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’

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

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.

DNA/RNA Backbone Structure

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

Pseudorotation Phase Cycle of

Deoxyribose

“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 = J

1’-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

V

total

= V

bondlength

+ V

bondangles

, V

dihedral

+ V

electrostatics

+ V

NOE

+ V

jcoupling

V

NOE

= 

Structure Determination

k

2

(r-r

l

)

2 when r<rl

0

when r

l

< r <r

u

k

3

(r-r

u

)

2 when r

u

< r

4k

2

(r-r

u

)

2 when r>r

u

all NOEs

r

u

r

l

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 structures
of [d(AGCTTGCCTTGAG)-
[CTCAAGGCAAGCT)]

RMSD = 0.9Å
267 distance restraints
130 torsion angle restraints