The objective of the proposed research is to understand the molecular basis for Ca2+-dependent regulation of biochemical activity. The experimental approach is to utilize CaM's interactions with both skeletal and smooth muscle myosin light chain kinase (MLCK) utilizing enzymatically active truncation mutants. The applicant will investigate how substrate and ATP modulate these interactions, and look for evidence of a 2Ca2+.CaM.MLCK intermediate inactivated complex. The applicant will investigate the interaction of the fibronectin domain N-terminal to the catalytic core in smooth muscle MLCK with the catalytic core and how it might be modulated by CaM binding to MLCK. CaM's interaction with phosphorylase kinase (PhK), utilizing CaM-binding peptides from the catalytic subunit PhK(302-306). CaM's interaction with the smooth muscle protein caldesmon (CaD) utilizing CaM-binding peptides from the CaD sequence. TnC/TnI interactions in both skeletal muscle and cardiac forms, with an emphasis on improving the current model for skeletal interactions and understanding the structural basis for the differences in function with the cardiac system. The applicant will also expand these studies to the ternary TnC/TnI/TnT complex using the cardiac system. Biochemical regulation is key to healthy function. Breakdown of regulatory mechanisms if uncorrected generally leads to pathological conditions and to uncontrolled proliferation or cell death. Understanding the underlying molecular mechanisms of regulation is thus of fundamental importance in health research.
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