Folding of Protein L with Implications for Collapse in the Denatured State Ensemble

Abstract

A fundamental question in protein folding is whether the coil to globule collapse transition occurs during the initial stages of folding (burst-phase) or simultaneously with the protein folding transition. Single molecule fluorescence resonance energy transfer (FRET) and small angle X-ray scattering (SAXS) experiments disagree on whether Protein L collapse transition occurs during the burst-phase of folding. We study Protein L folding using a coarse-grained model and molecular dynamics simulations. The collapse transition in Protein L is found to be concomitant with the folding transition. In the burst-phase of folding, we find that FRET experiments overestimate radius of gyration, R g , of the protein due to the application of Gaussian polymer chain end-to-end distribution to extract R g from the FRET efficiency. FRET experiments estimate ≈ 6Å decrease in R g when the actual decrease is ≈ 3Å on Guanidinium Chloride denaturant dilution from 7.5M to 1M, and thereby suggesting pronounced compaction in the protein dimensions in the burst-phase.The ≈ 3Å decrease is close to the statistical uncertainties of the R g data measured from SAXS experiments, which suggest no compaction, leading to a disagreement with the FRET experiments.

Denaturants Alter the Flux through Multiple Pathways in the Folding of PDZ Domain

“…8,9 The theoretical basis for the success of the MTM has been explained elsewhere. 10 Applications of MTM to probe folding of a variety of proteins have yielded quantitative agreement with experiments 7,8,11–13 which attests to the efficacy of the MTM.…”

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

“…Therefore, one might conclude that the collapse transition is virtually nonexistent for IDPs and abrupt and concomitant with the ratelimiting folding transition for autonomously folding proteins. The discrepancies in interpretations regarding the collapse transition for protein folding and for IDPs have led to numerous debates (4,9,15,(20)(21)(22)(23).…”

“…Both techniques have distinct strengths and weaknesses (15,(20)(21)(22)(23). Strengths of smFRET measurements include the ultralow protein concentrations, at which experiments can be conducted, the ability to resolve distinct conformational populations, and the advantage of following motions across timescales that range from the nanosecond to the millisecond regimes.…”

“…SAXS measurements of labeled vs. unlabeled molecules rule out the dyes as the source of the discrepancy. Once we rule out specific errors with the smFRET measurements, which are presented in Discussion, we are left with three other possible sources for the observed discrepancies: (i) the nature of the averaging that goes into the calculation of R G is likely to make this quantity relatively insensitive to small changes in solvent quality (20), especially for heteropolymers that transition between coil-like ensembles corresponding to ν ∼ 0.59 and ν ∼ 0.5 (42); (ii) because R E quantifies the average distance between a pair of residues, as opposed to an average over all interresidue distances, it is possible that this quantity is more sensitive to fluctuations due the dangling ends of chains (43); and (iii) it is also possible that the inferred values of R E are subject to errors due to assumptions of a Gaussian chain model for the distribution of interdye distances. Each of these factors contributes to the discrepancies between inferences drawn from analysis of SAXS vs. smFRET data.…”

Computational Approaches to Understanding the Biological Behaviour of Intrinsically Disordered Proteins

“…A coarse grained potential is essentially a description of interactions where the degrees of freedom of individual atoms are not considered independently, but a chemical group is described as a spherical mass with an overall charge. Coarsegrained simulations use longer time steps and are capable of sampling systems of much larger dimensions than atomistic force fields, and are therefore useful in investigating higher ordered growth in IDP aggregation [75][76][77][78][79][80][81][82] . A recent work by Kurcinski et al 83 utilized a coarse-grained approach to characterize the various modes of complex formation and interactions of the IDP, phosphorylated kinase-inducible domain (pKID) and its interacting domain KIX.…”