We propose to develop """"""""targeted"""""""" heterobifunctional cross-linking reagents in order to study the interaction of calmodulin (CaM) with other proteins. Since CaM plays an important regulatory role in its interactions with numerous other proteins, a study of this type will provide valuable insight into the influence of the various domains of CaM on regulation. Preliminary studies suggested that the cross-linking reagents should possess the following features: (1) it will be useful to have """"""""targeted"""""""" heterobifunctional reagents in which one terminus has a reporter group that will attack specific sites rather than a multitude of sites in CaM; (2) it will be important to select photostable radiolabels; (3) it will be preferable to use perfluorinated aryl azides as the photoactive group since this group cross-links with good efficiency at wavelengths not affecting the protein and produces a hydrolytically stable covalent bond in the photochemical cross-linking event; and (4) it will be necessary to incorporate some feature in the """"""""linking arm"""""""" between the two termini that would be cleavable and that would facilitate purification of the cross-linked peptides. In order to fulfill these expectations, we will synthesize various families of cross-linking reagents including """"""""yard stick"""""""" reagents for mapping intramolecular distances in CaM and heterobifunctional reagents for intermolecular studies of the interaction of CaM with other proteins including Ca+2,Mg+2-ATPase and myosin light chain kinase. These latter reagents will possess an electrophilic succinimidyl (NHS) ester that will intercept Lys residues on CaM, a phenothiazine that will direct or target the NHS ester, a perfluorinated aryl azide, an 35S radiolabel, and a cleavable diol linking arm. The unique features of these reagents will permit us to direct the initial covalent attachment to specific sites in CaM, to cross-link these modified CaM derivatives to other proteins, and to purify and sequence the photomodified domain in the second protein. These studies will provide the basis both for furthering our understanding of the.regulatory role of CaM and the basis on which to develop other families of """"""""targeted"""""""" cross-linking reagents for studying other protein-protein interactions.
| Rajagopalan, K; Chavan, A J; Haley, B E et al. (1993) Synthesis and application of bidentate photoaffinity cross-linking reagents. Nucleotide photoaffinity probes with two photoactive groups. J Biol Chem 268:14230-8 |
