Detection of functional ligand-binding events using synchrotron x-ray scattering.

Small-molecule ligands that change the structure of a protein are likely to affect its function, whereas those causing no structural change are less likely to be functional. Wide-angle x-ray scattering (WAXS) can be easily carried out on proteins and small molecules in solution in the absence of chemical tags or derivatives. The authors demonstrate that WAXS is a sensitive probe of ligand binding to proteins in solution and can distinguish between nonfunctional and productive binding. Furthermore, similar ligand-binding modes translate into similar scattering patterns. This approach has high potential as a novel, generic, low-throughput assay for functional ligand binding.

Molecular crowding inhibits intramolecular breathing motions in proteins.

In aqueous solution some proteins undergo large-scale movements of secondary structures, subunits or domains, referred to as protein "breathing", that define a native-state ensemble of structures. These fluctuations are sensitive to the nature and concentration of solutes and other proteins and are thereby expected to be different in the crowded interior of a cell than in dilute solution. Here we use a combination of wide angle X-ray scattering (WAXS) and computational modeling to derive a quantitative measure of the spatial scale of conformational fluctuations in a protein solution. Concentration-dependent changes in the observed scattering intensities are consistent with a model of structural fluctuations in which secondary structures undergo rigid-body motions relative to one another. This motion increases with decreasing protein concentration or increasing temperature. Analysis of a set of five structurally and functionally diverse proteins reveals a diversity of kinetic behaviors. Proteins with multiple disulfide bonds exhibit little or no increase in breathing in dilute solutions. The spatial extent of structural fluctuations appears highly dependent on both protein structure and concentration and is universally suppressed at very high protein concentrations.

Skeletal muscle performance determined by modulation of number of myosin motors rather than motor force or stroke size.

Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.