Presentation on theme: "Structure Determination by NMR I. Choose a biologically important question. II. Determine if and how NMR can address the question. III. Synthesize or extract."— Presentation transcript:
Structure Determination by NMR I. Choose a biologically important question. II. Determine if and how NMR can address the question. III. Synthesize or extract the molecule to study. IV. Design the NMR study. V. Make the NMR sample(s). VI. Acquire and process the NMR data. VII. Extract information relevant to your question or hypothesis. VIII. Report your findings.
Hepatitis B Virus The Disease Member of the hepatocellular DNA virus family 300 million people worldwide are carriers. Symptom of infection vary but usually involve inflamation of the liver and sometimes liver damage. 90% of the people who contract the virus will go through an acute phase of infection and then recover with lasting immunity. 10% of the people who contract the disease do not resolve the primary infection and become carriers. Those that have the chronic infection have a 100-fold or greater risk of hepatocellular carcinoma (liver cancer).
The Hepatitis B Virus Genome 5’ + - RNA Protein Plus strand + 5’-GGCAGAGGTGAAA-3’ 3’-CCGTCTCCACTTT-5’ Direct Repeat Sequence ~3.2 kilobases
The Hepatitis B Virus Direct Repeat Sequence 5’-GGCAGAGGTGAAA-3’ 3’-CCGTCTCCACTTT-5’ I. Performs a critical role in the initiation of viral DNA synthesis which is not completely understood. II. Deletion or mutation of just one residue can be catastrophic to virus. III. Small enough to be studied by NMR. IV. Are there any unique structural features that can give us insight into biological activity? V. The sequence will have an extra base-pair on each end.
Review of DNA Structure
NMR Study of DR1 COSY resonance assignments torsion angles sugar conformation NOESY resonance assignments interproton distances Chemical exchange imino proton exchange rates, i.e. base pair opening
Resonance Assignments A combination of COSY and NOESY. Use known characteristics of molecule. sequence, identity of terminal bases, etc. Confirm base-pair formation. Initially assume it has a regular structure, e.g. B-DNA.