| Popis: |
Hypertrophic cardiomyopathies (HCM) and dilated cardiomyopathies (DCM) are common inherited cardiovascular diseases, often resulting from single point mutations in genes encoding sarcomeric proteins. Genetic and clinical studies have identified several hundred mutations, including severe disease causing mutations in β-myosin heavy chain (MHC). Despite the clinical significance, few single molecule studies exist for mutated β-cardiac myosin, primarily due to difficulties of heterologous protein expression and instrumental limitations. Previous studies have used mouse α-cardiac myosin or biopsies from patients. Those studies are not optimal to understand the molecular mechanism of HCM/DCM because there are significant differences between mouse α- and human β-MHC. Furthermore, biopsy samples from patients are often inhomogeneous mixtures of wildtype (wt) and mutants. This may explain why there have been many inconsistencies between the previous studies.Here, we demonstrate the first single molecule studies of recombinant human cardiac myosin. We expressed homogenous and fully functional wt human cardiac α- and β-S1 with human light chains bound. Then, we characterized the actin-myosin interaction using in vitro motility and laser beam trapping assays. From the in vitro motility assay, we measured the maximum velocity from wt α- and β-S1. Using the laser trap, we measured stroke sizes, ATP binding rates (low [ATP]) and ADP release rates (high [ATP]). Furthermore, we expressed several HCM (R403Q, S453C) and DCM (S532P) causing mutants, and obtained preliminary in vitro motility and trap data. We have built a modern version optical trap that can resolve the ∼10 nm stroke size and ∼10 ms strongly bound state of cardiac β-S1 at high [ATP]. We have further improved the resolution by implementing real-time feedback control in the system to accurately determine fine changes caused by the single mutations. |
