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

Biophysics Student Seminar Series

introducing

Solid State NMRBo Zhao

Zimeng Li

Introduction to NMR

Application

Solid State NMR

Research

Introduction to NMR

Application

Solid State NMR

Research

Physics

• Nuclear Magnetic Resonance

Biology

• Structure• Dynamics

Introduction to NMR

Physical Origin

Measurement

Introduction to NMR

• Rabi (1938)• Spin is internal property of particles• Spin can generate magnetic field• Protons and Neutrons have spin 1/2

Physical Origin

Xu, Modern Physics (1993)

Physical Origin

Spin ½ System

Nuclei Unpaired Protons

Unpaired Neutrons

Net Spin γ (MHz/T) Abundance

1H 1 0 1/2 42.58 99.98%2H 1 1 1 6.54 0.0184%

14N 1 1 1 3.08 99.636%15N 1 0 1/2 -4.316 0.34%12C 0 0 0 N/A 99%13C 0 1 1/2 10.71 1%19F 1 0 1/2 40.08 100%31P 1 0 1/2 17.25 100%

?

• External field – energy splitting

Spin ½ System

Hornak, The Basics of NMR (1997)

1H [ppm]

• Internal property• External factor

– High Field (1964)– Electron shielding– Spin coupling

• Chemical Shift

Spin ½ System

?e

p

Spin coupling

Chemical Shift Anisotropy

Shi, NMR Introduction course (2003)Trausch et al., Chemical Physics Letters (2008)

Spin ½ System

Chemical Shift Anisotropy

Dipole-Dipole coupling

𝜈

• More shielding -> lower chemical shift.

Chemical Shift Anisotropy

𝜎 ↑ ,𝐵𝑒𝑓𝑓 ↓,𝜈↓

𝜎 𝑧𝑧

𝜎 𝑥𝑥

𝜎 𝑦𝑦

𝐵 h𝑠 𝑖𝑒𝑙𝑑

𝐵𝑒𝑓𝑓 =𝐵0−𝐵 h𝑠 𝑖𝑒𝑙𝑑

Rossum, Solid State NMR and proteins (2009)J. Duer, Solid State NMR spectroscopy (2002)

• More shielding -> lower chemical shift.

• Dependent on angular orientation

More shielded

Chemical Shift Anisotropy

𝜎 ↑ ,𝐵𝑒𝑓𝑓 ↓,𝜈↓

Spin ½ System

Chemical Shift Anisotropy

Dipole-Dipole Coupling

Nuclear Pair Internuclear distance

Dipolar coupling

1H,1H 10 1201H,13C 1 301H,13C 2 3.8

• Dipolar coupling causes huge line broadening

Dipole-Dipole Coupling

J. Duer, Solid State NMR spectroscopy (2002)

• (1952) Purcell and Bloch

Spin ½ System

Equilibrium

Spin ½ System

Equilibrium

B0

B1 B1

Equilibrium

𝐵0

M

J. Duer, Solid State NMR spectroscopy (2002)

𝜔=𝛾 ∙𝐵Spin ½ System

Goldstein, Classical Mechanics

𝑤0

𝐵0

M

𝐵0

M

𝐵1

Spin ½ System

M • Resonance • Maximum signal

𝜔0

𝜔 h𝜈=ℏ𝜔

h𝜈0 h𝜈

Physics Origin

Measurement

Introduction to NMR

h𝜈

• Conventional – Continuous Wave• Modern – Pulse Signal

Measurement

𝐵0

𝜈

𝜈

Hornak, The Basics of NMR (1997)

• Conventional – Continuous Wave• Modern – Pulse Signal

Measurement

𝐵0

𝜈

𝜈+𝛿

𝜈−𝛿

𝜈1𝐻−𝛿𝜈1𝐻𝜈1𝐻+𝛿

Anisotropy of 1H

Measurement

Shi, NMR Introduction course (2003)

Introduction to NMR

Application

Solid State NMR

Research

• Protein 3D structure and function study at atomic resolution

• (1976) R. R. Ernst: Multi dimensional NMR• (1979) K. Wuthrich: Solve protein structure

Application

Markley, the Scientists – magazine of life science (2005)

• Protein Dynamics/Protein folding intermediates

Application

A B ABk k

1 1,

Frank, et al. Nature (2010)

• Fast structure determination/recognition of macromolecular compound

• Medical Imaging

Application

Solid Solution

Dipolar Coupling (10-100kHz) Scalar Coupling (10-100Hz)

Anisotropic interactions Isotropic interactions

13C detection 1H detection

Sensitivity low Sensitivity high

Require special techniques to improve linewidth

Natural tumbling of molecules

Solution vs. Solid State NMR

Application

Application

Introduction to NMR

Solid State NMR

Research

Problems

General Techniques

OS-NMR

MAS-NMR

• Powder Spectra13C NMR of glycine

Problems with SSNMR

Adapted from M. Edén, Concepts in Magnetic Resonance 18A, 24.D. Lide, G. W. A. Milne, Handbook of Data on Organic Compounds: Compounds 10001-15600 Cha-Hex. (CRC Press, 1994).

Solid Liquid

Problems with SSNMR

• Goal: simplify solid state spectra

Adapted from R. Tycko, Annu. Rev. Phys. Chem. 52, 575 (2001).

Problems

General Techniques

MAS-NMR

Solid State NMR

OS-NMR

• Developed in 1976• Suppresses 1H-1H and 1H-S coupling• Resolves dilute spins based on chemical

environment• Gives dipolar coupling information

Separated Local Field

R. K. Hester, J. L. Ackerman, B. L. Neff, J. S. Waugh, Physical Review Letters 36, 1081 (1976).

• 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 Polarization

• First published in 1973• Transfer population information from I to S• Detect off of dilute species

– Cleaner spectra– More sensitive

Cross Relaxation

Barth-Jan van Rossum: Solid-state NMR and proteins, a pictorial introduction

Problems

General Techniques

MAS-NMR

Solid State 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

– 1H-1H coupling ~40kHz

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

3.6kHz

Static

MAS decoupling

Problems

General Techniques

MAS-NMR

Solid State NMR

OS-NMR

Physical Orientation Lipids

Oriented Sample NMR

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

Replacing “tumbling” with Rf irradiation

• Polarization Inversion Spin Exchange at the Magic Angle– Developed in 1994

• Form of SLF with enhanced sensitivity• Further suppression of 1H- 1H coupling

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

PISEMA

SLF

PISEMA

Modified SLF

PISEMA vs SLF

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).

Polar Index Slant Angle Wheel

S. Kim, T. A. Cross, Journal of Magnetic Resonance 168, 187 (2004).G. A. Cook, S. J. Opella, Methods Mol Biol 637, 263 (2010).

PISA Wheel

Limitations of PISEMA

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

• 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).

SAMMY

Limitations of SAMMY

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

• 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).

SAMPI4

Application

Introduction to NMR

Solid State NMR

Research

Sensitivity Enhancement

Spectroscopic Assignment

Structure Calculations

• What is mosaic spread?

Reducing the effects of mosaic spread

Sensitivity Enhancement

C. R. Sanders, K. Oxenoid, Biochimica et Biophysica Acta (BBA) - Biomembranes 1508, 129 (2000).

M. J. Duer, Solid-state NMR spectroscopy: principles and applications. (Blackwell Science, 2001).

A. A. Nevzorov, The Journal of Physical Chemistry B 115, 15406 (2011/12/29, 2011).

Sensitivity Enhancement

Static Slow diffusion Fast diffusion

Uniaxial Diffusion

Sensitivity Enhancement

Spectroscopic Assignment

Structure Calculations

Research

Spectroscopic Assignment

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

Assigning peaks in uniformly labeled proteins

Spectroscopic Assignment

A. A. De Angelis, S. C. Howell, A. A. Nevzorov, S. J. Opella, Journal of the American Chemical Society 128, 12256 (2006/09/01, 2006).

R. W. Knox, G. J. Lu, S. J. Opella, A. A. Nevzorov, Journal of the American Chemical Society 132, 8255 (2010/06/23, 2010).

• Can identify coupling up to 6.7Å away

• Previous methods onlyidentify coupling < 5Å

Sensitivity Enhancement

Spectroscopic Assignment

Structure Calculations

Research

Determining structure from “shiftless” data

Y. Yin, A. A. Nevzorov, Journal of Magnetic Resonance 212, 64 (2011).

Structure Calculations

C. H. Wu, S. J. Opella, J Chem Phys 128, 052312 (Feb 7, 2008).Y. Yin, A. A. Nevzorov, Journal of Magnetic Resonance 212, 64 (2011).

Structure Calculations

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

Thank you!

• 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?

Discussion Questions

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

MAOSS

Compare methods of solving Protein Structure

Discussion

NMR X-ray Crystallography

No crystal needed Crystal

Can be used in solution Solid only

Not good for large proteins, smaller molecules are comparable to X-ray Generally higher solution

Can measure dynamics Stationary

In vivo possible (imaging) In vitro

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

PISA Wheel