Single myosin lever arm orientation in a muscle fiber detected with photoactivatable GFP.
Biochemistry
; 48(4): 754-65, 2009 Feb 03.
Article
em En
| MEDLINE
| ID: mdl-19127992
ABSTRACT
Myosin 2 is the molecular motor in muscle. It binds actin and executes a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. Myosin 2 has evolved to function optimally under crowded conditions where rates and equilibria of macromolecular reactions undergo major shifts relative to those measured in dilute solution. Hence, an important research objective is to detect in situ the lever arm orientation. Single-molecule measurements are preferred because they clarify ambiguities that are unavoidable with ensemble measurements; however, detecting single molecules in the condensed tissue medium where the myosin concentration exceeds 100 muM is challenging. A myosin light chain (MLC) tagged with photoactivatable green fluorescent protein (PAGFP) was constructed. The recombinant MLC physically and functionally replaced native MLC on the myosin lever arm in a permeabilized skeletal muscle fiber. Probe illumination volume was minimized using total internal reflection fluorescence microscopy, and PAGFP was sparsely photoactivated such that polarized fluorescence identified a single probe orientation. Several physiological states of the muscle fiber were characterized, revealing two distinct orientation populations in all states called straight and bent conformations. Conformation occupancy probability varies among fiber states with rigor and isometric contraction at extremes where straight and bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where the myosin concentration is low suggests molecular crowding in the A-band promotes occupancy of the straight myosin conformation [Burghardt, T. P., et al. (2007) Biophys. J. 93, 2226]. The latter may have a role in contraction because it provides additional free energy favoring completion of the cross-bridge power stroke.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Fibras Musculares Esqueléticas
/
Cadeias Leves de Miosina
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Proteínas de Fluorescência Verde
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos