Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin

Abstract

The folding of small protein ubiquitin (Ub), which plays an indispensable role in targeting proteins for degradation and DNA damage response, is complex. A number of experiments on Ub folding have reached differing conclusions regarding the relation between collapse and folding, and whether intermediates are populated. In order to resolve these vexing issues, we elucidate the denaturant-dependent thermodynamics and kinetics of Ub folding at low and neutral pH as a function of guanidinium chloride and urea using coarse-grained molecular simulations. The changes in the fraction of the folded Ub, and the radius of gyration (R) as a function of the denaturant concentration, [C], are in quantitative agreement with experiments. Under conditions used in experiments, R of the unfolded state at neutral pH changes only by ≈17% as the [GdmCl] decreases from 6 to 0 M. We predict that the extent of compaction of the unfolded state increases as temperature decreases. A two-dimensional folding landscape as a function of R and a measure of similarity to the folded state reveals unambiguously that the native state assembly is preceded by collapse, as discovered in fast mixing experiments on several proteins. Analyses of the folding trajectories, under mildly denaturing conditions ([GdmCl] = 1.0 M or [Urea] = 1.0 M), shows that Ub folds by collision between preformed secondary structural elements involving kinetic intermediates that are primarily stabilized by long-range contacts. Our work explains the results of small angle X-ray scattering (SAXS) experiments on Ub quantitatively, and establishes that evolved globular proteins in the unfolded ensemble are poised to collapse as the solvent conditions for the biopolymer changes from good solvent to Θ-solvent like conditions on denaturant dilution. In the process, we explain the discrepancy between SAXS and single molecule fluorescent resonant energy transfer (smFRET) experiments, which have arrived at a contradicting conclusion concerning the collapse of polypeptide chains.

Understanding nonequilibrium scaling laws governing collapse of a polymer

“…This means that when C s (t)P (C d , t) at different times are plotted against C d /C s (t) they should fall on top of each other. This verification is presented in Figure 4 where in the main frame we show plots of the (unscaled) distributions P (C d , t) at different times, and in the inset the corresponding scaling plot using the form (15). Coincidentally, here, the tail of the distribution shows an exponential decay as observed in coarsening of particle [79] and spin systems [80,81].…”

“…This transition belongs to a class of phase transitions that can be understood by investigating various associated scaling laws [5][6][7][8]. From a general point of view, the understanding of the collapse transition in homopolymers can be extended to investigate other conformational transitions experienced by different types of macromolecules, e.g., in a protein the collapse of the backbone may occur simultaneously or precede its folding to a native state [11][12][13][14][15].…”

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

“…Both experiments and molecular dynamics simulations point to formation of the N-terminal β-hairpin and its adjacent α-helix as the initial step in ubiquitin’s folding process ( 62 – 65 ). Our unfolded state data at 1 bar support the propensity of residues 1–18 to form this antiparallel β-hairpin but do not show a significant increase in transient population of ubiquitin’s native α-helix relative to the pressure-denatured state.…”

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

“…One alternative, herein denoted the "heteropolymer-decoupling hypothesis," posits that the heteropolymeric nature of proteins leads to variation in the relationship between R g and R ee , a relationship that is fixed (i.e., independent of chain length) at a ratio of 6.3 for a homopolymer SARW. Recent simulations suggest that this ratio may not be fixed for unfolded proteins, which are more complex than homopolymers (29,39,42,43). This "decoupling" offers a possible explanation for the discrepancy between SAXS (which is sensitive to R g ) and FRET (which is sensitive to R ee ).…”

“…In contrast, consensus is lacking regarding the behavior of IDPs at lower or no denaturant. Specifically, while numerous FRET (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and computational studies (11,14,18,23,(26)(27)(28)(29) have argued that the expanded, disordered ensemble detected at high denaturant contracts significantly [typically 25-50% upon transfer to low or no denaturant (ν < 0.5)] (11,14,18,23,(26)(27)(28)(30)(31)(32)(33)(34)(35), a similar number of SAXS studies report little or no contraction under these same conditions (10,(36)(37)(38)(39)(40)(41).…”