Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration.
The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.
Curmi
P M
PM
Cardiovascular Research Institute, University of California, San Francisco 94143.
Stone
D B
DB
Schneider
D K
DK
Spudich
J A
JA
Mendelson
R A
RA
D000199:Actins / Q000378:metabolism*; D003198:Computer Simulation; D004023:Dictyostelium; D004926:Escherichia coli; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D009502:Neutrons; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism; D012542:Scattering, Radiation
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D009218:Myosins