Sunday, 12 February 2012 16:05
Written by Administrator
The anticipated 700 MHZ NMR instrumentation is an optimal compromise of cost-effective, high-field and high-resolution NMR equipment suitable for biomolecular studies.
The UPAT’s 700 MHz NMR (SEE-STRUCT Module) with a cryogenically cooled probe for enhanced sensitivity and four channels, in concert with uniformly or selectively enriched recombinant proteins (in 13C, 15N and 2H), allows the set up of various applications in the field of structural biology and drug design. Such an instrument will permit the:
- High-resolution, comparable to X-ray, structure determination of protein drug-targets with molecular weight above 10 kDa, and atomic-level insight in protein and protein-protein/drug complexes dynamics, interaction and determination of binding affinity.
- Novel NMR experiments for fast acquisition of 3D, 4D, 5D, nD, using automated spectra collection and analysis of projections of N-dimensional spectra (K. Wüthrich et. al. Proc Natl Acad Sci USA 2005, 102;10876-10881). This application measures the information content of high dimensional spectra in low dimensional projection spectra, which results in a drastic reduction in acquisition time (while the acquisition of a complete set of heteronuclear 2D/3D experiments requires up to 3 weeks for a protein sample of 80-100 residues, this technique requires less than a week).
- Coupled fast acquisition and automated assignment techniques with unsupervised structure determination of proteins that reduces the time of the entire procedure from months to weeks.
- Time-resolved kinetics and fast acquisition of 2D fingerprint, diagnostic, 1H-15N HSQC spectra (~1 sec), eventually crucial for monitoring protein-protein/peptide/small-molecules transient complexes, folding/unfolding processes and mechanistic studies, protein-drug screening applicable in drug-design process.
- Use of reduced protein amounts and investigation of low concentration protein samples (since numerous proteins exhibit low expression yields, while others tend to homo- or hetero-oligomerize or aggregate in medium to high concentrations, i.e. >0.4-0.6 mM).
The UPAT’s existing 400 MHz NMR instrument, will still be exploited in the fine-tuning of the peptide/protein sample experimental conditions and the characterization of the small molecules. The 700 MHz NMR machine will be dedicated to high-resolution structural studies, high-throughput protein samples and screening of drug candidates and structure-based drug-design efforts.
Additionally, it will allow researchers from the entire Greek Academia or Industry and all the other Southern-East Europe countries (like Bulgaria, Turkey or others) to have access to this modern instrumentation. Access will be provided not only on a protein sample screening basis prior to NMR studies or X-ray crystallography, but also on a joint collaboration basis.
It will also allow the researchers working with small molecules to perform daily walk-in analysis of their samples. Finally, and even more importantly, cutting-edge structural biology projects will be eventually performed.