| Autor: |
Wright M; Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University., Redford S; Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University., Vehar J; Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University., Courtney KC; Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University., Billington N; Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University; Microscope Imaging Facility, West Virginia University., Liu R; Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University; rong.liu@hsc.wvu.edu. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2024 Aug 23 (210). Date of Electronic Publication: 2024 Aug 23. |
| DOI: |
10.3791/67135 |
| Abstrakt: |
Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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