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‐NH 2 of different lengths ( n = 0–10) were determined in dependence on temperature in D 2 O 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 (log A versus log N ) 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.