Conformational studies of TOAC-labeled bradykinin analogues in model membranes |
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Authors: | R F F Vieira F Casallanovo E M Cilli A C M Paiva S Schreier and C R Nakaie |
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Institution: | (1) Department of Biophysics, Universidade Federal de São Paulo, São Paulo, SP, Brazil;(2) Department of Biochemistry, Institute of Chemistry, Universidade de São Paulo, São Paulo, SP, Brazil |
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Abstract: | Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg1 (TOAC0-BK) or replacing Pro3 (TOAC3-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism(CD) approach was used to examine the conformational propertiesof the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionicN-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC3-BK's EPR spectral lineshapes are broader than those of TOAC0-BK, indicating amore restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC0-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidlyinterconverting forms), while TOAC3-BK's spectra correspondto a more ordered structure. SDS binding induces drastic changesin BK and TOAC0-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. Incontrast, TOAC3-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC3-BK to acquire the correct receptor-bound conformation, leading to loss of biological activity. On the other hand, the greater flexibility of TOAC0-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity. |
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Keywords: | bradykinin CD EPR micelle peptide conformation spin label structure-activity relationship TOAC |
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