Hydrogen Exchange Methods Research Articles

Effect on intrinsic peroxidase activity of substituting coevolved residues from Ω-loop C of human cytochrome c into yeast iso-1-cytochrome c

Naturally-occurring variants of human cytochrome c (Cytc) that induce thrombocytopenia IV occur within Ω-loop C (residues 40–57). These variants enhance the peroxidase activity of human Cytc apparently by facilitating access to the heme by destabilizing Ω-loops C and D (residues 70–85). Given the importance of peroxidase activity in the early stages of apoptosis, we identified three sites with the EVmutation algorithm in or near Ω-loop C that coevolve and differ between yeast iso-1-Cytc and human Cytc. We prepared iso-1-Cytc variants with all possible combinations of the S40T, V57I and N63T substitutions to determine if these residues decrease the peroxidase activity of iso-1-Cytc to that of human Cytc producing an effective off state for a peroxidase signaling switch. At pH 6 and above, all variants significantly decreased peroxidase activity. However, the correlation of peroxidase activity with local and global stability, expected if cooperative unfolding of Ω-loops C and D is required for peroxidase activity, was generally poor. The m-values derived from the guanidine hydrochloride dependence of the kinetics of imidazole binding to horse Cytc, which is well-characterized by native-state hydrogen exchange methods, and K72A/K73A/K79A iso-1-Cytc show that local structural fluctuations and not subglobal cooperative unfolding of Ω-loops C and D are sufficient to permit binding of a small molecule like peroxide to the heme. A 2.46 Å structure of N63T iso-1-Cytc identifies a change to a hydrogen bond network linking Ω-loops C and D that could modulate the local fluctuations needed for the intrinsic peroxidase activity of Cytc.

Native State Hydrogen Exchange-Mass Spectrometry Methods to Probe Protein Folding and Unfolding.

Native state hydrogen exchange (HX) methods provide high-resolution structural data on the rare and transient opening motions in proteins under native conditions. Mass spectrometry-based HX methods (HX-MS) have gained popularity because of their ability to delineate population distributions, which allow a direct determination of the mechanism of inter conversion of the partially folded states under native conditions. Various technological advancements have provided further impetus to the development of HX-MS-based experiments to study protein folding. Classical HX-MS studies use proteolytic digestion to produce fragments of the protein subsequent to HX in solution, in order to obtain structural data. New chemical fragmentation methods, which achieve the same result as proteolysis and cause minimal change to the HX pattern in the protein, provide an attractive alternative to proteolysis. Moreover, when used in conjunction with proteolysis, chemical fragmentation methods have significantly increased the structural resolution afforded by HX-MS studies, even bringing them at par with the single amino acid resolution observed in NMR-based measurements. Experiments based on one such chemical fragmentation method, electron transfer dissociation (ETD), are described in this chapter. The ETD HX-MS method is introduced using data from a protein which is inherently resistant to proteolytic digestion as example of how such an experiment can provide high-resolution structural data on the folding-unfolding transitions of the protein under native conditions.

Hydrogen Exchange Mass Spectrometry.

Hydrogen exchange (HX) methods can reveal much about the structure, energetics, and dynamics of proteins. The addition of mass spectrometry (MS) to an earlier fragmentation-separation HX analysis now extends HX studies to larger proteins at high structural resolution and can provide information not available before. This chapter discusses experimental aspects of HX labeling, especially with respect to the use of MS and the analysis of MS data.

Observing and Characterizing Early Folding Intermediates of E.Coli Rnase H using Kinetic and Equilibrium Approaches

E. coli ribonuclease H has been an important model system for protein folding for many years. It has long been known that it folds through an intermediate (Icore) en route to the native state. However, because folding to Icore occurs on the fast millisecond timescale, and there is very little Icore populated under equilibrium conditions, key questions have remained unanswered: What is the nature of the folding pathway en route to Icore, and how well packed is Icore?In this work, a new ultrarapid mixing technique coupled with time-correlated single photon counting detection of intrinsic fluorescence allows observation of the millisecond folding to Icore, including new early intermediates formed along the way. Mutagenesis and hydrogen exchange methods provide structural information about these new early intermediates. Additionally, two equilibrium models of Icore are made and characterized, providing new insight into the nature of this intermediate. This work increases our knowledge about the folding pathway and energy landscape of an important protein folding model system, at a time- and size-scale that is amenable to comparison with protein folding simulations.

Protein conformational dynamics of crambin in crystal, solution and in the trajectories of molecular dynamics simulations

Atomic displacement parameters--B factors of the eight crambin crystal structures obtained at 0.54-1.5 angstroms resolution and temperatures of 100-293K have been analyzed. The comparable contributions to the B factor values are the intramolecular motions which are modeled by the harmonic vibration calculations and derived from the molecular dynamics simulation (MD) as well as rigid body changes in the position of a protein molecule as a whole. In solution for the average NMR structure of crambin the amplitudes of the backbone atomic fluctuations of the most residues of the segments with the regular backbone conformations are close to the amplitudes of the small scale harmonic vibrations. For the same residues the probability of the medium scale fluctuations fixed by the hydrogen exchange method is very low. The restricted conformational mobility of those segments is coupled with the depressed amplitudes of the fluctuation changes of the tertiary structure registered by the residue accessibility changes in an ensemble of NMR structures that forms the average NMR structure of crambin. The amplitudes of temperature fluctuations of backbone atoms and the tertiary structure raise in the segment with the irregular conformations, turn and loops. In the same segments the amplitudes of the calculated harmonic vibrations also increase, but to a lesser extent and especially in the interhelical loop with the most strong and complicated fluctuation changes of the backbone conformation. In solution for the NMR structure in this loop the conformational transitions occur between the conformational substates separated by the energy barriers, but they are not observed even in the long 100 ns trajectories from the MD simulation of crambin. These strong local fluctuation changes of the structure may play a key role in the protein functioning and modern performance improvements in the MD simulation techniques are oriented to increase the probability of protein appearance in the trajectories from the MD simulations.

Comparison of apoA-I helical structure and stability in discoidal and spherical HDL particles by HX and mass spectrometry

Elucidation of apoA-I secondary structure in spherical plasma HDL particles is essential for understanding HDL structure and function at the molecular level. To provide this information, we have applied hydrogen exchange (HX) and mass spectrometry methods to compare apoA-I secondary structure in discoidal (two apoA-I molecules/particle) and spherical (five apoA-I molecules/particle) HDL particles. The HX kinetics indicate that the locations of helical segments within the apoA-I molecules are the same in both discoidal and spherical HDL particles (approximately 10 nm hydrodynamic diameter). Helix stabilities in both types of particles are 3–5 kcal/mol, consistent with the apoA-I molecules being in a highly dynamic state with helical segments unfolding and refolding in seconds. For the spherical HDL, apoA-I fragments corresponding to residues 115–158 exhibit bimodal HX kinetics consistent with this segment adopting an inter-converting (on the timescale of tens of minutes) helix-loop configuration. The segment adopting this configuration in the 10 nm disc is shorter because the surface area available to each apoA-I molecule is apparently larger. Loop formation in the central region of the apoA-I molecule contributes to the ability of the protein to adapt to changes in available space on the HDL particle surface. Overall, apoA-I secondary structure is largely unaffected by a change in HDL particle shape from disc to sphere.

The H/D-Exchange Kinetics of the Escherichia coli Co-Chaperonin GroES Studied by 2D NMR and DMSO-Quenched Exchange Methods

We studied hydrogen/deuterium-exchange reactions of peptide amide protons of GroES using two different techniques: (1) two-dimensional 1H–15N transverse-optimized NMR spectroscopy and (2) the dimethylsulfoxide-quenched hydrogen-exchange method combined with conventional 1H–15N heteronuclear single quantum coherence spectroscopy. By using these techniques together with direct heteronuclear single quantum coherence experiments, we quantitatively evaluated the exchange rates for 33 out of the 94 peptide amide protons of GroES and their protection factors, and for the remaining 61 residues, we obtained the lower limits of the exchange rates. The protection factors of the most highly protected amide protons were on the order of 106–107, and the values were comparable in magnitude to those observed in typical small globular proteins, but the number of the highly protected amide protons with a protection factor larger than 106 was only 10, significantly smaller than the numbers reported for the small globular proteins, indicating that significant portions of free heptameric GroES are flexible and natively unfolded. The highly protected amino acid residues with a protection factor larger than 105 were mainly located in three β-strands that form the hydrophobic core of GroES, while the residues in a mobile loop (residues 17–34) were not highly protected. The protection factors of the most highly protected amide protons were orders of magnitude larger than the value expected from the equilibrium unfolding parameters previously reported, strongly suggesting that the equilibrium unfolding of GroES is more complicated than a simple two-state or three-state mechanism and may involve more than a single intermediate.

Hydrogen exchange method to identify the protein targets of drugs

I is essential to identify a drug's target(s) to better understand the mechanism of action and anticipate possible side effects. Identifying these target(s) is a significant challenge in drug discovery. The most commonly used approach to identify the protein targets of small bioactive molecules is to capture binding proteins by affinity chromatography. Although well established, the affinity-based methods have considerable drawbacks. Small bioactive molecules need to be attached covalently to a solid support, which may result in masking critical recognition site(s) for the target proteins, and extensive washes are required to minimize the contamination of non-specifically bound proteins. This decreases the likelihood of identifying weakly bound proteins that may play significant roles in the pharmacological actions of small bioactive molecules. We propose here a new approach that does not require the immobilization of drugs. Our method measures the thermodynamic stability of proteins in the presence and absence of a small molecule drug by monitoring the changes of hydrogen-deuterium exchange rates of histidine residues in varying concentrations of protein denaturant. Because binding of a small molecule to a protein usually enhances the thermodynamic stability of the protein, the experiment should identify the target proteins. The results from our proof-of-concept study will be presented, and the strategy for the target identification in a proteome-scale sample will be discussed. Masaru Miyagi, J Anal Bioanal Tech 2013, 4:5 http://dx.doi.org/10.4172/2155-9872.S1.013

Advances in the Mass Spectrometry of Membrane Proteins: From Individual Proteins to Intact Complexes

Rapid advances in structural genomics and in large-scale proteomic projects have yielded vast amounts of data on soluble proteins and their complexes. Despite these advances, progress in studying membrane proteins using mass spectrometry (MS) has been slow. This is due in part to the inherent solubility and dynamic properties of these proteins, but also to their low abundance and the absence of polar side chains in amino acid residues. Considerable progress in overcoming these challenges is, however, now being made for all levels of structural characterization. This progress includes MS studies of the primary structure of membrane proteins, wherein sophisticated enrichment and trapping procedures are allowing multiple posttranslational modifications to be defined through to the secondary structure level in which proteins and peptides have been probed using hydrogen exchange, covalent, or radiolytic labeling methods. Exciting possibilities now exist to go beyond primary and secondary structure to reveal the tertiary and quaternary interactions of soluble and membrane subunits within intact assemblies of more than 700 kDa.

Protein Vivisection Reveals Elusive Intermediates in Folding

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the β5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

Characterization of the N-terminal tail domain of histone H3 in condensed nucleosome arrays by hydrogen exchange and NMR.

The N-terminal tail domains (NTDs) of histones play important roles in the formation of higher-order structures of chromatin and the regulation of gene functions. Although the structure of the nucleosome core particle has been determined by X-ray crystallography at near-atomic resolution, the histone tails are not observed in this structure. Here, we demonstrate that large quantities of nucleosome arrays with well-defined DNA positioning can be reconstituted using specific DNA sequences and recombinant isotope-labeled histones, allowing for the investigation of NTD conformations by amide hydrogen exchange and multidimensional nuclear magnetic resonance (NMR) methods. We examined the NTD of Drosophila melanogaster histones H3 in condensed nucleosome arrays. The results reveal that the majority of the amide protons in the NTD of H3 are protected from exchange, consistent with the NTDs having formed folded structures. Our study demonstrates hydrogen exchange coupled with NMR can provide residue-by-residue characterization of NTDs of histones in condensed nucleosome arrays, a technique that may be used to study NTDs of other histones and those with post-translational modifications.

How Proteins Fold Deciphered by Hydrogen Exchange

Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates that live for milliseconds cannot be isolated and solved. It has been possible to determine the structure and properties of intermediates during kinetic folding by hydrogen exchange (HX) methods. Having reached the native state proteins repeatedly unfold and refold. The normally invisible intermediates can be characterized by HX. Three concepts emerge. Proteins act like accretions of cooperative unfolding/refolding units called foldons. They account for the unit steps in folding pathways (and other functions). During folding the stepwise formation of foldons is guided by pre‐existing structure in a sequential stabilization process. These two factors generate predetermined stepwise pathways that progressively assemble the foldon units of the target protein. Experimental results that have been interpreted differently, in terms of multiple independent pathways, are due to chance misfolding errors that cause different population fractions to fold differently and so appear to represent different pathways. The integration of these three concepts ‐ foldon units, sequential stabilization, and optional errors ‐ explains much of the vast literature on the folding behavior of protein molecules.

The Foldon Substructure of Staphylococcal Nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔGHX) and the kinetic unfolding and refolding rates (kop and kcl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6×10−6 s−1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1–β2–β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.

Protein folding and misfolding: mechanism and principles.

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding.

Branching in the sequential folding pathway of cytochrome c

Previous results indicate that the folding pathways of cytochrome c and other proteins progressively build the target native protein in a predetermined stepwise manner by the sequential formation and association of native-like foldon units. The present work used native state hydrogen exchange methods to investigate a structural anomaly in cytochrome c results that suggested the concerted folding of two segments that have little structural relationship in the native protein. The results show that the two segments, an 18-residue omega loop and a 10-residue helix, are able to unfold and refold independently, which allows a branch point in the folding pathway. The pathway that emerges assembles native-like foldon units in a linear sequential manner when prior native-like structure can template a single subsequent foldon, and optional pathway branching is seen when prior structure is able to support the folding of two different foldons.

An On-pathway Intermediate in the Folding of a PDZ Domain

The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.

Detection of a hidden folding intermediate in the focal adhesion target domain: Implications for its function and folding

The focal adhesion target (FAT) domain of focal adhesion kinase has a four-helix bundle structure. Based on a hydrogen exchange-constrained computer simulation study and some indirect experimental results, it has been suggested that a partially unfolded state of the FAT domain with the N-terminal helix unfolded plays an important role in its biological function. Here, using a native-state hydrogen exchange method, we directly detected an intermediate with the N-terminal helix unfolded in a mutant (Y925E) of the FAT domain. In addition, kinetic folding studies on the FAT domain suggest that this intermediate exists on the native side of the rate-limiting transition state for folding. These results provide more direct evidence of the existence of the proposed intermediate and help to understand the folding mechanism of small single domain proteins.

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to "zipper up," while having an extrapolated folding rate of k(f) = 2 x 10(5) s(-1). These properties make it likely to attain the elusive "downhill folding" limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. The stability and its denaturant dependence for the hydrogen bonds in the central part of protein equal the values calculated for whole-molecule unfolding. Like-wise, their closing and opening rates indicate that these hydrogen bonds are broken and reformed in a single cooperative event representing the folding transition from the fully unfolded state to the native state. Additionally, closing rates for these hydrogen bonds agree with the extrapolated barrier-limited folding rate observed near the melting transition. Therefore, even in the absence of denaturant, where DeltaG(eq) approximately -6 kcal.mol(-1) (1 cal = 4.18 J) and tau(f) approximately 6 mus, folding remains cooperative and barrier-limited. Given that this prime candidate for downhill folding fails to do so, we propose that protein folding will remain barrier-limited for proteins that fold cooperatively.

An experimental strategy to evaluate the thermodynamic stability of highly dynamic binding sites in proteins using hydrogen exchange.

Quantitative characterization of the stability of highly dynamic regions in proteins is a significant goal because it represents a cornerstone to an understanding of the role of dynamics in function. Due to experimental constraints, however, monitoring the local stability of highly dynamic regions using standard hydrogen exchange (HX) methods is not a viable approach. Here, an experimental strategy is outlined that takes advantage of the coupling between stability as monitored by HX and binding affinity as monitored by isothermal titration calorimetry. It is shown that the stability of dynamic regions, which are part of binding sites, can be inferred from the response of the system to Gly mutations at surface-exposed sites. When applied to the analysis of the highly dynamic RT loop of SEM5 C-terminal SH3 domain, this approach reveals that the energetic consequences of the observed conformational heterogeneity are significant.

Hydrogen exchange methods to study protein folding

The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling methods can specify the structure, stability and kinetics of folding intermediates that exist for less than 1 s during kinetic folding. Native state HX methods can detect and characterize folding intermediates that exist as infinitesimally populated high energy excited state forms under native conditions. The results obtained in these ways suggest principles that appear to explain the properties of partially folded intermediates and how they are organized into folding pathways. The application of these methods is detailed here.