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Rapid methods for deriving structural and functional Information for proteins by NMR

Reference	BB/C004728/1
Principal Investigator / Supervisor	Dr Martin Parker
Co-Investigators / Co-Supervisors	Dr C Craven
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	152,785
Status	Completed
Type	Research Grant
Start date	01/04/2005
End date	31/07/2007
Duration	28 months

Abstract

A unique feature of NMR spectroscopy is that is can provide a set of residue specific probes with which to study both protein structure and protein ligand interactions. The prime source for these probes is the 1H-15N HSQC spectrum. Despite many technological developments, the assignment of HSQC crosspeaks to specific residues in the protein remains a significant challenge, especially for large systems. We have developed a Combinatorial Selective Labelling (CSL) method for the efficient assignment of the majority of HSQC crosspeaks, which uses a small number of samples that can be rapidly and cost effectively produced in parallel in a commercially available cell-free system. The CSL method uses 5 samples, each containing a particular combination of 16 labelled amino acid types, which are individually either 100 per centage 13C 15N or 50 per cent 15N 50 per centage 14N labelled. For each sample a 1H-15N HSQC spectrum and a 1H-15N 2D HNCO spectrum are acquired. The relative peak intensities in the HSQC spectra yield the amino acid type of each peak; there are 2x2x2x2 equals 16 possible 100 per cent 15N 50 per centage 15N labelling patterns contained within the sample set (sample 1 is the fully labelled control). For a particular crosspeak, the amino acid type of the preceding residue is established by examining the presence or absence of peaks in the HNCO spectra. Thus all 16x16 amino acid pairs are identifiable simultaneously in the 5 samples. We demonstrated the method is practicable using GFP, but its poor solution characteristics led to low sensitivity in the spectra and it was not possible to investigate problems due to labelling noise. We will use a combination of assays of the amino acid solutions and correlate this data with the observed labelling ratios in protein G and SH3, which have excellent NMR characteristics. We will explore a strategy for providing additional side chain methyl assignments for Ala, Val, Ile and Leu. This involves adding 13C, 15N deuterium-labelled (methyl-protonated) ala, Val, Ile and Leu into the control sample and running a (H)C(CO)NH-TOCSY. This experiment correlates the methyl group spins with the 15N, NH spins of the following residues. Within the framework of the CSL method, the methyl groups of these residues are assignable so long as they are followed by a uniquely assignable NH group. If an amino acid pair appears n times in the sequence the CSL assignment will be n-fold degenerate. The number of unique pairs drops as the size of the protein increases. In addition to reducing spectral complexity, segmental isotope labelling would increase the assignment level. To exploit this technology, intein vector constructs will be engineered and tested to enable expression of label chimeras in a cell-free system. These methods will be applied to a challenging and medically important problem the interaction of human bactericidal permeability increasing protein (BPI) with lipopolysaccharide (LPS), a component of the outer envelope of gram-negative bacteria. The binding mode of LPS remains to be elucidated, and correct understanding of the interaction is important for the design of therapeutics. The assignment methods will be applied to BPI and a titration with LPS performed in order to map the binding site(s) onto the X-ray structure. BPI is 456 residues long, making it problematic for standard assignment methods, and is an ideal candidate for cell-free expression, being toxic to bacteria.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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