If you have any problems related to the accessibility of any content (or if you want to request that a specific publication be accessible), please contact us at email@example.com.
Characterization of Smooth Muscle Myosin Light Chain Kinase Using Crystallization and Transient Kinetics
Biochemistry and Molecular Biology
Biochem and Molecular Biology
AltmetricsView Usage Statistics
Crystallization experiments and transient kinetic methods were used to characterize the first 75 amino acid segment of smooth muscle myosin light chain kinase (MLCK N1-75). As of now, the exact mechanism by which MLCK interacts with actomyosin complexes in smooth muscle is not well-understood because there is no highresolution 3-dimensional structure of MLCK available. In this project, the crystallization of the actin-binding domain of MLCK (called N1-75) with glutathione S-transferase (GST) was attempted in order to provide such a model. Polyethylene glycol and ammonium sulfate, within a pH range of 6.5 to 8.0, seemed to be the most promising precipitants to induce crystallization. Observation of crystallization setups with these paramenters showed no definitive results, and crystallization was not successful. Future experiments should attempt to increase protein quality before crystallization by removing the GST tag and achieving higher protein concentration. In addition to the crystallization experiments, transient kinetic methods were used to characterize MLCK N1-75 interactions with actin and smooth muscle myosin subfragment 1 (S1). It was found that MLCK N1-75, when mixed with actin, decreased the rate at which actin binds S1. This data suggests that MLCK N1-75 may be competing with S1 for the same binding site on actin.