ISSN:
0887-3585
Keywords:
calmodulin
;
peptides
;
fluorescence spectroscopy
;
photoaffinity labeling
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2+-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, α-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal do main was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long α-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met 124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthsized. After photocross-linking the Bpa redidue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphi peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal sturcture of the uncommplexed protein.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340060311
Permalink
