In an effort to establish the geometrical relationships among the monomeric units within F-actin, and between actin and myosin in actomyosin, we will attempt to introduce intermolecular crosslinks of known length between specific amino acid sidechains in F-actin. Photoactivatable crosslinkers will be used; novel compounds willb e synthesized, and they will be incorporated into sites that have not previously been labeled. The first crosslinkers to be used will contain the photoactivatable moeity benzophenone having an arm terminating in an amino group, and will be bound to a limited number of carboxyl groups in actin that we have recently found can be specifically labeled. The specific sidechains to which both ends of the bifunctional crosslinkers bind will be identified, and it is anticipated that location of the crosslinked residues within the tertiary structure of actin. Little is known about the products of reaction of activated benophenone with proteins, and we will incorporate benzophenone- iodoacetamide into Cys-374 of actin, irradiate it, forming a variety of intramolecular crosslinks, and analyze the products. HPLC wil be used to obtain small benzophenone-containing peptides. The residues labeled will be identified by sequence analysis, and mass spectrometry will be used to identify the exact products formed. The ractive carboxyls will also be labeled with fluorescent compounds (eosin and fluorescene), having arms with amino groups. If specific and limited incorporation (as occurs with glycine ethyl ester) can be achieved, fluorescence energy transfer studies will be carried out to measure distances between sites that have not previously been accessible for this type of measurement. In another series of experiments, the products of limited enzymic digestion of myosin S-1 will be analyzed. Various proteases and reaction conditions will be used to generate different fragments, and the exact peptide bonds that are split will be identified by sequence analyses at both ends of the released peptides. It is anticipated that these studies will give insights into the precise locations of sidechains that move during conformational changes in myosin.
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