Monitoring Hydrogen Exchange During Protein Folding by Fast Pressure Jump NMR Spectroscopy.
ABSTRACT: A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a pressure-sensitized ubiquitin variant that contains two Val to Ala mutations. Increased protection against hydrogen exchange with solvent is monitored as a function of time during the folding process. Results for 53 backbone amides show narrow clustering with protection occurring with a time constant of ca. 85 ms, but slower protection is observed around a reverse turn near the C-terminus of the protein. Remarkably, the native NMR spectrum returns with this slower time constant of ca. 150 ms, indicating that the almost fully folded protein retains molten globule characteristics with severe NMR line broadening until the final hydrogen bonds are formed. Prior to crossing the transition state barrier, hydrogen exchange protection factors are close to unity, but with slightly elevated values in the ?1-?2 hairpin, previously shown to be already lowly populated in the urea-denatured state.
Project description:Characterization of the polymorphic structural range of A? oligomers is important to the understanding of the mechanisms of toxicity. Yet for highly polymorphic ensembles, experimental structural elucidation is difficult. Here, we use a combination of NMR solvent protection experiments and computational structural screening to identify major species in the amyloid conformational ensemble. We examined the polymorphic pentamer and fibril seeds of A?42 and its mutants and compared the theoretical backbone amide protection obtained from simulations with experimental hydrogen/deuterium (H/D) exchange protection ratio. We observed that highly flexible pentamers do not share structural similarities with fibril seed oligomers, except the turn regions. We found that a novel amyloid structural motif of a triple ?-sheet, with the N-terminal residues interacting with the core (Lys(17)-Glu(22)) ?-sheet region, correlates with H/D exchange protection. The triple ?-sheet A?42 oligomer has a minimal exposure of hydrophobic residues and is further stabilized by the E22Q (Dutch) mutation in Alzheimer disease. The experimental H/D exchange solvent protection ratio implies that triple ?-sheet fibrils and globulomers could coexist in the A?42 ensemble, pointing to a broad heterogeneous aggregate population. Our results suggest that an approach that combines computational modeling with NMR protection data can be a useful strategy for obtaining clues to the preferred conformational species of the assemblies in solution and help in alleviating experimental difficulties and consequently possible errors in the exchange data for A?42 fibrils.
Project description:Hydrogen-exchange rates for an OB-fold subdomain fragment of staphylococcal nuclease have been measured at pH 4.7 and 4 degrees C, conditions close to the minimum of acid/base catalyzed exchange. The strongest protection from solvent exchange is observed for residues from a five-stranded beta-barrel in the NMR structure of the protein. Protection factors, calculated from the experimental hydrogen-exchange rates, range between 1 and 190. Similarly small protection factors have in many cases been attributed to "molten globule" conformations that are supposed to lack a specific tertiary structure. The present results suggest that marginal protection from solvent exchange does not exclude well-defined structure.
Project description:We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.
Project description:The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side-chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side-chain 1 Hϵ protons and thus present a method to gauge the strength of arginine side-chain interactions. The method employs 13 C-detection and the one-bond deuterium isotope shift observed for 15 Nϵ to generate two exchanging species in 1 H2 O/2 H2 O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side-chains in enzymes, in phase separation, and in protein interaction interfaces in general.
Project description:Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic ?-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ?5 h for the unstructured N-terminus to 600 h for amide protons in the two ?-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two ?-strands in amylin fibrils. There is variation in protection within the ?-strands, particularly for strand ?1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related ?-hairpins in the protofilament structure, with strand ?1 positioned on the surface and ?2 in the interior.
Project description: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.
Project description:Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.
Project description:The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.
Project description: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.
Project description:Conformational fluctuations of proteins are crucially important for their functions. However, changes in the location and dynamics of hydrated water in many proteins accompanied by the conformational transition have not been fully understood. Here, we used phase-modulated clean chemical exchange NMR approach to investigate pressure-induced changes in water-to-amide proton exchange occurring at sub-second time scale. With the transition of ubiquitin from its native conformation (N1) to an alternative conformation (N2) at 250 MPa, proton exchange rates of residues 32-35, 40-41, and 71, which are located at the C-terminal side of the protein, were significantly increased. These observations can be explained by the destabilization of the hydrogen bonds in the backbone and partial exposure of those amide groups to solvent in N2. We conclude that phase-modulated clean chemical exchange NMR approach coupled with pressure perturbation will be a useful tool for investigations of more open and hydrated protein structures.