Ashton, A.W.; (2005) Computer modelling of multi domain proteins based on X-ray neutron solution scattering data. Doctoral thesis , University of London.

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Abstract

The study of multi domain proteins and their complexes in solution using X-ray and neutron solution scattering data is a valuable tool for structural biologists. This thesis describes how a novel automated computer modelling technique combined with high quality solution scattering data and biochemical knowledge can be used to give biologically significant information. In this study the use of this technique as applied to two biochemical systems is described. The first system to be studied was the interaction between tissue factor (TF) and blood coagulation Factor Vila (FVIIa), an interaction essential as an initiator of the blood coagulation cascade. FVIIa is a four domain plasma protein consisting of a serum protease domain with two glycosylation sites, two epidermal growth factor domains and a C-terminal Gla domain (a domain rich in y-carboxyglutamic residues). TF has two extracellular Fn-III domains which are bound to the cell membrane by a transmembrane region. In initial studies we were able to show how FVIIa complexed with sTF, formed a compact conformation in solution, confirming the findings of biochemical studies which had implicated numerous residues involved in complex formation. Modelling allowed us to propose domain models for free FVIIa and the complex between sTF and FVIIa which was subsequently confirmed by the crystal structure for this complex. The second protein to be studied was the pentameric ring structure of serum amyloid P (SAP). Although SAP is a plasma protein with no known biological function, SAP is found in all amyloid deposits and is believed to play a protective role in preventing normal body responses to removing such plaques. Previous studies had shown that SAP could exist as a pentamer or a decamer in solution. Even though the crystal structure of the pentameric form was known there were no details of how the decamer was formed. Using solution scattering data, along with the high resolution crystal structure of the stable pentameric ring, it was possible both to produce a model for the decamer, and to assess critically the parameters used in the modelling procedure. This study has advanced the method of scattering analysis. The ease of modelling solution scattering data makes it a valuable technique which is now becoming a routine procedure in structural and functional analysis of proteins.

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