Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

Abstract

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

“…2 is derived for homopolymers in the infinitely long chain limit. Following Peran and coworkers [28], for finite-length chains, a lower bound of |i − j| > 15 was used to exclude deviations from infinitely long chain scaling behaviour at short sequence-separations and an upper bound of |i − j < |nres − 5 was used to exclude deviations due to "dangling ends." With these restrictions, finite-length homopolymers are expected to be well fit by Eq.…”

“…In contrast to infinitely long homopolymers which follow uniform power-law scaling of internal distances, Sic1 and pSic1 follow good-solvent scaling at short sequence separations and poor-solvent scaling at long sequence separations, leading to uncoupling between the rootmean-squared (rms) end-to-end distance (R ee ) and the rms radius of gyration (R g ). This heteropolymer effect has previously been hypothesized to explain apparently discrepant structural inferences between SAXS and smFRET [26][27][28].…”

“…To infer the root-mean-squared radius of gyration R g from R ee requires an additional assumption about the polymeric nature of system under study, namely the ratio G = R 2 ee /R 2 g . It has recently been shown that finite-length heteropolymeric chains can take on values of G that deviate from the values derived for infinitely long homopolymers in either the θ-state (Gaussian chains, G = 6) or excluded-volume (EV)-limit (self-avoiding walks, G ≈ 6.25) [26][27][28]. Application of polymer-theoretic values of G to the smFRET inferred R ee results in R g 24-27Å for Sic1 and 26-29Å for pSic1 (SI Appendix Table S3 ).…”

Information theoretic measures for quantifying sequence–ensemble relationships of intrinsically disordered proteins

“…These chemical details cannot be readily captured using coarse-grained approximations for heteropolymers ( Ruff and Holehouse, 2017 ). Recent studies have highlighted the importance of chemical heterogeneity on decoupling size and shape fluctuations and also the fluctuations of R g and R e ( Fuertes et al , 2017 ; Song et al , 2017 ; Peran et al , 2019 ). This type of decoupling raises caution about inferring SERs purely from the scaling of ensemble-averaged values of R g , R e or asphericity.…”

Controlling Structure and Dimensions of a Disordered Protein via Mutations

“…For example, this can be observed in studies of denatured states of folded proteins. 14 – 16 It should, therefore, be possible to tune the dimensions of natural IDPs through small perturbations of basic thermodynamic factors including backbone conformational entropy, hydrophobicity, and charge–charge interactions, that (de)stabilize pockets of structure.…”