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A self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering.

ABSTRACT: Characterization of the conformational properties of unfolded proteins is essential for understanding the mechanisms of protein folding and misfolding. This information is also fundamental to determining the relationship between flexibility and function in the highly diverse families of intrinsically disordered proteins. Here we present a self-consistent model of conformational sampling of chemically denatured proteins in agreement with experimental data reporting on long-range distance distributions in unfolded proteins using small-angle x-ray scattering and nuclear magnetic resonance pulse-field gradient-based measurements. We find that standard statistical coil models, selected from folded protein databases with secondary structural elements removed, need to be refined to correct backbone dihedral angle sampling of denatured proteins, although they appear to be appropriate for intrinsically disordered proteins. For denatured proteins, pervasive increases in the sampling of more-extended regions of Ramachandran space {50 degrees

SUBMITTER: Bernado P

PROVIDER: S-EPMC2776250 | biostudies-other | 2009 Nov

REPOSITORIES: biostudies-other

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A self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering.

Bernadó Pau P Blackledge Martin M

Biophysical journal 20091101 10

Characterization of the conformational properties of unfolded proteins is essential for understanding the mechanisms of protein folding and misfolding. This information is also fundamental to determining the relationship between flexibility and function in the highly diverse families of intrinsically disordered proteins. Here we present a self-consistent model of conformational sampling of chemically denatured proteins in agreement with experimental data reporting on long-range distance distribu ...[more]

PMID: 19917239

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Project description:Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D2O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10(-4) cm(3)mol/g(2) for Mb in phosphate buffer, 1.6 ×10(-4) cm(3)mol/g(2) for BPTI in phosphate buffer and 9.2 ×10(-4) cm(3)mol/g(2) for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.

Dataset Information

A self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering.

Publications

A self-consistent description of the conformational behavior of chemically denatured proteins from NMR and small angle scattering.

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