BCMB / CHEM 8190 Biomolecular NMRGRADUATE 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/

SyllabusI. Introduction

A. M 1/9 Prestegard Magnetic properties of nuclei and electrons – precession 5-38 L*

B. W 1/11 Prestegard RF pulses and spin relaxation -Bloch equations 39-50 L*, 653 L*

II. InstrumentationA. M 1/16 MLK Jr. Holiday (no class)B. W 1/18 Prestegard Instrumental considerations -

a look at probes 65-76 L*C. 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

Unstructured Regions in Proteins% with rmsd > 4 Å

0

2

4

6

8

10

12

14

16

18

0 0.1 0.2 0.3 0.4 0.5 0.6

Protein fraction

Freq

uenc

y

NMRXray

Can NMR Make a Unique Contribution ?Membrane Proteins, Protein-Protein Interactions, Ligand Binding

• 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

NMR Recognition

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 Proteinassign resonances, collect NOE’s, calculate structure

- - - C N C C N - - -α

Hα

Cβ

= =- -

O O

H

H H

HH

- - - C N C C N - - -α

Hα

Cβ

= =

- -

O O

H

H H

HH

(HB)CBCA(CO)NH

HNCACB

Extension to 3D: Through-bond Correlations in PeptidesIsotope Labeling is Key

IKURA M; KAY LE; BAX A, (1990) BIOCHEMISTRY 29:4659-4667

1D2D

3D

f3 (1H)

f1 (13C)

f2 (15N)

HN(CO)CA

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, 1-16.

• 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 PropertiesB0

μ = γ I Iz = hm E = -μ⋅B0

B0

m = -1/2

m = 1/2

ν = γB0/2π Mz = γhΣipimiN0

= N0γ h B0 I(I+1)

3kT

22

p = exp(-Ei/kT)/Z

for I = 1/2

E

NMR Active Isotopes Exist for Nearly Every Element

http://bouman.chem.georgetown.edu/NMRpt/NMRPerTab.html

Select an element by clicking on it:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 H H He Li Be B C N O F Ne Na Mg Al Si P S Cl Ar K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe Cs Ba * Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn Fr Ra ** Rf Ha Sg Ns Hs Mt *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

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

Spin ½ Nuclei are Most Useful in Biomolecualr NMR

Polypeptides are Rich in NMR Active Nuclei

Me – 1H

Amide 1HCα 1H

Amide 15N

Cβ 13C

C’ 13C 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

N

S

Stern Gerlach experiment: Na atom - 1 unpaired electronTwo spots implies quantized moments: +/- 1/2protons and neutrons are also spin 1/2 particles

e- Beam in fieldgradient

Current Loop Model for Magnetic MomentsM

iArea, S

M = i 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

r

Values of Particle Magnetogyric Ratios

Electron: g ≈ 2, γe = -17.7 x 1010 T-1s-1

Proton: expect 1/mp dependence, 1/2000 and positive2.7 x 108 T-1s-1

Neutron: similar mass to proton-1.8 x 108 T-1s-1

Heavier Nuclei: the Shell Model

Simple Rules:

a) spherical particle in a box potentialψ = Rnl(r) Yl

m(θ,φ), E(n,l)ladder of energy levels like H atom, but all ls allowed

b) strong coupling of spin and orbit angular momentumquantized total: j = l +/- 1/2 for spin 1/2 particlelarger j, lower energy (usually)

V(r)

r0r0 0

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 total

2s 1/2 2 20

3/2 41d

5/2 6

1/2 2 81p

3/2 4

1s 1/2 2 2

Examples of Nuclear Properties

• 168O - most abundant in Earth’s crust. 8 protons, 8

neutrons - 2 magic #s. All particles paired - no moment.

• 136C - 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