Kinetics of Contact Formation and End-to-End Distance Distributions of Swollen Disordered Peptides

Abstract

Unstructured polypeptide chains are subject to various degrees of swelling or compaction depending on the combination of solvent condition and amino acid sequence. Highly denatured proteins generally behave like random-coils with excluded volume repulsion, whereas in aqueous buffer more compact conformations have been observed for the low-populated unfolded state of globular proteins as well as for naturally disordered sequences. To quantitatively account for the different mechanisms inducing the swelling of polypeptides, we have examined three 14-residues peptides in aqueous buffer and in denaturant solutions, including the well characterized AGQ repeat as a reference and two variants, in which we have successively introduced charged side chains and removed the glycines. Quenching of the triplet state of tryptophan by close contact with cysteine has been used in conjunction with Förster resonance energy transfer to study the equilibrium and kinetic properties of the peptide chains. The experiments enable accessing end-to-end root mean-square distance, probability of end-to-end contact formation and intrachain diffusion coefficient. The data can be coherently interpreted on the basis of a simple chain model with backbone angles obtained from a library of coil segments of proteins and hard sphere repulsion at each Calpha position. In buffered water, we find that introducing charges in a glycine-rich sequence induces a mild chain swelling and a significant speed-up of the intrachain dynamics, whereas the removal of the glycines results in almost a two-fold increase of the chain volume and a drastic slowing down. In denaturants we observe a pronounced swelling of all the chains, with significant differences between the effect of urea and guanidinium chloride.

Measuring distances within unfolded biopolymers using fluorescence resonance energy transfer: The effect of polymer chain dynamics on the observed fluorescence resonance energy transfer efficiency

“…͑3͒ would be common in FRET studies of short polypeptides. For example, Soranno et al 37 reported on measurements of the kinetics of end-to-end collisions in polypeptides with N = 14. They interpreted their data in terms of an effective one-dimensional diffusion model and estimated the relative end-to-end diffusion coefficient to be in the range D end to end = 10-20 Å 2 / nm.…”

“…They interpreted their data in terms of an effective one-dimensional diffusion model and estimated the relative end-to-end diffusion coefficient to be in the range D end to end = 10-20 Å 2 / nm. Using the typical value 37 ͱ͗R 2 ͘ = 20 Å, the polymer reconfiguration time can be estimated as R = ͗R 2 ͘ / 6D end to end =3-6 ns. Therefore the value of R for these short peptides is comparable to lifetimes of dyes often employed in FRET and thus the effect of polymer dynamics on the observed FRET efficiency may be important.…”

Measurement of energy landscape roughness of folded and unfolded proteins

“…In particular, intrapeptide contact formation has been investigated extensively using fluorescence and triplet quenching (15)(16)(17)(18)(19). These experiments observe intrachain loop formation from an equilibrium distribution of initial conformations and provide important information on polypeptide backbone diffusion, including the effects of excluded volume and chain stiffness.…”

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] …”