Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Multi-state unfolding of the alpha subunit of tryptophan synthase, a TIM barrel protein: insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry

The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured. The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS.     

Folding subdomains of thioredoxin characterized by native-state hydrogen exchange

Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (DeltaG(op)) was determined at each of the 24 amide hydrogen sites. The values of DeltaG(op) for the amide hydrogens belonging to residues in the helices alpha(1), alpha(2), and alpha(4) are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of DeltaG(op) provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the beta strands, which form the core of the protein, appear to have higher values of DeltaG(op) than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the beta strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central beta strands-beta(3,) beta(2), and beta(4)-would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions.     

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly (15)N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the beta-barrel and to a lesser degree in the alpha-helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a beta-hairpin formed by strands 2 and 3 of the beta-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the beta-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.     

A dynamic bundle of four adjacent hydrophobic segments in the denatured state of staphylococcal nuclease.

In an earlier study of the denatured state of staphylococcal nuclease (Wang Y, Shortle D, 1995, Biochemistry 34:15895-15905), we reported evidence of a three-strand antiparallel beta sheet that persists at high urea concentrations and is stabilized by a local "non-native" interaction with four large hydrophobic residues. Because the amide proton resonances for all of the involved residues are severely broadened, this unusual structure is not amenable to conventional NMR analysis and must be studied by indirect methods. In this report, we present data that confirm the important role of interactions involving four hydrophobic residues (Leu 36, Leu 37, Leu 38, and Val 39) in stabilizing the structure formed by the chain segments corresponding to beta 1-beta 2-beta 3-h, interactions that are not present in the native state. Glycine substitutions for each of these large hydrophobic residues destabilizes or disrupts this beta structure, as assessed by HN line sharpening and changes in the CD spectrum. The 13C resonances of the carbonyl carbon for several of the residues in this structure indicate conformational dynamics that respond in a complex way to addition of urea or changes in sequence. Studies of hydrogen exchange kinetics in a closely related variant of staphylococcal nuclease demonstrate the absence of the stable hydrogen bonding between the strands expected for a native-like three-strand beta sheet. Instead, the data are more consistent with the three beta strand segments plus the four adjacent hydrophobic residues forming a dynamic, aligned array or bundle held together by hydrophobic interactions.     

Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199-215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.     

GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.     

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.     

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.     

An obligatory intermediate controls the folding of the alpha-subunit of tryptophan synthase, a TIM barrel protein

The proposed kinetic folding mechanism of the alpha-subunit of tryptophan synthase (alphaTS), a TIM barrel protein, displays multiple unfolded and intermediate forms which fold through four parallel pathways to reach the native state. To obtain insight into the secondary structure that stabilizes a set of late, highly populated kinetic intermediates, the refolding of urea-denatured alphaTS from Escherichia coli was monitored by pulse-quench hydrogen exchange mass spectrometry. Following dilution from 8 M urea, the protein was pulse-labeled with deuterium, quenched with acid and mass analyzed by electrospray ionization mass spectrometry (ESI-MS). Hydrogen bonds that form prior to the pulse of deuterium offer protection against exchange and, therefore, retain protons at the relevant amide bonds. Consistent with the proposed refolding model, an intermediate builds up rapidly and decays slowly over the first 100 seconds of folding. ESI-MS analysis of the peptic fragments derived from alphaTS mass-labeled and quenched after two seconds of refolding indicates that the pattern of protection of the backbone amide hydrogens in this transient intermediate is very similar to that observed previously for the equilibrium intermediate of alphaTS highly populated at 3 M urea. The protection observed in a contiguous set of beta-strands and alpha-helices in the N terminus implies a significant role for this sub-domain in directing the folding of this TIM barrel protein.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Temperature-dependence of protein hydrogen bond properties as studied by high-resolution NMR

The temperature-dependence of a large number of NMR parameters describing hydrogen bond properties in the protein ubiquitin was followed over a range from 5 to 65 degrees C. The parameters comprise hydrogen bond (H-bond) scalar couplings, h3JNC', chemical shifts, amide proton exchange rates, 15N relaxation parameters as well as covalent 1JNC' and 1JNH couplings. A global weakening of the h3JNC' coupling with increasing temperature is accompanied by a global upfield shift of the amide protons and a decrease of the sequential 1JNC' couplings. If interpreted as a linear increase of the N...O distance, the change in h3JNC' corresponds to an average linear thermal expansion coefficient for the NH-->O hydrogen bonds of 1.7 x 10(-4)/K, which is in good agreement with overall volume expansion coefficients observed for proteins. A residue-specific analysis reveals that not all hydrogen bonds are affected to the same extent by the thermal expansion. The end of beta-sheet beta1/beta5 at hydrogen bond E64-->Q2 appears as the most thermolabile, whereas the adjacent hydrogen bond I3-->L15 connecting beta-strands beta1 and beta2 is even stabilized slightly at higher temperatures. Additional evidence for the stabilization of the beta1/beta2 beta-hairpin at higher temperatures is found in reduced hydrogen exchange rates for strand end residue V17. This reduction corresponds to a stabilizing change in free energy of 9.7 kJ/mol for the beta1/beta2 hairpin. The result can be linked to the finding that the beta1/beta2 hairpin behaves as an autonomously folding unit in the A-state of ubiquitin under changed solvent conditions. For several amide groups the temperature-dependencies of the amide exchange rates and H-bond scalar couplings are uncorrelated. Therefore, amide exchange rates are not a sole function of the hydrogen bond "strength" as given by the electronic overlap of donors and acceptors, but are clearly dependent on other blocking mechanisms.     

NMR study of the reconstitution of the β-sheet of thioredoxin by fragment complementation

The study of complementary protein fragments is thought to be generally useful to identify early folding intermediates. A prerequisite for these studies is the reconstitution of the native-like structure by fragment complementation. Structural analysis of the complementation of the domain-sized proteolytic fragments of E. coli thioredoxin, using a combination of H-exchange and 2D NMR experiments as a fingerprint technique, provide evidence for the extensive reconstitution of a native β-sheet, with local conformational adjustments near the cleavage site. Remarkably, the antiparallel β-strand between the fragments shows a native-like protection of the amide protons to solvent exchange. Our results indicate that these fragments can be useful to study the early events in the still little understood formation of β-sheets. © 1995 Wiley-Liss, Inc.     

Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding.     

Sequence-specific 1H-NMR assignments and folding topology of human CD59.

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted beta 1-beta 5 in sequence order), arranged into separate two-stranded (beta 1 and beta 2) and three-stranded (beta 3-beta 5) antiparallel beta-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the beta 4 and beta 5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.     

Hydrogen exchange kinetics of RNase A and the urea:TMAO paradigm

A key paradigm in the biology of adaptation holds that urea affects protein function by increasing the fluctuations of the native state, while trimethylamine N-oxide (TMAO) affects function in the opposite direction by decreasing the normal fluctuations of the native ensemble. Using urea and TMAO separately and together, hydrogen exchange (HX) studies on RNase A at pH* 6.35 were used to investigate the basic tenets of the urea:TMAO paradigm. TMAO (1 M) alone decreases HX rate constants of a select number of sites exchanging from the native ensemble, and low urea alone increases the rate constants of some of the same sites. Addition of TMAO to urea solutions containing RNase A also suppresses HX rate constants. The data show that urea and TMAO independently or in combination affect the dynamics of the native ensemble in opposing ways. The results provide evidence in support of the counteraction aspect of the urea:TMAO paradigm linking structural dynamics with protein function in urea-rich organs and organisms. RNase A is so resistant to urea denaturation at pH* 6.35 that even in the presence of 4.8 M urea, the native ensemble accounts for >99.5% of the protein. An essential test, devised to determine the HX mechanism of exchangeable protons, shows that over the 0-4.8 M urea concentration range nearly 80% of all observed sites convert from EX2 to EX1. The slow exchange sites are all EX1; they do not exhibit global exchange even at urea concentrations (5.8 M) well into the denaturation transition zone, and their energetically distinct activated complexes leading to exchange gives evidence of residual structure. Under these experimental conditions, the use of DeltaG(HX) as a basis for HX analysis of RNase A urea denaturation is invalid.     

Protein dynamics viewed by hydrogen exchange

To examine the relationship between protein structural dynamics and measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nuclease was compared with that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursion that removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motion and the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.