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Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching.

ABSTRACT: Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage ?, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D(2)O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded protein. The scattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of random conformations. With BPTI added to a concentration of 65 mg/mL, there was a clear change in the scattering profile representing an increase in the mass fractal dimension of the unfolded protein, from 1.7 to 1.9, as expected if crowding favors more compact conformations. The crowding protein also inhibited aggregation of the unfolded protein. At 130 mg/mL BPTI, however, the fractal dimension was not significantly different from that measured at the lower concentration, contrary to the predictions of models that treat the unfolded conformations as convex particles. These results are reminiscent of the behavior of polymers in concentrated melts, suggesting that these synthetic mixtures may provide useful insights into the properties of unfolded proteins under crowding conditions.

SUBMITTER: Johansen D

PROVIDER: S-EPMC3037566 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching.

Johansen Daniel D Jeffries Cy M J CM Hammouda Boualem B Trewhella Jill J Goldenberg David P DP

Biophysical journal 20110201 4

Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage λ, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D(2)O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded p ...[more]

PMID: 21320458

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Project description:Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) play important roles in many aspects of normal cell physiology, such as signal transduction and transcription, as well as pathological states, including Alzheimer's, Parkinson's, and Huntington's disease. Unlike their globular counterparts that are defined by a few structures and free energy minima, IDP/IDR comprise a large ensemble of rapidly interconverting structures and a corresponding free energy landscape characterized by multiple minima. This aspect has precluded the use of structural biological techniques, such as X-ray crystallography and nuclear magnetic resonance (NMR) for resolving their structures. Instead, low-resolution techniques, such as small-angle X-ray or neutron scattering (SAXS/SANS), have become a mainstay in characterizing coarse features of the ensemble of structures. These are typically complemented with NMR data if possible or computational techniques, such as atomistic molecular dynamics, to further resolve the underlying ensemble of structures. However, over the past 10-15 years, it has become evident that the classical, pairwise-additive force fields that have enjoyed a high degree of success for globular proteins have been somewhat limited in modeling IDP/IDR structures that agree with experiment. There has thus been a significant effort to rehabilitate these models to obtain better agreement with experiment, typically done by optimizing parameters in a piecewise fashion. In this work, we take a different approach by optimizing a set of force field parameters simultaneously, using machine learning to adapt force field parameters to experimental SAXS scattering profiles. We demonstrate our approach in modeling three biologically IDP ensembles based on experimental SAXS profiles and show that our optimization approach significantly improve force field parameters that generate ensembles in better agreement with experiment.

Dataset Information

Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching.

Publications

Effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching.

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