Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

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164

217

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on nativelike interfoldon interactions orders the pathway.D o proteins fold through varied and multiple tracks, or do they fold through predetermined intermediates according to understandable biophysical principles (1)? This question is fundamental for the interpretation of a large amount of biophysical and biological research. The question could be resolved if it were possible to define the intermediate structures and pathways that unfolded proteins move through on their way to the native state. Unfortunately, transient intermediates cannot be studied by the usual crystallographic and NMR methods. The range of kinetic and spectroscopic methods has been applied to many proteins, but these methods do not yield the necessary structural information.We used a developing technology, hydrogen exchange pulse labeling measured by MS (HX MS), to study the folding of a cysteine-free variant of Escherichia coli ribonuclease H1 (RNase H), a mixed α/β protein that has served as a major proteinfolding model (2-5). Previous studies showed that RNase H folds in a fast, unresolved burst phase (15 ms dead time) to an intermediate termed "I core " and then much more slowly (in seconds) to the native state (3). HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2-4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6, 7).The HX MS technique used here is able to follow the entire folding trajectory of RNase H in considerable structural and temporal detail. The analysis monitors every amide site, evaluates the folding coopera...

Section: Discussionmentioning

confidence: 99%

“…Given only kinetic phase data and no structural information, it is not possible to distinguish a multiple pathway interpretation from a single pathway with an optional barrier that slows one population fraction and not another (26,27).…”

Section: Discussionmentioning

confidence: 99%

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

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164

217

“…Despite a dearth of experimental verification, this view is still current, and experimental as well as theoretical folding results are often phrased in this language. Some spectrometry-based experiments have been taken to suggest more than one folding pathway (55-59), but it has been shown that this kind of data cannot distinguish alternative parallel pathways from a given pathway with alternative misfolding barriers (60,61).…”

Section: Discussionmentioning

confidence: 99%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

Self Cite

107

Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

“…Additional work is needed to discriminate the mutual effects of the driving force (which could be defined as the derivative of the free energy relative to a generalized folding coordinate) from trapping forces (derivatives of the free energy relative to local degrees of freedom) in folding. The trapping forces can contribute to the kinetic heterogeneity of protein folding and be a source of optional misfolded errors in the mechanism of predetermined folding pathways recently proposed (34). The methodology developed here to discriminate slow and fast residues by analyzing their MSD could be useful to analyze folding.…”

Section: Dynamics In a Multiple-minima Potential And Varying Conformamentioning

confidence: 99%

How main-chains of proteins explore the free-energy landscape in native states

Senet

Maisuradze²,

Foulie³

et al. 2008

Understanding how a single native protein diffuses on its freeenergy landscape is essential to understand protein kinetics and function. The dynamics of a protein is complex, with multiple relaxation times reflecting a hierarchical free-energy landscape. Using all-atom molecular dynamics simulations of an ␣/␤ protein (crambin) and a ␤-sheet polypeptide (BS2) in their ''native'' states, we demonstrate that the mean-square displacement of dihedral angles, defined by 4 successive C ␣ atoms, increases as a power law of time, t ␣ , with an exponent ␣ between 0.08 and 0.39 (␣ ‫؍‬ 1 corresponds to Brownian diffusion), at 300 K. Residues with low exponents are located mainly in well-defined secondary elements and adopt 1 conformational substate. Residues with high exponents are found in loops/turns and chain ends and exist in multiple conformational substates, i.e., they move on multiple-minima free-energy profiles.protein has a multiple-minima free-energy landscape with typical activation barriers varying by at least 1 order of magnitude (1-3). Biological function is coupled to the fluctuations between these different local minima; in enzymes, conformational fluctuations imply a wide distribution of rate constants for catalytic reactions (4-7). Understanding how a single native protein explores its free-energy landscape in the course of time is thus required for a complete microscopic description of its function.In recent years, several techniques have been developed to study the temporal variations of structural fluctuations of a single molecule: In single-molecule fluorescence studies, the dynamics of an entire protein is revealed by recording the fluctuations of local fluorescent probes (8). In such an experiment, 1 or 2 degree(s) of freedom influencing the response of local probes directly are recorded and analyzed. For instance, the temporal fluctuations of the distance between the side chains of 2 residues and/or their orientation can be measured because the fluorescence of local probes located at these positions strongly depends on these degrees of freedom (4,8). A statistical analysis of these fluctuations provides a complete distribution of the conformational states visited and the distribution of kinetic constants of a protein (8).However, a protein itself is a heterogeneous molecule (9, 10); one may therefore expect that the dynamics recorded at 1 point along the sequence by measuring the motions of a local probe would not be similar to the dynamics recorded elsewhere. What are the relations between the local dynamics of each residue and the secondary and tertiary structures? By what kind of diffusive mechanism does a protein explore its main-chain conformational space? In the present work, we address these questions by using molecular dynamics (MD) simulations in explicit water using GROMACS software (11) at 1 bar and 300 K for 2 significantly different molecules (see Materials and Methods); 46-residue ␣/␤ protein crambin (PDB ID code 1CCM) (12) and 20-residue all-␤ polypeptide (BS2) (13). Crambin is hydrop...