S. Walter Englander

Protein Folding Intermediates and Pathways Studied by Hydrogen Exchange

In order to solve the immensely difficult protein-folding problem, it will be necessary to characterize the barriers that slow folding and the intermediate structures that promote it. Although protein-folding intermediates are not accessible to the usual structural studies, hydrogen exchange (HX) methods have been able to detect and characterize intermediates in both kinetic and equilibrium modes--as transient kinetic folding intermediates on a subsecond time scale, as labile equilibrium molten globule intermediates under destabilizing conditions, and as infinitesimally populated intermediates in the high free-energy folding landscape under native conditions. Available results consistently indicate that protein-folding landscapes are dominated by a small number of discrete, metastable, native-like partially unfolded forms (PUFs). The PUFs appear to be produced, one from another, by the unfolding and refolding of the proteins intrinsically cooperative secondary structural elements, which can spontaneously create stepwise unfolding and refolding pathways. Kinetic experiments identify three kinds of barrier processes: (a) an initial intrinsic search-nucleation-collapse process that prepares the chain for intermediate formation by pinning it into a condensed coarsely native-like topology; (b) smaller search-dependent barriers that put the secondary structural units into place; and (c) optional error-dependent misfold-reorganization barriers that can cause slow folding, intermediate accumulation, and folding heterogeneity. These conclusions provide a coherent explanation for the grossly disparate folding behavior of different globular proteins in terms of distinct folding pathways.

Hydrogen exchange and mass spectrometry: A historical perspective.

Protein molecules naturally emit streams of information-rich signals in the language of hydrogen exchange concerning the intimate details of their stability, dynamics, function, changes therein, and effects thereon, all resolved to the level of their individual amino acids. The effort to measure protein hydrogen exchange behavior, understand the underlying chemistry and structural physics of hydrogen exchange processes, and use this information to learn about protein properties and function has continued for 50 years. Recent work uses mass spectrometric analysis together with an earlier proteolytic fragmentation method to extend the hydrogen exchange capability to large biologically interesting proteins. This article briefly reviews the advances that have led us to this point and the understanding that has so far been achieved.

Mechanisms and uses of hydrogen exchange.

Recent work has largely completed our understanding of the hydrogen-exchange chemistry of unstructured proteins and nucleic acids. Some of the high-energy structural fluctuations that determine the hydrogen-exchange behavior of native macromolecules have been explained; others remain elusive. A growing number of applications are exploiting hydrogen-exchange behavior to study difficult molecular systems and elicit otherwise inaccessible information on protein structure, dynamics and energetics.

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid- and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms.

Stable submolecular folding units in a non-compact form of cytochrome c☆

Studies of structure, dynamics, and stability of cytochrome c (cyt c) at low pH in a non-compact pre-molten globule state indicate that the protein contains submolecular folding units that are independently stable. In high salt, acid cyt c (pD 2.2; where D is deuterium) is nearly as compact as the native form. Nuclear magnetic resonance (n.m.r.) line broadening typical of the molten globule form is seen, indicating loosened packing and increased mobility not only for side-chains but also for the main chain. As NaCl concentration is decreased below 0.05 M, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In the extended form, tertiary structural hydrogen bonds are largely broken (hydrogen exchange rate), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (circular dichroism). The helices involve the same amino acid residues that are helical in the native state (hydrogen exchange labeling monitored by 2-dimensional n.m.r.). The equilibrium constant for helix formation at 20 degrees C (0.02 M-NaCl, pD 2.2) is about 10 (hydrogen exchange rate), even though the individual helical segments when isolated have little or no structure. Additional experiments were done to check assumptions and calibrate parameters that underlie the hydrogen exchange analysis of protein folding. These results indicate that the native-like helical segments in the expanded non-globular form of cyt c exist as part of somewhat larger submolecular folding units that possess significant equilibrium stability. Results from equilibrium and kinetic studies of protein folding support the generality of this conclusion. This view is contrary to the two-state paradigm for equilibrium folding and inconsistent with the idea that side-chain packing constraints determine folding motifs. The result suggests an extension of the thermodynamic hypothesis for protein structure to kinetic folding processes, so that the amino acid code for equilibrium and kinetic folding may be the same, and also seems pertinent to the biological evolution of contemporary protein structures.

Structure and properties of α-synuclein and other amyloids determined at the amino acid level

The structure of α-synuclein (α-syn) amyloid was studied by hydrogen-deuterium exchange by using a fragment separation–MS analysis. The conditions used made it possible to distinguish the exchange of unprotected and protected amide hydrogens and to define the order/disorder boundaries at close to amino acid resolution. The soluble α-syn monomer exchanges its amide hydrogens with water hydrogens at random coil rates, consistent with its natively unstructured condition. In assembled amyloid, long N-terminal and C-terminal segments remain unprotected (residues 1–≈38 and 102–140), although the N-terminal segment shows some heterogeneity. A continuous middle segment (residues ≈39–101) is strongly protected by systematically H-bonded cross-β structure. This segment is much too long to fit the amyloid ribbon width, but non-H-bonded amides expected for direction-changing loops are not apparent. These results and other known constraints specify that α-syn amyloid adopts a chain fold like that suggested before for amyloid-β [Petkova et al. (2002) Proc. Natl. Acad Sci. USA 99, 16742–16747] but with a short, H-bonded interlamina turn. More generally, we suggest that the prevalence of accidental amyloid formation derives mainly from the exceptional ability of the main chain in a structurally relaxed β-conformation to adapt to and energy-minimize side-chain mismatching. Seeding specificity, strain variability, and species barriers then arise because newly added parallel in-register chains must faithfully reproduce the same set of adaptations.

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.

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.

Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding.

Do stable intermediates form very early in the protein folding process? New results and a quantity of literature that bear on this issue are examined here. Results available provide little support for early intermediate accumulation before an initial search-dependent nucleation barrier.

Future directions in folding: The multi‐state nature of protein structure

All possible protein folding intermediates exist in equilibrium with the native protein at native as well as non‐native conditions, with occupation determined by their free energy level. The study of these forms can illuminate the fundamental principles of protein structure and folding. Hydrogen exchange methods can be used to detect and characterize these partially unfolded forms at native conditions and as a function of mild denaturant and temperature. This information illuminates the requirements that govern the ability of kinetic and equilibrium methods to study folding intermediates.