Temperature-dependent loop formation kinetics in flexible peptides studied by time-resolved fluorescence spectroscopy

Looping rates in short polypeptides can be determined by intramolecular fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by tryptophan. By this methodology, the looping rates in glycine-serine peptides with the structure Trp-(Gly-Ser)n-Dbo-NH2 of different lengths (n = 0–10) were determined in dependence on temperature in D2O and the activation parameters were derived. In general, the looping rate increases with decreasing peptide length, but the shortest peptide (n=0) shows exceptional behavior because its looping rate is slower than that for the next longer ones (n=1,2). The activation energies increase from 17.5 kJ mol−1 for the longest peptide (n=10) to 20.5 kJ mol−1 for the shortest one (n=0), while the pre-exponential factors (log⁡(A/s−1)) range from 10.20 to 11.38. The data are interpreted in terms of an interplay between internal friction (stiffness of the biopolymer backbone and steric hindrance effects) and solvent friction (viscosity-limited diffusion). For the longest peptides, the activation energies resemble more and more the value expected for solvent viscous flow. Internal friction is most important for the shortest peptides, causing a negative curvature and a smaller than ideal slope (ca. –1.1) of the double-logarithmic plots of the looping rates versus the number of peptide chain segments (N). Interestingly, the corresponding plot for the pre-exponential factors (logA versus logN) shows the ideal slope (–1.5). While the looping rates can be used to assess the flexibility of peptides in a global way, it is suggested that the activation energies provide a measure of the “thermodynamic” flexibility of a peptide, while the pre-exponential factors reflect the “dynamic” flexibility.