BCMB / CHEM 8190 Biomolecular NMR
GRADUATE COURSE OFFERING IN NUCLEAR
MAGNETIC RESONANCE
"Biomolecular Nuclear Magnetic Resonance" is a
course intended for all graduate students with an
interest in applications of nuclear magnetic resonance
(NMR) to problems in structural and functional biology.
It will begin with a treatment of the fundamentals that
underlie magnetic resonance phenomena and develop
this into a basis for experimental design, interpretation
of data, and critical reading of the literature.
http://tesla.ccrc.uga.edu/courses/bionmr/
Syllabus
I.
Introduction
A.
B.
II.
Instrumentation
A.
B.
C.
•
•
M 1/9
Prestegard
Magnetic properties of nuclei
and electrons – precession
5-38 L*
W 1/11
Prestegard
RF pulses and spin relaxation Bloch equations
39-50 L*, 653 L*
M 1/16
MLK Jr. Holiday
(no class)
W 1/18
Prestegard
Instrumental considerations a look at probes
65-76 L*
M 1/23
Prestegard
Fourier transform methods
and data Processing
85-102 L*
Friday Labs are scheduled separately (C122) – intro to computer
systems 1/13, 1/20
Texts: "Spin Dynamics - Basics of Nuclear Magnetic Resonance",
M. H. Levitt (L), “Protein NMR Spectroscopy, Principles &
Practice“, J. Cavanagh, W. J. Fairbrother, A. G. Palmer III, N. J.
Skelton. (C)
Biomolecular NMR 2012
• Biomolecular NMR - short history ~ 1985 first
protein structure
• Compared to X-ray ~ 1953 first protein
structure
• Today ~ 13 % of structures in the PDB come
via NMR – higher for nucleic acids
• Unique structural applications – weak
associations, partially structured, membrane
associations, in-cell observation
• Diverse applications: drug screening,
metabolic monitoring, in vivo imaging
• NMR is still an evolving science
Can NMR Make a Unique Contribution ?
Membrane Proteins, Protein-Protein Interactions, Ligand Binding
Unstructured Regions in Proteins
% with rmsd > 4 Å
18
16
NMR
Xray
Frequency
14
12
10
8
6
4
2
0
0
0.1
0.2
0.3
Protein fraction
0.4
0.5
0.6
NMR Recognition
•
•
•
•
1952 - Bloch and Purcell – Nobel Prize in Physics
1991 – Richard Ernst – Nobel Prize in Chemistry
2002 – Kurt Wuthrich – Nobel Prize in Chemistry
2003 – Lauterbur and Mansfield – Nobel Prize in
Physiology and Medicine
Computers Were Primitive in 1966
Varian HR 220
Early
Superconducting
Magnets
Required a Lot of
Care And
Feeding
High Field (220 MHz), but Still 1D CW NMR
80’s Approach: 2D 1H-1H NMR: 9 kDa Protein
assign resonances, collect NOE’s, calculate structure
=
=
Extension to 3D: Through-bond Correlations in Peptides
Isotope Labeling is Key
3D
O H Hα O H
- - - C N Cα C N - - H - Cβ - H
H
f1 (13C)
(HB)CBCA(CO)NH
f3 (1H)
f2 (15N)
=
=
O H Hα O H
- - - C N Cα C N - - -
2D
HN(CO)CA
1D
H - Cβ - H
H
HNCACB
IKURA M; KAY LE; BAX A, (1990) BIOCHEMISTRY 29:4659-4667
Today Very Large Systems Can Be Studied:
Proteasome subunit – active site dynamics
Sprangers, R and Kay, LE, 2007. Probing supramolecular structure from
measurement of methyl H-1-C-13 residual dipolar couplings. Journal of
the American Chemical Society 129: 12668-+.
Ruschak AM and Kay LE, 2010, Methyl groups as probes of supra-molecular
structure, dynamics and function, J Biomol NMR 46:75-87
NMR is widely applicable to structure and function of biomolecules
• Judge, P. J. & Watts, A. (2011). Recent contributions from solid-state NMR to the
understanding of membrane protein structure and function. Current Opinion in
Chemical Biology 15, 690-695.
• Kang, C. B. & Li, Q. X. (2011). Solution NMR study of integral membrane proteins.
Current Opinion in Chemical Biology 15, 560-569.
• Dominguez, C., Schubert, M., Duss, O., Ravindranathan, S. & Allain, F. H. T.
(2011). Structure determination and dynamics of protein-RNA complexes by NMR
spectroscopy. Progress in Nuclear Magnetic Resonance Spectroscopy 58, 1-61.
• Montelione, G. T. & Szyperski, T. (2010). Advances in protein NMR provided by the
NIGMS Protein Structure Initiative: Impact on drug discovery. Current Opinion in
Drug Discovery & Development 13, 335-349.
• Pacheco-Torres, J., Lopez-Larrubia, P., Ballesteros, P. & Cerdan, S. (2011).
Imaging tumor hypoxia by Magnetic Resonance methods. Nmr in Biomedicine 24, 116.
• Powers, R. (2009). NMR metabolomics and drug discovery. Magnetic Resonance in
Chemistry 47, S2-S11.
• Tycko, R. (2011). Solid-State NMR Studies of Amyloid Fibril Structure. In Annual
Review of Physical Chemistry, Vol 62 (Leone, S. R., Cremer, P. S., Groves, J. T. &
Johnson, M. A., eds.), Vol. 62, pp. 279-299.
• Tzeng, S. R. & Kalodimos, C. G. (2011). Protein dynamics and allostery: an NMR
view. Current Opinion in Structural Biology 21, 62-67.
Spin Properties
B0
μ=γI
Iz = hm
E = -μ⋅B0
for I = 1/2
m = -1/2
ν = γB0/2π
E
p = exp(-Ei/kT)/Z
Mz = γhΣipimiN0
2 2
= N0γ h B0 I(I+1)
B0
m = 1/2
3kT
NMR Active Isotopes Exist for Nearly Every Element
http://bouman.chem.georgetown.edu/NMRpt/NMRPerTab.html
Select an element by clicking on it:
1
H
Li
Na
K
Rb
Cs
Fr
2
Be
Mg
Ca
Sr
Ba
Ra
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
B
Al
Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga
Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In
* Hf Ta W Re Os Ir Pt Au Hg Tl
** Rf Ha Sg Ns Hs Mt
H
C N O F
Si P S Cl
Ge As Se Br
Sn Sb Te I
Pb Bi Po At
*La Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu
**Ac Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr
He
Ne
Ar
Kr
Xe
Rn
Spin ½ Nuclei are Most Useful in Biomolecualr NMR
Isotope: 13C
Spin:
1/2
Natural abunance:
1.108%
Magnetogyric ratio (rad/T s): 6.7283 x 10^7
Relative receptivity:
1.76 x 10^-4
Magnetic moment
1.2166
Quadrupole moment Q/m(2)
0
Resonance frequency
25.144 MHz
Polypeptides are Rich in NMR Active Nuclei
Cβ 13C
Amide 1H
Cα 1H
Amide 15N
C’ 13C
Me – 1H
Cα 13C
Nuclear Properties
•
•
•
•
Not all nuclei have magnetic moments, Why?
Not all nuclei are equally abundant, Why?
Spins vary, Why?
Magnetogyric ratios vary, Why?
Fundamental Particle Properties
e-
S
Beam in field
gradient
N
Stern Gerlach experiment: Na atom - 1 unpaired electron
Two spots implies quantized moments: +/- 1/2
protons and neutrons are also spin 1/2 particles
Current Loop Model for Magnetic Moments
M
r
M=iS
i
Area, S
i in Coulomb s-1
S in m2
M in JT-1 (Tesla)
Estimates: i = -ev/(2πr), S = πr2, M (or μ) = -erv/2
μ = -e(r x v)/2, L = me r x v, μ = -e/(2me) L = γ L = γh/(2π)l
γ = -g (e/(2me)), g = Lande g factor
Values of Particle Magnetogyric Ratios
Electron: g ≈ 2,
γe = -17.7 x 1010 T-1s-1
Proton: expect 1/mp dependence, 1/2000 and positive
2.7 x 108 T-1s-1
Neutron: similar mass to proton
-1.8 x 108 T-1s-1
Heavier Nuclei: the Shell Model
V(r)
Simple Rules:
r0
0
r0
a) spherical particle in a box potential
ψ = Rnl(r) Ylm(θ,φ), E(n,l)
ladder of energy levels like H atom, but all ls allowed
b) strong coupling of spin and orbit angular momentum
quantized total: j = l +/- 1/2 for spin 1/2 particle
larger j, lower energy (usually)
Shell Model Rules Continued
c) Treat protons and neutrons separately and fill
from bottom up assuming 2j + 1 degeneracy
d) Assume particle pair strongly within levels: only
unpaired spins count - total spin angular momentum
given by j of level for unpaired spin
e) sign of moment depends on sign of moment for
fundamental particle but changes sign when moment
subtracts instead of adds to l in giving j
Energy Level Diagram
n+1
j
degeneracy
2s
1/2
2
3/2
4
5/2
6
1/2
2
3/2
4
1/2
2
total
20
1d
8
1p
1s
2
Examples of Nuclear Properties
•
16 O
8
•
13 C
6
- most abundant in Earth’s crust. 8 protons, 8
neutrons - 2 magic #s. All particles paired - no
moment.
- 6 protons, 7 neutrons. All protons paired. One
neutron unpaired in 1p1/2. j = l - 1/2. Therefore,
negative moment subtracts from l. Positive γ spin 1/2
particle