Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions.
Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact. Compared to pPDM-S1, the fluorescence intensity of pPDM-S1 *MgADP is increased 50%(compared to 8 and 31% increases, respectively, for MgADP and MgATP binding to S1). Compared to acto-pPDM-S1, the absolute value of the product of the net effective electric charge at the actin binding interface of acto-pPDM-S1 *MgADP increases 7.3 esu2 (compared to a 0.9 esu2 decrease and an 11.0 esu2 increase, respectively, for MgADP and MgATP binding to acto-Sl).The interaction free energy for the ligands MgADP and actin, is -2.0 kcal/mol for pPDM-S1, compared to -1.2 kcal/mol for unmodified S1.
Kirshenbaum
K
K
Department of Biochemistry, University of the Pacific School of Dentistry, San Francisco, California 94115-2399.
Papp
S
S
Highsmith
S
S
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D007700:Kinetics; D008301:Maleimides; D015394:Molecular Structure; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013816:Thermodynamics
D000199:Actins; D003432:Cross-Linking Reagents; D008301:Maleimides; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; C011328:N,N'-4-phenylenedimaleimide; D003545:Cysteine