Introduction to NMR
Biophysics Student Seminar Series
Application
Solid State NMR introducing
Research
Bo Zhao
Zimeng Li

Introduction to NMR
Application
Solid State NMR
Research

Introduction to NMR

Introduction to NMR
Physical Origin
Measurement

Physical Origin
• Rabi (1938)
• Spin is internal property of particles
• Spin can generate magnetic field
• Protons and Neutrons have spin 1/2
Xu, Modern Physics (1993)

Nuclei
1 H
2 H
14 N
15 N
12 C
13 C
19 F
31 P
Physical Origin
Unpaired
Protons
1
1
1
1
0
0
1
1
Unpaired
Neutrons
0
1
1
0
0
1
0
0
Net Spin γ (MHz/T) Abundance
1/2
1
1
1/2
0
1/2
1/2
1/2
42.58
6.54
3.08
N/A
10.71
40.08
17.25
99.98%
0.0184%
99.636%
-4.316
0.34%
99%
1%
100%
100%
Spin ½ System

Spin ½ System
• External field – energy splitting
?
Hornak, The Basics of NMR (1997)

Spin ½ System
• Internal property
• External factor
– High Field (1964)
– Electron shielding
– Spin coupling
• Chemical Shift 𝛿 = 𝑣−𝑣 𝑟𝑒𝑓 𝑣 𝑟𝑒𝑓 ppm
Chemical Shift Anisotropy
Spin coupling e
?
p
1 H [ppm]
Shi, NMR Introduction course (2003)
Trausch et al., Chemical Physics Letters (2008)

Spin ½ System
Chemical Shift Anisotropy
Dipole-Dipole coupling 𝜈

Chemical Shift Anisotropy
• More shielding -> lower chemical shift.
𝐵 𝑒𝑓𝑓
= 𝐵
0
− 𝐵 𝑠ℎ𝑖𝑒𝑙𝑑 𝜎 𝑧𝑧
𝐵 𝑠ℎ𝑖𝑒𝑙𝑑 𝜎 𝑦𝑦 𝜎 𝑥𝑥 𝜎 ↑, 𝐵 𝑒𝑓𝑓
↓, 𝜈 ↓
Rossum, Solid State NMR and proteins (2009)
J. Duer, Solid State NMR spectroscopy (2002)

Chemical Shift Anisotropy
• More shielding -> lower chemical shift.
• Dependent on angular orientation 𝜎 ↑, 𝐵 𝑒𝑓𝑓
↓, 𝜈 ↓
More shielded

Spin ½ System
Chemical Shift Anisotropy
Dipole-Dipole Coupling

Dipole-Dipole Coupling
• Dipolar coupling causes huge line broadening
Nuclear Pair
1 H, 1 H
1 H, 13 C
1 H, 13 C
Internuclear distance [Å]
10
1
2
Dipolar coupling [𝒌𝑯𝒛]
120
30
3.8
J. Duer, Solid State NMR spectroscopy (2002)

Spin ½ System
• (1952) Purcell and Bloch
Equilibrium

𝐵
0
M
Spin ½ System
B
0
B
1
B
1
Equilibrium Equilibrium
J. Duer, Solid State NMR spectroscopy (2002)

• 𝜔 = 𝛾 ∙ 𝐵
M
𝐵
0 𝑤
0
Spin ½ System
Goldstein, Classical Mechanics

𝐵
0 𝜔
0
Spin ½ System
• Resonance 𝜔 = 𝜔
0
• Maximum signal
𝐵
1 𝜔 ℎ𝜈 = ℏ𝜔 ℎ𝜈
0 ℎ𝜈

Introduction to NMR
Physics Origin
Measurement

Measurement
• Conventional – Continuous Wave
• Modern – Pulse Signal
𝐵
0 𝜈 ℎ𝜈 𝜈
Hornak, The Basics of NMR (1997)

Measurement
• Conventional – Continuous Wave
• Modern – Pulse Signal
𝐵
0
Anisotropy of 1 H 𝜈
1 𝐻
− 𝛿 𝜈
1 𝐻 𝜈
1 𝐻
+ 𝛿 𝜈 + 𝛿 𝜈 𝜈 − 𝛿

Measurement
Shi, NMR Introduction course (2003)

Introduction to NMR
Application
Solid State NMR
Research

Application
• Protein 3D structure and function study at atomic resolution
• (1976) R. R. Ernst: Multi dimensional NMR
• (1979) K. Wuthrich: Solve protein structure
Markley, the Scientists – magazine of life science (2005)

Application
• Protein Dynamics/Protein folding intermediates
A B
 k k

1

AB
Frank, et al. Nature (2010)

Application
• Fast structure determination/recognition of macromolecular compound
• Medical Imaging

Application
Solution vs. Solid State NMR
Solution Solid
Dipolar Coupling (10-100kHz) Scalar Coupling (10-100Hz)
Anisotropic interactions
13 C detection
Sensitivity low
Isotropic interactions
1 H detection
Sensitivity high
Require special techniques to improve linewidth
Natural tumbling of molecules

Application
General Techniques
Solid State NMR
OS-NMR

Problems with SSNMR
• Powder Spectra
13 C NMR of glycine
Solid Liquid
D. Lide, G. W. A. Milne, Handbook of Data on Organic Compounds:

Problems with SSNMR
• Goal: simplify solid state spectra
Adapted from R. Tycko, Annu. Rev. Phys. Chem. 52, 575 (2001).

Solid State NMR
Problems
General Techniques
MAS-NMR
OS-NMR

Separated Local Field
• Developed in 1976
• Suppresses 1 H1 H and 1 H-S coupling
• Resolves dilute spins based on chemical environment
• Gives dipolar coupling information
R. K. Hester, J. L. Ackerman, B. L. Neff, J. S. Waugh, Physical Review Letters 36, 1081 (1976).

Cross Polarization
• Hartmann-Hahn Condition
– Detailed in 1962
– Between heteroatoms
– Same Larmor frequency
– Allows for cross relaxation
L. W. Jelinski, M. T. Melchior, Applied Spectroscopy Reviews 35, 25 (2004/05/24, 2004).

Cross Relaxation
• First published in 1973
• Transfer population information from I to S
• Detect off of dilute species
– Cleaner spectra
– More sensitive
Barth-Jan van Rossum: Solid-state NMR and proteins, a pictorial introduction

Solid State NMR
Problems
General Techniques
MAS-NMR
OS-NMR

Magic Angle Spinning
Simulating the “tumbling” of molecules http://www.rs2d.com/english/images/protasis/doty/doty.jpg

Magic Angle Spinning
• Proposed in 1958
• Coupling dependent on
– At magic angle, 54.7356°, equals zero
• Spin sample to decouple
– 1 H1 H coupling ~40kHz

Static
MAS decoupling
3.6kHz
E. R. Andrew, Philosophical Transactions of the Royal Society of London.
Series A, Mathematical and Physical Sciences 299, 505 (March 18, 1981,
1981).

Solid State NMR
Problems
General Techniques
MAS-NMR
OS-NMR

Physical Orientation Lipids
Replacing “tumbling” with Rf irradiation
G. Orädd, G. Lindblom, Magnetic Resonance in Chemistry 42, 123 (2004).
C. R. Sanders, K. Oxenoid, Biochimica et Biophysica Acta (BBA) -
Biomembranes 1508, 129 (2000). http://avantilipids.com

PISEMA
• Polarization Inversion Spin Exchange at the
Magic Angle
– Developed in 1994
• Form of SLF with enhanced sensitivity
• Further suppression of 1 H1 H coupling
C. H. Wu, A. Ramamoorthy, S. J. Opella, Journal of Magnetic Resonance, Series A 109, 270 (1994).

PISEMA vs SLF
SLF
Modified SLF
PISEMA
C. H. Wu, A. Ramamoorthy, S. J. Opella, Journal of Magnetic Resonance, Series A 109, 270 (1994).

D. S. Thiriot, A. A. Nevzorov, S. J. Opella, Protein Sci 14, 1064 (Apr, 2005).

PISA Wheel

Limitations of PISEMA
A. A. Nevzorov, S. J. Opella, Journal of Magnetic Resonance 185, 59 (2007).

SAMMY
• Compliments PISEMA
– Developed in 2003
• Averages out homonuclear spin-spin interaction
• More uniform over wide range linewidths
A. A. Nevzorov, S. J. Opella, Journal of Magnetic Resonance 164, 182 (2003).

Limitations of SAMMY
A. A. Nevzorov, S. J. Opella, Journal of Magnetic Resonance 185, 59 (2007).

SAMPI4
• Slight modification of SAMMY
– Developed in 2007
• Combines pros of PISEMA and SAMMY
– Sensitivity of PISEMA
– Range of SAMMY
• Can be implemented generally
A. A. Nevzorov, S. J. Opella, Journal of Magnetic Resonance 185, 59 (2007).

Application
Spectroscopic Assignment
Solid State NMR

• What is mosaic spread?
Reducing the effects of mosaic spread

Static Slow diffusion
Uniaxial Diffusion
Fast diffusion
A. A. Nevzorov, The Journal of Physical Chemistry B 115, 15406 (2011/12/29, 2011).

Research
Sensitivity Enhancement
Spectroscopic Assignment
Structure Calculations

Assigning peaks in uniformly labeled proteins
D. S. Thiriot, A. A. Nevzorov, S. J. Opella, Protein Sci 14, 1064 (Apr, 2005).

• Can identify coupling up to
6.7Å away
• Previous methods only identify coupling < 5Å

Research
Sensitivity Enhancement
Spectroscopic Assignment
Structure Calculations

Determining structure from “shiftless” data
Y. Yin, A. A. Nevzorov, Journal of Magnetic Resonance 212, 64 (2011).

Acknowledgement:
• Dr. Sharon Campbell
• Dr. Barry Lentz
• Dr. Alexander Nevzorov

Discussion Questions
• Why SSNMR is important?
• What do you think the next development for solid state NMR is?
• Can you briefly compare the two major structure determination techniques: NMR and
X-ray crystallography?

MAOSS
C. Glaubitz, A. Watts, Journal of Magnetic Resonance 130, 305 (1998).

Discussion
Compare methods of solving Protein Structure
NMR X-ray Crystallography
No crystal needed
Can be used in solution
Not good for large proteins, smaller molecules are comparable to X-ray
Can measure dynamics
In vivo possible (imaging)
Crystal
Solid only
Generally higher solution
Stationary
In vitro

PISA Wheel
D. S. Thiriot, A. A. Nevzorov, S. J. Opella, Protein Sci 14, 1064 (Apr, 2005).