Fluorescence study of conformational flexibility of RNase S-peptide: distance-distribution, end-to-end diffusion, and anisotropy decays.
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 A 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 A between residues 8 and 18 in the presence of 50% TFE is, however, significantly smaller than 15.3 A found for the S-protein complex. The half-width of the distance distribution increased from about 9 to 18 A for residues 1-8 and from about 6 to 14 A for segment 8-18 with the loss of helical structure. The half-widths of 9 A in the case of 1-8 segment when peptide is helical suggests the presence of considerable conformational heterogeneity. Also, the 14 A 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 x 10(-7) cm2/s at 2 degrees C for both segments and increased to 1-2 x 10(-6) cm2/s at 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Maliwal
B P
BP
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Lakowicz
J R
JR
Kupryszewski
G
G
Rekowski
P
P
D003619:Dansyl Compounds / Q000737:chemistry; D004058:Diffusion; D004735:Energy Transfer; D005454:Fluorescence Polarization; D009038:Motion; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D003619:Dansyl Compounds; D010446:Peptide Fragments; C023560:ribonuclease S-peptide; D014364:Tryptophan; D012260:Ribonucleases