Fluorescence Study of Conformational Flexibility of RNase S-Peptide: Distance-Distribution End-to-End Diffusion and Anisotropy Decays

摘要

Frequency-domain fluorescence resonance energy transfer and anisotropy measurements were performed to characterize conformational dynamics of an analog of the RNase S-peptide (residues 1–20). Trp was used as a donor by replacing Phe 8, and a dansyl acceptor group was introduced at position 1 or 18. The distance-distribution parameters, half width of the distribution, end-to-end diffusion coefficient, and to some extent anisotropy decays were sensitive to changes in the S-peptide conformation. The observed mean distance of about 13–14 Å between residues 1 and 8 in the presence of 50% TFE and when bound to RNase S-protein is in reasonable accord with the X-ray structure of RNase. The mean distance of 9.3 Å between residues 8 and 18 in the presence of 50% TFE is, however, significantly smaller than 15.3 Å found for the S-protein complex. The half-width of the distance distribution increased from about 9 to 18 Å for residues 1–8 and from about 6 to 14 Å for segment 8–18 with the loss of helical structure. The half-widths of 9 Å in the case of 1–8 segment when peptide is helical suggests the presence of considerable conformational heterogeneity. Also, the 14 Å half-width for segment 8–18 when it is random-coil is smaller than that expected for a random coil 11-residue segment. The donor-to-acceptor diffusion coefficients were less than 1 × 10⁻⁷ cm²/s at 2°C for both segments and increased to 1–2 × 10⁻⁶ cm²/s at 35 °C. The anisotropy decays reveal the S-peptide to be rather rigid at 2°C irrespective of the conformation and the peptide becomes flexible upon raising the temperature to 35°C. The results also indicate small but significant differences between two segments in their conformational dynamics. Overall, the results suggest that the specific amino acid sequence will significantly influence the relationship between distance distribution parameters and conformational dynamics in case of short peptides. Also, these results suggest that distance-distribution measurements and anisotropy decays are a valuable tool to characterize conformational dynamics of short peptides.