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

Sequence-dependent correlated segments in the intrinsically disordered region of ChiZ

“…Sequence analysis and coarse-grained modeling have identified some generic determinants, in particular charged residues, for disorder and mean sizes of IDPs (16)(17)(18)(19). In recent years, small angle x-ray/neutron scattering (SAXS/SANS) (20), fluorescence techniques including nanosecond fluorescence correlation spectroscopy (nsFCS) and single-molecule fluorescence resonance energy transfer (smFRET) (21)(22)(23), nuclear magnetic resonance (NMR) spectroscopy (24), and all-atom molecular dynamics (MD) simulations (25) have become key biophysical tools to characterize conformation ensembles of IDPs. Among these, nsFCS, NMR, and MD can also probe conformational dynamics, each with strengths on particular timescales.…”

Intramolecular Diffusion in α-Synuclein: It Depends on How You Measure It

“…However, there seem to be discrepancies in the range of observed dynamics depending on how diffusion is measured. Measurements made by fluorescence resonance energy transfer (FRET) tend to be faster than those made by contact quenching for foldable proteins under folding conditions (5,9,11,16). This may be because FRET is sensitive over a range of distances between 10 and 100 Å (depending on the pair of probes), whereas contact quenching is sensitive to distances between 4 and 8 Å .…”

Ligand Modulation of the Conformational Dynamics of the A2AAdenosine Receptor Revealed by Single-Molecule Fluorescence

“…This value represents the mean distance from the Cβ atom of V229 to the center of geometry of non-hydrogen atoms in the dye's aromatic group obtained from three replicate simulations ( Figure S4). Since the typical quenching distance for BODIPY-FL is 1.0 nm or less 29,42 , we defined a quenching cutoff distance of dq = (1+ <l>) = 1.81 nm from the Cβ atom of V229 to the center of mass of the side chain of each quenching candidate residue in A2AR ( Figure S5).…”