Nuclear Magnetic Resonance Studies of Biological Systems

 

 

Principal Investigator

Marco Casarotto

John Curtin School of Medical Research

Co-Investigators

Angela Dulhunty

Peter Gage

Gary Ewart

Francis Shannon

John Curtin School of Medical Research

Projects

MDSS

             
   

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography remain the two most powerful techniques for the structural elucidation of biological molecules. Of these, NMR is not only capable of yielding high resolution structural information of biomolecules in solution but can also provide dynamic and biochemical information not accessible by other techniques. Over the past few years great advances have taken place in NMR and molecular biology enabling the study of biomolecules approaching molecular weights of up to 100 kD.

One of the primary roles of the biological NMR laboratory is to foster and develop collaborative ties with members of the ANU community. A number of projects are in progress involving drug binding, mechanistic studies of enzyme systems and the structural study of membrane related proteins. Since many of the projects are collaborative efforts with other members in the university, an integrated approach involving other techniques complementary to NMR such as molecular biology, kinetics and molecular modelling are employed.

   
         
                 

     
                 

 

What are the results to date and the future of the work?

One enzyme under investigation is dihydrofolate reductase which is an important target for anticancer, antibacterial and antimalarial drugs. Its role in the cell is to catalyse the conversion of dihydrofolate to tetrahydrofolate using NADPH as the coenzyme. Although this enzyme has been the focus of numerous kinetic and structural studies, the manner in which it functions remains unknown. Our goal is to combine protein engineering and NMR to examine the drug binding and mechanistic properties of this enzyme and ultimately be in a position to design novel therapeutic agents.

Structural information of membrane associated proteins is often difficult to obtain, however NMR spectroscopy can be a versatile tool in exploring such systems. As part of the "Membrane Biology Program" we have undertaken NMR studies on a number of different and challenging membrane related proteins.

                 
- Appendix A

 
                 

       

One of these involves the dihydropyridine receptor (DHPR) which is important for muscle excitation-contraction. In skeletal muscle it is known that DHPR interacts with the ryanodine receptor (RyR) triggering release of Ca2+ from the sarcoplasmic reticulum. A cytoplasmic loop region of DHPR has been identified as being essential for RyR activation and excitation-contraction coupling. We are using high resolution NMR spectroscopy to probe the solution structure of several loop fragments of DHPR ranging from the entire loop of 12 kD to small fragments of approximately 20 amino acids. Results indicate that specific peptides with well defined structures are capable of interacting with RyR. The region of RyR which interacts with DHPR has been recently identified raising the prospect of examining protein-protein type interactions between these two membrane systems using NMR methods.

The viral proteins Vpu and Vpr of the immunodeficiency virus have been reported to possess ion channel activity. It is thought that these proteins play some role in replication and/or pathogenesis. Peptide fragments of these viral proteins are capable of maintaining their ion-pore capabilities and are being structurally examined in a variety of media by NMR.

What computational techniques are used?

All of our structure calculations are performed using the program XPLOR and carried out on our SGI O2 R5000 computers. The NMR data is stored on the ANUSF Mass Data Storage System.

       

 

 

 

Appendix A -