Extremely slow intramolecular diffusion in unfolded protein L

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k B T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k B T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues. photochemical trigger | subdiffusion M ajor advances have been made in recent years in understanding dynamic aspects of protein conformational changes, particularly protein folding; however, many issues remain to be solved (1). Among these are the properties of the unfolded protein ensemble and the role of residual structure of denatured proteins in promoting folding (2), the heterogeneity of microscopic folding pathways (3), and the existence of multiple distinct, but only transiently populated, intermediates (4). Particularly for fast-folding proteins, the idea of downhill folding, i.e., the absence of a significant barrier, has been suggested as an alternative mechanism (5, 6), but it is not clear to what extent fast-folding proteins make use of this mechanism. On the other hand, technical progress has made it possible to observe multiple folding and unfolding events in millisecond all-atom molecular dynamics simulations. Such simulations have shown that some proteins always follow the same folding pathway, whereas others have several different pathways (7). Moreover, individual folding events occur with submicrosecond transit times through a distinct transition state but are separated by long waiting times, which yield the experimentally observed folding times (8).The idea of motion on a rugged energy landscape (9-11) has been used widely to describe conformational changes in protei...

Section: Resultsmentioning

confidence: 99%

Measurement of energy landscape roughness of folded and unfolded proteins

Milanesi

Waltho

Hunter

et al. 2012

“…Although a collapse to a very dense state (ν ¼ 1∕3 and R G ∕R H ¼ 0.77) favors folding by reducing the conformational entropy, it could drastically slow down the dynamics of the chain (57) by processes such as internal friction, which have been shown to increase with increasing compaction of unfolded proteins (16,17,33,58). However, especially during the early stages of the folding process, many interactions have to be sampled to find the correct contacts that incrementally decrease the energy of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…Theoretical concepts from polymer physics (7)(8)(9) have frequently been used to describe the properties of unfolded polypeptide chains (4,10,11) with the goal to establish the link between protein folding and collapse (12)(13)(14)(15). However, the methodology to test many of these concepts experimentally has only become available rather recently (2,16,17). A considerable body of experimental and theoretical work suggests that the dimensions of unfolded proteins depend on parameters such as amino acid composition (4), temperature (18), and solvent quality (3,10,15,19).…”

mentioning

confidence: 99%

Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

Hofmann

Soranno

Borgia

et al. 2012

426

599

The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 AE 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 AE 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.protein folding | single-molecule FRET | coil-globule transition | polymer theory I t has become increasingly clear that the structure and dynamics of unfolded proteins are essential for understanding protein folding (1-3) and the functional properties of intrinsically disordered proteins (IDPs) (4-6). Theoretical concepts from polymer physics (7-9) have frequently been used to describe the properties of unfolded polypeptide chains (4, 10, 11) with the goal to establish the link between protein folding and collapse (12-15). However, the methodology to test many of these concepts experimentally has only become available rather recently (2,16,17). A considerable body of experimental and theoretical work suggests that the dimensions of unfolded proteins depend on parameters such as amino acid composition (4), temperature (18), and solvent quality (3,10,15,19). The continuous collapse of polymers has been treated exhaustively by a number of theories (20-24) based on general principles that relate the dimensions and the length of a chain to its free energy. However, a prerequisite for the quantitative application of these theories and their comparison to experimental results is that the dimensions of the Θ-state are known, which serves as an essential reference state. At the Θ-point*, chain-chain and chain-solvent interactions balance such that the polymer is at a critical point, at which the thermodynamic phase boundaries disappear. As a result, the polypeptide chain obeys the same length scaling as an ideal chain without excluded volume and intrachain interactions. However, the Θ-conditions for protein chains are unknown. Besides its importance for obtaining the corre...

“…This observation suggests that polypeptide reconfiguration during the conformational searching that ultimately organizes the native structure is not limited by diffusional searching of the polypeptide chain through free solvent but rather by the difficulty of conformational reorganization within and between condensed polyglobular regions. This behavior studied in smaller proteins is commonly attributed to so-called internal friction (6,28). Significant differences should be appreciated.…”

Section: Significancementioning

confidence: 99%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

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...