Single Myosin Lever Arm Orientation in a Muscle Fiber Detected with Photoactivatable GFP

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 ~70° 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 unavoidable with ensemble measurements, however, detecting single molecules in the condensed tissue medium where myosin concentration exceeds 100 µM is challenging. A photoactivatable green fluorescent protein (PAGFP) tagged myosin light chain (MLC) 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 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 or bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where myosin concentration is low suggests molecular crowding in the Aband promotes occupancy of the straight myosin conformation (Burghardt 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. KeywordsPhotoactivatable green fluorescent protein; photoselected activation; myosin regulatory light chain; single molecule biophysics; molecular crowding; myosin lever arm orientation; human cardiac myosin regulatory light chain Myosin 2 is the actin-dependent molecular motor driving sarcomeric shorting and muscle contraction by transducing chemical free energy from MgATP hydrolysis into directed movement (1,2). The MgATP hydrolysis is coupled to a series of conformational changes in myosin, resulting in a cycle of attachment to the actin filament, strain development, release, and re-attachment. Myosin structural intermediates from the cycle summarized in Scheme 1 † This work was supported by NIH-NIAMS grant R01AR049277 and the Mayo Foundation. *Corresponding author: Telephone: 507 284 8120, Fax 507 284 9349, email: E-mail: burghardt@mayo.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 February 3. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptwere uniquely identified by near ultra-violet circular dichroism spectra and nucleotide sensitive tryptophan fluorescence intensity changes (3)....

Mapping microscope object polarized emission to the back focal plane pattern

“…Samples observed included fluorescent nanospheres and single photoactivatable green fluorescent proteins (PAGFP) tagged myosin light chains exchanged onto myosin cross-bridges in skeletal muscle fibers. The latter is an interesting biological application on crowded proteins, where emission from a single brightly fluorescent probe competes with background from many more probes in the low-fluorescence state 2. Our results show good agreement between observed and calculated patterns for the fluorescent nanosphere sample where sample intensity dominates the pattern.…”

“…Recently, photoactivatable GFP-tagged MLC (MLC-PAGFP) was introduced to the fiber 2. Sparse photoactivation of the PAGFP permitted detection of single-lever-arm orientation in the myosin dense muscle fiber.…”

“…Rabbit psoas muscle fibers were prepared, and the native myosin regulatory light chain (RLC) exchanged with PAGFP-tagged human cardiac RLC (HCRLC-PAGFP) as described 1,2…”

“…Nevertheless, accurate spatial representation of the fluorescent source might be secondary to efficient light detection in some applications although resolution and light detection efficiency tend to improve together with increasing numerical aperture (NA) of the microscope objective. A single-molecule fluorescence polarization application in which the sources are sufficiently separated or selectively activated only needs microscope collection efficiency 1,2. In this case, an image of the objective back focal plane (BFP) light intensity distribution contains all the dipole orientation information in a pattern that is interpretable by inspection 3,4…”

Single-molecule fluorescence characterization in native environment

“…We exchanged PA-GFP-tagged human cardiac RLC (HCRLC-PAGFP) with native light chain on myosin cross-bridges in permeabilized muscle fibers. Sparse photo-activation of the PA-GFP permitted detection of single-myosin lever-arm orientation across the fiber sample (Burghardt et al 2009b). …”