Pulsed hydrogen exchange and electrospray charge-state distribution as complementary probes of protein structure in kinetic experiments: implications for ubiquitin folding

The possible involvement of "hidden" kinetic intermediates in the apparent two-state folding of some proteins is currently a matter of debate. This study uses time-resolved electrospray ionization (ESI) mass spectrometry with on-line pulsed hydrogen-deuterium exchange (HDX) for monitoring the refolding of acid/methanol-denatured ubiquitin. It is demonstrated that the ESI charge-state distribution (CSD) and the extent of HDX represent nonredundant probes of the protein structure in solution. When considered in isolation, the data provided by both of these probes are consistent with a two-state behavior, involving only denatured ubiquitin D and refolded protein F. However, a careful comparison of the CSD and HDX kinetics reveals the presence of an additional species, exhibiting a CSD like the folded protein but showing non-native HDX characteristics. This kinetic intermediate, D*, is in rapid equilibrium with D, such that the overall reaction is consistent with the mechanism D <--> D* --> F. The results of this work suggest that the occurrence of transient intermediates may be more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior.     

Automated prediction of protein association rate constants

The association rate constants (k(a)) of proteins with other proteins or other macromolecular targets are a fundamental biophysical property. Observed rate constants span over ten orders of magnitude, from 1 to 10(10) M(-1)s(-1). Protein association can be rate limited either by the diffusional approach of the subunits to form a transient complex, with near-native separation and orientation but without short-range native interactions, or by the subsequent conformational rearrangement to form the native complex. Our transient-complex theory showed promise in predicting k(a) in the diffusion-limited regime. Here, we develop it into a web server called TransComp (http://pipe.sc.fsu.edu/transcomp/) and report on the server's accuracy and robustness based on applications to over 100 protein complexes. We expect this server to be a valuable tool for systems biology applications and for kinetic characterization of protein-protein and protein-nucleic acid association in general.     

Measuring dissociation rate constants of protein complexes through subunit exchange: experimental design and theoretical modeling

Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, k(off), provide structural and functional information on protein complexes. However, because all existing methods for measuring k(off) require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here, we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE), to measure k(off) using metabolic stable isotope labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. The subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that k(off) determines, and hence can be estimated from, the observed rate of subunit exchange. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method.     

Characterizing protein structure in amorphous solids using hydrogen/deuterium exchange with mass spectrometry

Elucidating protein structure in amorphous solids is central to the rational design of stable lyophilized protein drugs. Hydrogen/deuterium (H/D) exchange with electrospray ionization mass spectrometry was applied to lyophilized powders containing calmodulin (17 kDa) and exposed to D(2)O vapor at controlled relative humidity (RH) and temperature. H/D exchange was influenced by RH and by the inclusion of calcium chloride and/or trehalose in the solid. The effects were not exhibited uniformly along the protein backbone but occurred in a site-specific manner, with calcium primarily influencing the calcium-binding loops and trehalose primarily influencing the alpha-helices. The results demonstrate that the method can provide quantitative and site-specific structural information on proteins in amorphous solids and on changes in structure induced by protein cofactors and formulation excipients. Such information is not readily available with other techniques used to characterize proteins in the solid state, such as Fourier transform infrared, Raman, and near-infrared spectroscopy.     

Re-examination of rhodopsin structure by hydrogen exchange

The hydrogen exchange behavior of rhodopsin was re-examined by studies of the protein in the disc membrane and after solubilization in octyl glucoside. The methods used measure either the peptide hydrogens alone (hydrogen-deuterium exchange by infrared spectroscopy) or all slowly exchanging hydrogens (hydrogen-tritium exchange by hel filtration). Under mild exchange conditions, disc membranes and solubilized lipid-free proteins show very similar exchange behavior, indicating the absence of slowly exchanging lipid protons. At high temperature, exchange of an additional large group of very slow peptide NH can be detected. The total number of slow hydrogens significantly exceeds the amide content, and apparently includes slowly exchanging protons from perhaps 40% of the protein's non-amide side chains. This is thought to require the involvement of many polar side chains in internal H-bonding. The exchange rates of the non-amide side chains sites have not been determined. However, to the extent that these contribute to the fast time region of the measured kinetic H-exchange curve, previously identified with exposed, non-H-bonded peptides, the estimate of freely exposed rhodopsin peptides must be reduced. The fraction of free peptides could range from a remarkably high value of 70% down to about 45%.     

Role of native-state structure in rubredoxin native-state hydrogen exchange

Amide exchange rates were measured for Pyrococcus furiosus (Pf) rubredoxin substituted with either Zn(II), Ga(III), or Ge(IV). Base-catalyzed exchange rate constants increase up to 3000-fold per unit charge for the highly protected amides surrounding the active site metal, yielding apparent residue-specific conformational energy decreases of more than 8 kcal/mol in a comparison of the Zn(II)- and Ge(IV)-substituted proteins. However, the exchange kinetics for many of the other amides of the protein are insensitive to these metal substitutions. These differential rates are inversely correlated with the distance between the amide nitrogen and the metal in the X-ray structure, out to a distance of at least 12 A, consistent with an electrostatic potential-dependent shifting of the amide nitrogen pK. This strongly correlated distance dependence is consistent with a nativelike structure for the exchange-competent conformations. The electric field potential within the interior of the rubredoxin structure gives rise to a change of as much as a million-fold in the rate for the exchange-competent state of the individual amide hydrogens. Nevertheless, the strength of these electrostatic interactions in Pf rubredoxin appears to be comparable to those previously reported within other proteins. As a result, contrary to the conventional analysis of hydrogen exchange data, for exchange processes that occur via nonglobal transitions, the residual conformational structure will often modulate the observed rates. Although this necessarily complicates the estimation of the conformational equilibria of these exchange-competent states, this dependence on residual structure can provide insight into the conformation of these transient states.     

EX1 hydrogen exchange and protein folding

Slow amide hydrogen exchange is an increasingly popular tool for investigating structure and function in proteins. The kinetic model for slow hydrogen exchange has two limits, called EX2 and EX1, wherein the thermodynamics and kinetics of protein motions, respectively, are reported by the exchange data. While many laboratories have demonstrated that EX2 exchange can indeed provide accurate results regarding the thermodynamics of protein stability, the potential of EX1 exchange to follow the kinetics of protein unfolding and folding is only beginning to be realized. EX1 hydrogen exchange has advantages over more traditional folding experiments: it provides single-residue resolution, as well as whole-molecule information, the latter of which can be interpreted in terms of the cooperativity of unfolding. However, key questions remain regarding the interpretation of EX1 hydrogen exchange.     

Primary structure effects on peptide group hydrogen exchange

The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo-and polypeptides and, suprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured aligo- and polypeptides. The application of this approach to protein studies is discussed. © 1993 Wiley-Liss, Inc.     

Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7

The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (deltadeltaG(ui) = 4.4 kJ/mol) but the native state is only slightly destabilised (deltadeltaG(nu) = 1.8 kJ/mol) at 10 degrees C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Phi-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

The contribution of electrostatics to hydrogen exchange in the unfolded protein state

Although electrostatics have long been recognized to play an important role in hydrogen exchange (HX) with solvent, the quantitative assessment of its magnitude in the unfolded state has hitherto been lacking. This limits the utility of HX as a quantitative method to study protein stability, folding, and dynamics. Using the intrinsically disordered human protein α-synuclein as a proxy for the unfolded state, we show that a hybrid mean-field approach can effectively compute the electrostatic potential at all backbone amide positions along the chain. From the electrochemical potential, a fourfold reduction in hydroxide concentration near the protein backbone is predicted for the C-terminal domain, a prognosis that is in direct agreement with experimentally derived protection factors from NMR spectroscopy. Thus, impeded HX for the C-terminal region of α-synuclein is not the result of intramolecular hydrogen bonding and/or structure formation.     

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.     

The hydrogen exchange core and protein folding.

A database of hydrogen-deuterium exchange results has been compiled for proteins for which there are published rates of out-exchange in the native state, protection against exchange during folding, and out-exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359-360) is reexamined in a detailed comparison of the specific amide protons (NHs) and the elements of secondary structure on which they are located. For each pulsed exchange or competition experiment, probe NHs are shown explicitly; the large number and broad distribution of probe NHs support the validity of comparing out-exchange with pulsed-exchange/competition experiments. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native state, NHs first protected during folding, and NHs most protected in partially folded species. There is not a one-to-one correspondence of individual NHs. Proteins for which there are published data for native state out-exchange and theta values are also reviewed. The elements of secondary structure containing the slowest exchanging NHs in native proteins tend to contain side chains with high theta values or be connected to a turn/loop with high theta values. A definition for a protein core is proposed, and the implications for protein folding are discussed. Apparently, during folding and in the native state, nonlocal interactions between core sequences are favored more than other possible nonlocal interactions. Other studies of partially folded bovine pancreatic trypsin inhibitor (Barbar E, Barany G, Woodward C, 1995, Biochemistry 34:11423-11434; Barber E, Hare M, Daragan V, Barany G, Woodward C, 1998, Biochemistry 37:7822-7833), suggest that developing cores have site-specific energy barriers between microstates, one disordered, and the other(s) more ordered.     

Spatial distribution of dielectric shielding in the interior of Pyrococcus furiosus rubredoxin as sampled in the subnanosecond timeframe by hydrogen exchange

Experimental pK values of ionizable sidechains provide the most direct test for models representing dielectric shielding within the interior of a protein. However, only the strongly shifted pK values are particularly useful for discriminating among models. NMR titration studies have usually found only one or two such shifted pK values in each protein, so that the fitting of the experimental data to a uniform internal dielectric (epsilon(int)) model is not well constrained. The observed variation among proteins for such epsilon(int) estimates may reflect nonuniformity of dielectric shielding within each protein interior or qualitative differences between individual proteins. The differential amide kinetic acidities for a series of metal-substituted rubredoxins are shown to be consistent with Poisson-Boltzmann predictions of dielectric shielding that is relatively uniform for all of the amides that are sensitive to the metal charge, a region which corresponds to roughly 1/3 of the internal volume. The effective epsilon(int) values near 6 that are found in this study are significantly lower than many such estimates derived from sidechain pK measurements. The differing timeframes in which dielectric relaxation can respond to the highly transient peptide anion as compared to the longer lived states of the charged sidechains offers an explanation for the lower apparent dielectric constant deduced from these measurements.     

NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: the most conserved elements of structure are the most stable to unfolding.

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-helix between strands beta3 and beta4. Two additional alpha-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with DeltaGu(0) approximately 6 kcal/mol and m approximately 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of DeltaGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest DeltaGu(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the alpha-helices and beta-strands. The largest DeltaGu(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the DeltaGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.     

Determinants of protein hydrogen exchange studied in equine cytochrome c.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH(-)-ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 angstroms. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.