• Skip to primary navigation
  • Skip to main content
  • Skip to primary sidebar

SBRU

  • Home
  • Documents & Resources
        • Protein Prep
          • Before you begin
          • Biochemistry & assays
          • Crystallography
          • Electrophoresis & gel staining
          • Immunoassays & immunological techniques
          • Liquid handling
          • Protein expression & cloning
          • Protein purification techniques
        • Chromatography
          • Affinity chromatography
          • Gel filtration chromatography
          • HPLC
          • Hydrophobic interaction chromatography
          • Ion-exchange chromatography
          • Reversed Phase chromatography
        • Manuals
          • All manuals
          • IMAGIC-5
          • “O”
          • Pymol
          • Spider
        • Other Resources
          • Student Resources
          • Useful Structural Biology Links
        • Book MCB Equipment

  • Structural Biology Coursework
  • Research Projects
  • People and Publications
  • About the SBRU
    • Events
    • MCB Bookings
    • Contact Us
    • Sitemap
You are here: Home / Research Projects / Structure of angiotensin-converting enzyme mutants

Structure of angiotensin-converting enzyme mutants

X-ray structure determination of active site angiotensin-converting enzyme (ACE) mutants

Keywords: angiotensin-converting enzyme (ACE), kinetics, structure, mutagenesis

Aims

To determine a structural basis for key interactions between S2’ pocket of angiotensin-converting enzyme and domain-selective inhibitors using x-ray crystallography.

Background

Angiotensin-converting enzyme is a metalloprotease that plays a pivotal role in blood pressure, electrolyte and fluid homeostasis through the conversion of angiotensin I to the vasopressor, angiotensin II [cite]10.1021/bi00439a001[/cite]. Somatic ACE (sACE) contains two homologous domains: a C-domain and an N-Domain [cite source=pubmed]8390518[/cite]. The rational design of domain-specific ACE-inhibitors in the treatment of cardiovascular diseases is dependent upon a detailed structural knowledge of the molecule. Our group in collaboration with Professor Acharya solved the first crystal structure of the testis form of ACE (tACE) [cite]10.1038/nature01370[/cite] and, more recently, we have crystallised and solved the structure of an underglycosylated form of the protein.

Objectives and research plan

A truncated ACE protein, lacking the cytosolic tail and transmembrane domain, has been deglycosylated using an in vitro site-directed mutagenesis approach whereby selected Asn-residues were mutated to Gln [cite]10.1042/BJ20021842[/cite]. Furthermore, active site residues in the S1’ and S2’ pockets of tACE have been to their N-domain counterparts. Mutants will be expressed in Chinese Hamster Ovary (CHO) cells and purified using affinity chromatography methodology established in our laboratory. The Km and kcat kinetic parameters will be determined using domain-specific substrates and compared to that of wild-type ACE. Extensive crystallization trials will be carried out using commercially available crystal screen conditions (Hampton Research). In addition, ammonium sulfate, PEG (FLUKA), and MPD (Sigma) matrices will be used. Collection of X-ray diffraction data will be carried out on the crystals, locally at the University of the Western Cape and, if necessary, at the Daresbury synchrotron source in the UK. The structure will be solved using the molecular replacement method. Model building and refinement will be carried out.

Primary Sidebar

Contact Us

021 650 2817
trevor.sewell@uct.ac.za

Copyright © 2023 · Genesis Sample on Genesis Framework · WordPress · Log in