Biochemical and biophysical studies of nitric oxide synthase

Group leader: John Ingledew

Research overview

We study complex structural interactions (incorporating movement of hinged domains in nitric oxide synthase, assembly of complex signalling systems, assembly of DNA binding proteins on DNA, and movement in large intermembraneous complexes) by site-directed spin labelling (SDSL) coupled with emerging EPR spectroscopic techniques. Nitroxide spin labels have long been used as probes and reporter groups. In the last decade this technique has gained renewed impetus through the use of site directed mutagenesis to selectively place a small nitroxide radical by coupling to an engineered cysteine. Where an intrinsic paramagnetic or a second spin-label is present the dipole interaction between the two centres can be used to measure the inter-centre distance. Traditional cw EPR can exploit the dipolar interactions between paramagnetic centres to measure inter-centre distances up to about 20Å. More recently, pulsed double resonance techniques (pulsed-ELDOR, DEER) have been introduced which extend the distances over which interactions can be measured up to 80Å. Inter-spin distance measurements provide a relatively rapid and quantitative assessment of conformational change, assembly and dis-assembly in these large protein complexes. These are relatively new techniques. We are in the fortunate position at St.Andrews of having perhaps the best equipped lab. in the UK for advanced EPR techniques.  Instrumentation includes a ‘state of the art’ commercial instrument (Bruker E580 pulsed ESR EleXsys system, X-band and Q-band, ENDOR and ELDOR).  This was commissioned late summer 2005 as part of a Basic Technology Project with the development of SDSL methodologies as a major focus in a collaboration between St.Andrews and Dundee Universities. It is the first ELDOR instrumentation in the UK and is now routinely producing results at a level similar to the best international labs. Also under the Basic Technology program the Physics department (Dr G. Smith) is constructing a new pulsed EPR/ELDOR spectrometer at W-band as part of the EPSRC HiPER project [High Field, High Power, Pulsed ESR] that promises to combine improved orientational selectivity with dramatically improved sensitivity and time resolution.  Although, the full capabilities of the machine will only be apparent when it is fully operational in late 2006 initial indications are extremely promising and 30 fold+ sensitivity gains will be possible.  This will allow the use of lower protein concentrations and push the detectable interaction range up to 100Å.

Publications

Hagelueken, G , Ingledew, WJ , Huang, H , Petrovic-Stojanovska, B, Whitfield, C, ElMkami, H , Schiemann, O & Naismith, JH 2009, ' PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer-Membrane Protein Wza from Escherichia coli. ' Angewandte Chemie International Edition , vol 48, no. 16, pp. 2904-2906.
Marechal, A , Ingledew, WJ & Rich, PR 2008, ' Time-resolved FTIR study of CO recombination with horseradish peroxidase ' Biochemical Society Transactions , vol 36, pp. 1165-1168.
Rudolf, J, Makrantoni, V , Ingledew, WJ , Stark, MJR & White, MF 2006, ' The DNA repair helicases XPD and FancJ have essential iron-sulfur domains ' Molecular Cell , vol 23, no. 6, pp. 801-808.

Overview

Scientists associated with the thirty-two research groups that are affiliated with the Biomedical Sciences Research Complex perform highly innovative, multi-disciplinary research in eleven broad areas of biomedical research, employing state-of-the-art techniques to address key questions at the leading edge of the biomedical and biological sciences.

Follow the links on the left to view individual research groups associated with one or more of the eleven BSRC research areas.

Research areas

Scientists associated with the thirty-two research groups that are affiliated with the Biomedical Sciences Research Complex perform highly innovative, multi-disciplinary research in eleven broad areas of biomedical research, employing state-of-the-art techniques to address key questions at the leading edge of the biomedical and biological sciences.

Follow the links on the left to view individual research groups associated with one or more of the eleven BSRC research areas.

Research by academic schools

Research in the BSRC is conducted by thirty-two independent research groups based in the Schools of Biology, Chemistry, Physics and Astronomy, and Medicine. Follow the links on the left to view groups associated with each school.