Elucidation of physiologically relevant protein protein-interactions through fluorescence spectroscopy and allied biophysical methods is the theme of our research. The proposal describes two major areas of investigation involving a number of proteins in common. The first, dealing with the effects of cytoskeletal and contractile proteins on the structure and activity of phosphofructokinase (PFK), was prompted by the association of the enzyme with macromolecular components in the cell. Planned work examines the physiological significance of the troponin C-and calmodulin-induced phosphorylation of PFK that is catalyzed by the cAMP- dependent protein kinase. This includes investigation of the new phosphorylation site discovered by us (Ser376) and determination of in vivo phosphorylation patterns. The results will contribute to understanding of the neural and hormonal regulation of a major regulatory enzyme (PFK) in glycolysis. Fluorescence polarization and catalytic activity measurements will look at the interplay between PFK-stabilizing proteins (e.g. F-actin) and destabilizing proteins (e.g.troponin, troponin C, calmodulin, alpha-actinin) in the in vitro binding and activation of the enzyme. The second continuing area focuses on myosin light chain kinase (MLCK), a key component in the regulation of contraction in smooth muscle and generalized motility systems. Possible associations of novel myofilament proteins with MLCK and functional relationships between MLCK and other protein kinases will be determined in fluorometric measurements employing probes developed by us. Calmodulin, cAMP-dependent protein kinase, and phosphoprotein phosphatase PrP-1 play roles in both the PFK and MLCK systems. Our collaboration in the determination of the three-dimensional structures of specific peptide- calmodulin complexes will illuminate the interactions of MLCK, PFK, and other enzymes with calmodulin. Since some of the peptides are also biologically active, the results may suggest structures for receptor-bound hormones and neurotransmitters. The proposed work on phosphoprotein phosphatase PrP-1 is directed towards understanding of the slow conformational changes involved in the regulation of the enzyme. We will identify factors--including possible physiological mediators--that influence the transitions. This information may explain an apparent connection between the phosphatases and insulin action. Extension of our work on dityrosine formation in calmodulin and cardiac troponin C is pertinent to concerns about the effects of solar UV (B) irradiation of living cells. Continued development of fluorometric assay methods for protein kinases and phosphoprotein phosphatases expedites all aspects of our program and contributes to the advancement of the field. Methods: Fluorescence, HPLC, gas phase sequencing, analytical sedimentation, circular dichroism, (X-ray diffraction--collaboration).
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