Influence of multiple well defined conformations on small-angle scattering of proteins in solution.
A common structural motif for many proteins comprises rigid domains connected by a flexible hinge or linker. The flexibility afforded by these domains is important for proper function and such proteins may be able to adopt more than one conformation in solution under equilibrium conditions. Small-angle scattering of proteins in solution samples all conformations that exist in the sampled volume during the time of the measurement, providing an ensemble-averaged intensity. In this paper, the influence of sampling an ensemble of well defined protein structures on the small-angle solution scattering intensity profile is examined through common analysis methods. Two tests were performed using simulated data: one with the extended and collapsed states of the bilobal calcium-binding protein calmodulin and the second with the catalytic subunit of protein kinase A, which has two globular domains connected by a glycine hinge. In addition to analyzing the simulated data for the radii of gyration Rg, distance distribution function P(r) and particle volume, shape restoration was applied to the simulated data. Rg and P(r) of the ensemble profiles could be easily mistaken for a single intermediate state. The particle volumes and models of the ensemble intensity profiles show that some indication of multiple conformations exists in the case of calmodulin, which manifests an enlarged volume and shapes that are clear superpositions of the conformations used. The effect on the structural parameters and models is much more subtle in the case of the catalytic subunit of protein kinase A. Examples of how noise influences the data and analyses are also presented. These examples demonstrate the loss of the indications of multiple conformations in cases where even broad distributions of structures exist. While the tests using calmodulin show that the ensemble states remain discernible from the other ensembles tested or a single partially collapsed state, the tests performed using the simulated catalytic subunit of protein kinase A with noise added demonstrate that it can mask out the ensemble-dependent effects observed for the noiseless profiles.
Heller
William T
WT
Condensed Matter Sciences Division and Center for Structural Molecular Biology, Oak Ridge National Laboratory, USA. hellerwt@ornl.gov
D020816:Amino Acid Motifs; D000818:Animals; D002147:Calmodulin / Q000737:chemistry; D020134:Catalytic Domain; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D004331:Drosophila melanogaster; D051379:Mice; D008958:Models, Molecular; D015233:Models, Statistical; D008968:Molecular Conformation; D016011:Normal Distribution; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D012984:Software
D002147:Calmodulin; D011506:Proteins; D017868:Cyclic AMP-Dependent Protein Kinases