Native-state Hydrogen Exchange Experiments Research Articles

Network representation of conformational transitions between hidden intermediates of Rd-apocytochrome b562.

The folding kinetics of Rd-apocytochrome b562 is two-state, but native-state hydrogen exchange experiments show that there are discrete partially unfolded (PUF) structures in equilibrium with the native state. These PUF structures are called hidden intermediates because they are not detected in kinetic experiments and they exist after the rate-limiting step. Structures of the mimics of hidden intermediates of Rd-apocytochrome b562 are resolved by NMR. Based upon their relative stability and structural features, the folding mechanism was proposed to follow a specific pathway (unfolded → rate-limiting transition state → PUF1 → PUF2 → native). Investigating the roles of equilibrium PUF structures in folding kinetics and their interrelationship not only deepens our understanding of the details of folding mechanism but also provides guides in protein design and prevention of misfolding. We performed molecular dynamics simulations starting from a hidden intermediate and the native state of Rd-apocytochrome b562 in explicit solvent, for a total of 37.18 μs mainly with Anton. We validated our simulations by detailed comparison with experimental data and other computations. We have verified that we sampled the post rate-limiting transition state region only. Markov state model was used to analyze the simulation results. We replace the specific pathway model with a network model. Transition-path theory was employed to calculate the net effective flux from the most unfolded state towards the most folded state in the network. The proposed sequential folding pathway via PUF1 then more stable, more native-like PUF2 is one of the routes in our network, but it is not dominant. The dominant path visits PUF2 without going through PUF1. There is also a route from PUF1 directly to the most folded state in the network without visiting PUF2. Our results indicate that the PUF states are not necessarily sequential in the folding. The major routes predicted in our network are testable by future experiments such as single molecule experiment.

Partially Folded States of Staphylococcal Nuclease Highlight the Conserved Structural Hierarchy of OB-Fold Proteins

We have been interested in whether three proteins that share a five-stranded beta-barrel "OB-fold" structural motif but no detectable sequence homology fold by similar mechanisms. Here we describe native-state hydrogen exchange experiments as a function of urea for SN (staphylococcal nuclease), a protein with an OB-fold motif and additional nonconserved elements of structure. The regions of structure with the largest stability and unfolding cooperativity are contained within the conserved OB-fold portion of SN, consistent with previous results for CspA (cold shock protein A) and LysN (anticodon binding domain of lysyl tRNA synthetase). The OB-fold also has the subset of residues with the slowest unfolding rates in the three proteins, as determined by hydrogen exchange experiments in the EX1 limit. Although the protein folding hierarchy is maintained at the level of supersecondary structure, it is not evident for individual residues as might be expected if folding depended on obligatory nucleation sites. Rather, the site-specific stability profiles appear to be linked to sequence hydrophobicity and to the density of long-range contacts at each site in the three-dimensional structures of the proteins. We discuss the implications of the correlation between stability to unfolding and conservation of structure for mechanisms of protein structure evolution.

Investigation of the four cooperative unfolding units existing in the MICAL-1 CH domain

The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six α helices and one 3 10 helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, Δ G H2O, is calculated to be 7.11 ± 0.58 kcal mol − 1 from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, Δ G HX and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply.

Detection of a Hidden Folding Intermediate of the Third Domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.

Explicit-chain model of native-state hydrogen exchange: implications for event ordering and cooperativity in protein folding.

Native-state hydrogen exchange experiments on several proteins have revealed partially unfolded conformations with diverse stabilities. These equilibrium observations have been used to support kinetic arguments that folding proceeds via a sequential "pathway." This interpretative logic is evaluated here by analyzing the relationship between thermodynamic behavior and folding kinetics in a class of simplified lattice protein models. The chain models studied have varying degrees of cooperative interplay (coupling) between local helical conformational preference and favorable nonlocal interactions. When model cooperativity is high, as native conditions are weakened, "isotherms" of free energy of exchange for residues belonging to the same helix merge together before global unfolding. The point of merger depends on the model energetic favorability of the helix. This trend is similar to the corresponding experimental observations. Kinetically, we find that the ordering of helix formation in the very last stage of native core assembly tends to follow the stabilities of their converged isotherms. In a majority (but not all) of folding trajectories, the final assembly of helices that are thermodynamically more stable against exchange precedes that of helices that are less stable against exchange. These model features are in partial agreement with common experimental interpretations. However, the model results also underscore the ensemble nature of the folding process: the kinetics of helix formation is not a discrete, strictly "all-or-none" process as that envisioned by certain non-explicit-chain models. Helices generally undergo many cycles of partial formation and dissolution before their conformations are fixed in the final assembly stage of folding, a kinetic stage that takes up only approximately 2% of the average folding time in the present model; and the ordering of the helices' final assembly in some trajectories can be different from the dominant ordering stipulated by the exchange isotherms.

How Cytochrome c Folds, and Why: Submolecular Foldon Units and their Stepwise Sequential Stabilization

Native state hydrogen exchange experiments have shown that the cytochrome c (Cyt c) protein consists of five cooperative folding-unfolding units, called foldons. These are named, in the order of increasing unfolding free energy, the nested-Yellow, Red, Yellow, Green, and Blue foldons. Previous results suggest that these units unfold in a stepwise sequential way so that each higher energy partially unfolded form includes all of the previously unfolded lower free energy units. If this is so, then selectively destabilizing any given foldon should equally destabilize each subsequent unfolding step above it in the unfolding ladder but leave the lower ones before it unaffected. To perform this test, we introduced the mutation Glu62Gly, which deletes a salt link in the Yellow unit and destabilizes the protein by 0.8 kcal/mol. Native state hydrogen exchange and other experiments show that the stability of the Yellow unit and the states above it in the free energy ladder are destabilized by about the same amount while the lower lying states are unaffected. These results help to confirm the sequential stepwise nature of the Cyt c unfolding pathway and therefore a similar refolding pathway. The steps in the pathway are dictated by the concerted folding-unfolding property of the individual unit foldons; the order of steps is determined by the sequential stabilization of progressively added foldons in the native context. Much related information for Cyt c strongly conforms with this mechanism. Its generality is supported by available information for other proteins.

The folding pathway of barnase: the rate-limiting transition state and a hidden intermediate under native conditions.

The nature of the rate-limiting transition state at zero denaturant (TS(1)) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS(1) has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degrees C and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS(2)). Therefore, it exists after TS(2) in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rd-apocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.

Cytochrome c folding pathway: kinetic native-state hydrogen exchange.

Native-state hydrogen exchange experiments under EX1 conditions can distinguish partially unfolded intermediates by their formation rates and identify the amide hydrogens exposed and protected in each. Results obtained define a cytochrome c intermediate seen only poorly before and place it early on the major unfolding pathway. Four distinct unfolding steps are found to be kinetically ordered in the same pathway sequence inferred before.

Fast-folding protein kinetics, hidden intermediates, and the sequential stabilization model.

Do two-state proteins fold by pathways or funnels? Native-state hydrogen exchange experiments show discrete nonnative structures in equilibrium with the native state. These could be called hidden intermediates (HI) because their populations are small at equilibrium, and they are not detected in kinetic experiments. HIs have been invoked as disproof of funnel models, because funnel pictures appear to indicate (1) no specific sequences of events in folding; (2) a continuum, rather than a discrete ladder, of structures; and (3) smooth landscapes. In the present study, we solve the exact dynamics of a simple model. We find, instead, that the present microscopic model is indeed consistent with HIs and transition states, but such states occur in parallel, rather than along the single pathway predicted by the sequential stabilization model. At the microscopic level, we observe a huge multiplicity of trajectories. But at the macroscopic level, we observe two pathways of specific sequences of events that are relatively traditional except that they are in parallel, so there is not a single reaction coordinate. Using singular value decomposition, we show an accurate representation of the shapes of the model energy landscapes. They are highly complex funnels.