Stemberk, Vaclav (2011) Studies of fibrinogen and fibronectin binding to a Staphylococcus aureus surface protein. PhD thesis, University of York.
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Staphylococcus aureus is a common human pathogen and a major cause of infective endocarditis (IE), an infection of native as well as prosthetic heart valves. The fibronectin-binding protein of S. aureus (FnBPA) is a cell-wall attached adhesin that is closely linked with the development of IE owing to its ability to bind the plasma glycoproteins fibrinogen (Fg) and fibronectin (Fn). The FnBPA molecule contains adjacent Fn- and Fg-binding regions. Previous studies identified eleven intrinsically unstructured, homologous repeats from FnBPA (FnBRs) that bind Fn through an unusual tandem β-zipper mechanism. It was proposed that the Fg-binding region of FnBPA comprised two domains, N2 and N3, with predicted IgG folds. The main focus of this work is to characterise the FnBPA Fg interaction, to define the N2N3/FnBRs boundary and to determine whether simultaneous binding of Fg and Fn to FnBPA is affected by cooperativity or potential steric effects. Surface plasmon resonance in conjunction with isothermal titration calorimetry showed that the FnBPA N2N3 region retains the Fg binding activity of intact FnBPA. X ray crystallography of N2N3 in complex with the Fg peptide revealed the location of the Fg binding site on N2N3 and that a β zipper was formed upon Fg binding. A range of new biophysical data led to a redefined N2N3/FnBRs boundary, which, in the form of a ten residue long flexible linker, comprised more residues than originally thought. Simultaneous binding of Fg and the N terminal domain of Fn to an rFnBPA construct, consisting of the N2N3 region and the first FnBR, was demonstrated for the first time. However, simultaneous binding was not observed when intact Fg and Fn were used, providing the first preliminary evidence for the importance of steric hindrance in regulation of the ternary complex formation.
|Item Type:||Thesis (PhD)|
|Academic Units:||The University of York > Chemistry (York)|
|Depositing User:||Mr Vaclav Stemberk|
|Date Deposited:||17 Jan 2012 12:10|
|Last Modified:||08 Aug 2013 08:47|