Fluorescence study of conformational flexibility of RNase S-peptide: distance-distribution, end-to-end diffusion, and anisotropy decays.
Fluorescence study of conformational flexibility of RNase S-peptide: distance-distribution, end-to-end diffusion, and anisotropy decays.
复制标题
RNase S 肽构象灵活性的荧光研究：距离分布、端到端扩散和各向异性衰减。
DOI：
10.1021/bi00097a009
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Rekowski,P
中科院分区：
文献类型：
--
作者：
Maliwal,BP;Lakowicz,JR;Kupryszewski,G;Rekowski,P
Revised Manuscript Received August 23, 1993® abstract: 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 Phe8, 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 Á 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 Á between residues 8 and 18 in thepresence of 50% TFE is, however, significantly smaller than 15.3 Á found for the S-protein complex. The half-widthof the distance distribution increased from about 9 to 18 Á for residues 1-8 and from about 6 to 14 Á for segment 8-18 with the loss of helical structure. The half-widths of 9 Á in the case of 1-8 segment when peptide is helical suggests the presence of considerable conformational heterogeneity. Also, the 14 Á 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-acceptordiffusion coefficients were less than 1 X 10~ 7 cm2/s at 2 C for both segments and increased to 1-2 X 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 intheir conformational dynamics. Overall, the results suggest that the specific amino acid sequence will significantlyinfluence 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.Since the early seminal work of Anfinsen and co-workers, it has been recognized that the aminoacid sequence of a polypeptide chain contains much of the information required for correct folding (Anfinsen, 1973). The experimentally observed folding of proteins is a rapid and cooperative process which suggests that it cannot occur by a random search of all possible conformations. For a protein made up of 100 amino acids, it would take on the order of 1050 years or longer to search all possible conformations (Levinthal, 1968). It is now generally accepted that protein folding occurs via local folded intermediate sites (Wetlaufer, 1981; Kim & Baldwin, 1982; Gierasch & King, 1989; Barrick & Baldwin, 1993). However, it has been difficult to identify these transient structures during folding as the process is very rapid and cooperative. In principle, it should be possible to identify the peptide sequences which form the initiation sites and to characterize the secondary structurein the isolated short peptides. However, early efforts to observe secondary structure in isolated linear peptide fragments proved quite unsuccessful. At least part of this failure was due to rather insensitive techniques available
