20 Cal per mol, the activation energy for any contributing protein conformational process(es) is approximately equal to 0 to 5 Cal per mol. Most, if not all, of the peptide amide protons in a folded protein can exchange directly with solvent without the protein unfolding. The number of "slowly" exchanging protons at a given condition of pH and temperature is not related to a discrete structural unit, but rather to the distribution of observed rates within the broader distribution of actual rates. The large attenuation of hydrogen exchange rates in folded proteins, resulting in a distribution of first order rates over 6 orders of magnitude, is primarily due to the effects of restricted solvent accessibility of labile protons in the three-dimensional structure. Any protein conformational process, such as protein fluctuations, invoked to explain the solvent accessibility must be of low activation energy and attenuated by ethanol and other co-solvents (Woodward, C. K., Ellis, L. M., and Rosenberg, A. (1974) J. Biol. Chem. 250, 440-444).     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Pressure effect on the conformational fluctuation of apomyoglobin in the native state

We have investigated the effect of pressure on fluctuations of the native state of sperm whale apomyoglobin (apoMb) by H/D exchange, fluorescence, and limited proteolysis. The results from intrinsic fluorescence showed that a large fraction of apoMb molecules is in the native conformation in the pressure range from 0.1 to 150 MPa at 293 K and pH 6.0. The H/D exchange of protons of the individual backbone amino acids in this pressure range was monitored by NMR. The rate of H/D exchange was enhanced at high pressure, with the protection factors for some residues decreasing by factors of more than 100 compared to the values at 0.1 MPa. The amplitude of the decrease of the protection factor varied among the individual amino acids on the same secondary structure unit. This result suggests that H/D exchange in apoMb is explained best by the penetration model, in which solvent penetrates into the protein matrix via small motions. The result from limited proteolysis under high pressure showed that a pressure increase does not induce local unfolding of the secondary structure units of apoMb. Conformational fluctuations much smaller than local unfolding evidently provide pathways for water to diffuse into the protein interior, and are enhanced by an increase of pressure.     

Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments.

Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (<1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I(1)) with native-like secondary structure in the alpha-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I(1) and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I(2)). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I(1) have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.     

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.     

Deuterium/hydrogen exchange factors measured by solution nuclear magnetic resonance spectroscopy as indicators of the structure and topology of membrane proteins

Deuterium/hydrogen exchange factors (chi) were measured for the backbone amide sites of the membrane-bound forms of the 50-residue fd coat protein and the 23-residue magainin2 peptide in lipid micelles by solution nuclear magnetic resonance spectroscopy. By combining kinetic and thermodynamic effects, deuterium/hydrogen exchange factors overcome the principal limitations encountered in the measurements of kinetic protection factors and thermodynamic fractionation factors for membrane proteins. The magnitudes of the exchange factors can be correlated with the structure and topology of membrane-associated polypeptides. In fd coat protein, residues in the transmembrane helix have exchange factors that are substantially smaller than those in the amphipathic surface helix or the loop connecting the two helices. For the amphipathic helical peptide, magainin2, the exchange factors of residues exposed to the solvent are appreciably larger than those that face the hydrocarbon portion of membrane bilayers. These examples demonstrate that deuterium/hydrogen exchange factors can be measured by solution NMR spectroscopy and used to identify residues in transmembrane helices as well as to determine the polarity of amphipathic helices in membrane proteins.     

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.     

Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide.

The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions. The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T. (1991) Science 254, 539]. A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4. Protons at the hydrophobic interface show the most protection from exchange. The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues. This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils. The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths. These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.     

Hydrogen exchange in native and alcohol forms of ubiquitin.

Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by 1H NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

Backbone dynamics of rusticyanin: the high hydrophobicity and rigidity of this blue copper protein is responsible for its thermodynamic properties

Local dynamics and solute-solvent exchange properties of rusticyanin (Rc) from Thiobacillus ferrooxidans have been studied by applying heteronuclear ((1)H, (15)N) NMR spectroscopy. (15)N relaxation parameters have been determined for the reduced protein, and a model-free analysis has been applied. The high average value of the generalized order parameter, S(2) (0.93), indicates that Rc is very rigid. The analysis of cross correlation rates recorded in both the reduced and the oxidized forms conclusively proves that Rc possesses the same dynamic features in both oxidation states. The accessibility of backbone amide protons to the solvent at different time scales has also been studied by applying specific heteronuclear pulse sequences and by H(2)O/D(2)O exchange experiments. These experiments reveal that rusticyanin is extremely hydrophobic. The first N-35 amino acids, not present in the other BCPs, protect the beta-barrel core from its interaction with the solvent, and thus, this is one of the main factors contributing to the hydrophobicity. Both characteristics (high rigidity and hydrophobicity) are maintained in the metal ion surroundings.     

Effects of co-operative ligand binding on protein amide NH hydrogen exchange

Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP(+)). The substantial sizes of the residue-specific DeltaH and TDeltaS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TDeltaS energy) this is recovered, to a large extent, by the stronger binding (enthalpic DeltaH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in DeltaDeltaG(o) values for many residues being larger than the DeltaDeltaG(o) values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the DeltaDeltaG(o) values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in the ligand binding.