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