Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations

Abstract

Internal friction is an important contribution to protein dynamics at all stages along the folding reaction. Even in unfolded and intrinsically disordered proteins, internal friction has a large influence, as demonstrated with several experimental techniques and in simulations. However, these methods probe different facets of internal friction and have been applied to disparate molecular systems, raising questions regarding the compatibility of the results. To obtain an integrated view, we apply here the combination of two complementary experimental techniques, simulations, and theory to the same system: unfolded protein L. We use single-molecule Förster resonance energy transfer (FRET) to measure the global reconfiguration dynamics of the chain, and photoinduced electron transfer (PET), a contact-based method, to quantify the rate of loop formation between two residues. This combination enables us to probe unfolded-state dynamics on different length scales, corresponding to different parts of the intramolecular distance distribution. Both FRET and PET measurements show that internal friction dominates unfolded-state dynamics at low denaturant concentration, and the results are in remarkable agreement with recent large-scale molecular dynamics simulations using a new water model. The simulations indicate that intrachain interactions and dihedral angle rotation correlate with the presence of internal friction, and theoretical models of polymer dynamics provide a framework for interrelating the contribution of internal friction observed in the two types of experiments and in the simulations. The combined results thus provide a coherent and quantitative picture of internal friction in unfolded proteins that could not be attained from the individual techniques.single-molecule FRET | nanosecond FCS | PET quenching | Rouse model with internal friction | intrinsically disordered proteins

Transient Secondary and Tertiary Structure Formation Kinetics in the Intrinsically Disordered State of α‐Synuclein from Atomistic Simulations

“…These differences may in part be due to the use of a different force field, but it seems unlikely that this is the sole cause of difference of almost two orders of magnitude. Indeed, very recent single molecule spectroscopy measurements of aS ( B. Schuler, private communication ) and other unfolded and intrinsically disordered proteins show similarly fast reorganization dynamics at 10–50 ns time scale. Further, a similar effect was previously described for urea induced unfolding of a protein, where urea was found to destabilize the folded state not by accelerating unfolding, but rather by slowing down refolding …”

Perspective: Chain dynamics of unfolded and intrinsically disordered proteins from nanosecond fluorescence correlation spectroscopy combined with single-molecule FRET

“…27,28 However, characterizing the distance distribution usually requires independent information, and the analysis is very sensitive to the detailed shape of the distribution in the contact region. [29][30][31] …”

“…Interestingly, such IDPs typically exhibit global chain dynamics on similar time scales as chemically denatured proteins, in the range of ∼10-100 ns, 30,47,100,101 and they also show internal friction. 30,47,100 An interesting trend is illustrated by a plot of τ i versus the dimensions of the chains, which exhibits a decrease in τ i with increasing chain expansion (Fig. 3), independent of whether this expansion is caused by the presence of denaturants or by the repulsion between charged residues in polyelectrolytic IDPs.…”

“…4(a) 30 Experimental data 30 were taken at 3M GdmCl (black lines show a global fit of the three correlations with the reconfiguration time, τ r , as a shared fit parameter), and MD simulations 12 observed experimentally and calculated based on large-scale MD simulations of the same unfolded protein. 30 To identify the origin of internal friction, Echeverria et al 111 simulated unfolded Csp at different GdmCl concentrations and observed trends in remarkable agreement with the experimental data 47 [ Fig. 4(b)].…”

“…116 A direct comparison of the dynamics of several unfolded proteins from large-scale MD simulations 12 with single-molecule fluorescence experiments indicated a greater abundance of transient non-native sequence-distant hydrogen bonds, salt bridges, and hydrophobic contacts in proteins that are more compact and exhibit more internal friction. 30 These increased interactions were correlated with slowed dihedral angle transitions, suggesting that the two contributions may be difficult to separate.…”