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All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins.

ABSTRACT: The flexible and heterogeneous nature of carbohydrate chains often renders glycoproteins refractory to traditional structure determination methods. Small-angle X-ray scattering (SAXS) can be a useful tool for obtaining structural information of these systems. All-atom modeling of glycoproteins with flexible glycan chains was applied to interpret the solution SAXS data for a set of glycoproteins. For simpler systems (single glycan, with a well-defined protein structure), all-atom modeling generates models in excellent agreement with the scattering pattern and reveals the approximate spatial occupancy of the glycan chain in solution. For more complex systems (several glycan chains, or unknown protein substructure), the approach can still provide insightful models, though the orientations of glycans become poorly determined. Ab initio shape reconstructions appear to capture the global morphology of glycoproteins but in most cases offer little information about glycan spatial occupancy. The all-atom modeling methodology is available as a web server at http://salilab.org/allosmod-foxs.

SUBMITTER: Guttman M

PROVIDER: S-EPMC3840220 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins.

Guttman Miklos M Weinkam Patrick P Sali Andrej A Lee Kelly K KK

Structure (London, England : 1993) 20130301 3

The flexible and heterogeneous nature of carbohydrate chains often renders glycoproteins refractory to traditional structure determination methods. Small-angle X-ray scattering (SAXS) can be a useful tool for obtaining structural information of these systems. All-atom modeling of glycoproteins with flexible glycan chains was applied to interpret the solution SAXS data for a set of glycoproteins. For simpler systems (single glycan, with a well-defined protein structure), all-atom modeling generat ...[more]

PMID: 23473666

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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

All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins.

Publications

All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins.

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