Studying Protein Structure at the John Innes Centre

Introduction

To use new knowledge from our science programmes, in particular genomic and proteomic research, it is essential that we are able study protein structure and function. This requires technologies that extend from characterising gene expression to describing the structure and function of the gene's protein product.

Molecular structure analysis

JIC's X-ray crystallographic facilities include a rotating anode X-ray generator with confocal mirror optics, an image plate detector and a cryogenic cooling device. This is used for routine X-ray data collection. Use of synchrotron X-ray sources is largely restricted to specialist applications, such as collection of high-resolution data and experiments where a tunable X-ray source is required.

JIC has a 400 Mhz NMR spectrometer principally used in determining the structures of small molecules.

We are able to study systems containing paramagnetic centres using a 9 GHz EPR and ENDOR (Electron Nuclear Double Resonance) spectrometer (that can be used down to 1.8K) and a 9 and 35 GHz system, which provides higher sensitivity and resolution. Paramagnetic systems may be naturally occurring, such as transition metal ions or free radicals, or artificially introduced as 'spin labels' or 'spin probes'. Using these techniques we can determine the structural and functional features of liquid and frozen solutions, including soluble and membrane proteins.

The Chemistry Dept at the University of East Anglia has complementary equipment, including 95 GHz and pulsed machines. Together the two laboratories form the best-resourced UK centre for applying paramagnetic techniques in biology.

Reaction kinetics

JIC's facilities and expertise for the study of rapid reactions, principally the determination of enzyme reaction mechanisms, is unmatched in the UK.

A suite of spectroscopy techniques (including stopped-flow fluorescence and absorbance) are supplemented by rapid-quench and rapid-freeze techniques. All of these are optimized for work with oxygen-sensitive material and operate under anoxic conditions or at low defined O2 concentrations.