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Publication Detail
Modelling of the serine-proteinase fold by X-ray and neutron scattering and sedimentation analyses: Occurrence of the fold in factor D of the complement system
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Journal Article
  • Authors:
    Perkins SJ, Smith KF, Kilpatrick JM, Volanakis JE, Sim RB
  • Publication date:
  • Pagination:
    87, 99
  • Journal:
    Biochemical Journal
  • Volume:
  • Issue:
  • Status:
  • Print ISSN:
Solution scattering is a powerful means of determining the overall arrangement of domains in the multidomain proteins of complement. The serine-proteinase domain is central to all proteolytic events during complement activation. As models of this domain, bovine β-trypsin, trypsinogen, α-chymotrypsin and chymotrypsinogen A were studied by neutron and X-ray synchrotron solution scattering. At pH 7, all the X-ray and neutron M(r) values corresponded to monomeric proteins. The X-ray radii of gyration, R(G), of β-trypsin, trypsinogen, α-chymotrypsin and chymotrypsinogen A (measured in positive solute-solvent contrasts) were 1.59 nm, 1.78 nm, 1.71 nm and 1.76 nm (± 0.05-0.11 nm) in that order. Neutron contrast variation showed that the R(G) at infinite contrast, R(c), for these four proteins were 1.57 nm, 1.70 nm, 1.67 nm and 1.78 nm (±0.03 nm) in that same order. The radial inhomogeneity of neutron-scattering density, α, was positive at (5-13) x 10-5, and corresponds to the preponderance of hydrophilic residues near the protein surface. On trypsinogen activation, a small reduction in the R(G) value of 0.13 ± 0.07 nm was just detectable, while the R(G) of chymotrypsinogen A was unchanged after activation. The R(C) and α values of the four proteins can be calculated by using crystallographic co-ordinates. The reduced R(G) of β-trypsin relative to trypsinogen was explained in terms of the removal of the extended N-terminal hexapeptide of trypsinogen. The full X-ray and neutronscattering curves in positive and negative contrasts agreed well with scattering curves calculated from crystallographic coordinates to a nominal structural resolution of 4.5 nm, provided that the internal structure was considered in neutron modelling, and that the hydration was considered in X-ray modelling. Sedimentation-coefficient data also provide information on the disposition of domains in multidomain proteins. It was found that the hydrated X-ray sphere model could be directly utilized to calculate sedimentation coefficients. X-ray scattering on factor D showed from its R(G) of 1.78 nm that this is monomeric and very similar in structure to β-trypsin. The X-ray-scattering curve of factor D was readily modelled using the β-trypsin crystal structure after allowance for sequence changes. The success of these modellings provides a basis for the constrained modelling of solution scattering data for the multidomain proteins of complement.
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