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 VirusThe 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’

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

Review of DNA Structure

Review of DNA Structure

Review of DNA Structure

NMR Study of DR1

COSYresonance assignments

torsion anglessugar conformation

NOESYresonance assignmentsinterproton distances

Chemical exchangeimino 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.

DNA/RNA Backbone Structure

Bloomfield et.al. “Nucleic Acids; Structure,Properties, and Functions” 2000.

Pseudorotation Phase Cycle ofDeoxyribose

“Principles of Nucleic Acid Structure” Saenger, pg 19 (1984).

Preferred Pseudorotation Phase Angles

“Principles of Nucleic Acid Structure” Saenger, (1984).

B-DNA

A-DNA, RNA

Sequential Resonance Assignments

Interproton contacts less than 4Å in (a) B-DNA and (b) A-DNA.

“Biomolecular NMR Spectroscopy” J.N.S. Evans, pg 350 (1995).

2D NOESY of DR1

H8,H6-to-H1’,H5

H1’-to-H2’H2”

Bishop et.al., Biochemistry (1994).

Base-to-H1’ NOESY-walk

Bishop et.al., Biochemistry (1994).

Proton Chemical Shifts of DR1

~97% of all protons are assigned

Bishop et.al., Bioch (1994).

Distribution of Distance Constraints

502 NOE derived distance constraints.

Bishop et.al., Bioch (1994).

E.COSY H1’-to-H2’H2”

A18,H1’-A18,H2” Bishop et.al., Bioch (1994).

E.COSY A18,H1’-to-H2’H2”

5.9 Hz = J1’-2”

Linewidth ~4.9 Hz

Bishop et.al., Bioch (1994).

Coupling Constants and Conformations for Sugars

%S versus Base-pair

? ? ? ?Relative imino proton exchange rate.

Bishop et.al., Bioch (1994).

V

constraint

Vtotal = Vbondlength + Vbondangles, Vdihedral + Velectrostatics + VNOE + Vjcoupling

VNOE =

Structure Determination

k2(r-rl)2 when r<rl

0 when rl< r <ru

k3(r-ru)2 when ru

< r

4k2(r-ru)2 when r>ru

all NOEs

rurl

Structure Determination

NATO ASI Vol H87“NMR of Biol. Macr.”James et al., (1994).

Structure Determination

NATO ASI Vol H87“NMR of Biol. Macr.”James et al., (1994).

10 rMD structuresof [d(AGCTTGCCTTGAG)-[CTCAAGGCAAGCT)]

RMSD = 0.9Å267 distance restraints130 torsion angle restraints