Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.
We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region.
Kekic
M
M
Department of Anatomy and Histology, University of Sydney, Australia. rat@anatomy.su.oz.au
Huang
W
W
Moens
P D
PD
Hambly
B D
BD
dos Remedios
C G
CG
D000495:Allosteric Site; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002645:Chickens; D003545:Cysteine / Q000737:chemistry; D004735:Energy Transfer; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013552:Swine; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; C019057:2,6-mansyl chloride; D008239:Lysine; D003545:Cysteine