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The structural and functional characterisation of the SH3 domains of p47phox from the human neutrophil NADPH oxidase

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posted on 2014-12-15, 10:31 authored by Vicky A. Galbraith
Dysfunction of NADPH oxidase results in Chronic Granulomatous Disease, a syndrome characterised by a profound predisposition to bacterial and fungal infections. p47 phox is one of the cytosolic components of the NADPH oxidase enzyme complex. p47phox contains two src homology 3 (SH3) domains termed the amino-SH3 (N-SH3) domain, residues 158-217, and carboxyl-SH3 (C-SH3) domain, residues 225-286, named according to their relative positions in p47phox. SH3 domains are known to bind proline-rich regions and the peptides derived from these regions. This thesis details the studies undertaken to express, purify and characterise p47phox N-SH3, C-SH3 domains and the tandem SH3-SH3 domain and the investigations carried out with their binding proline-rich peptides. The affinity of the binding interactions of p47phox N-SH3 and C-SH3 domains with proline-rich peptides derived from the membrane-bound NADPH oxidase component p22phox (residues 149-162), p47phox (360-372) and the cytoskeletal protein vinculin (residues 359-379) have been quantified by fluorescence spectroscopy. Proton and nitrogen resonance assignments were made for p47phox C-SH3 domain and a family of structures was generated with rmsd of 1.8 angstroms of the beta-sheets of the family members. Carbon, nitrogen and proton resonance assignments have been made for p47phox N-SH3 domain in its complexed form with the proline-rich peptide derived from p22phox. A family of structures of p47phox N-SH3 domain has been generated with rmsd of 0.3 angstroms for beta-regions of backbone atoms and 0.6 angstroms for residues 3-57 backbone atoms.

History

Date of award

2000-01-01

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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