Nuclear Magnetic Resonance in Structural Biology Part I the physical principle the physical principle the spectrometer the spectrometer the NMR spectrum

Nuclear Magnetic Resonance in Structural BiologyNuclear Magnetic Resonance in Structural Biology

Part IPart I

• • the physical principlethe physical principle

•• the spectrometerthe spectrometer

•• the NMR spectrumthe NMR spectrum

•• the applications (what you can do)the applications (what you can do)

•• the samplethe sample

•• the limits (what you cannot do)the limits (what you cannot do)

• • protein structure calculationprotein structure calculation

• • referencesreferences

Nuclear Magnetic Resonance in Structural BiologyNuclear Magnetic Resonance in Structural Biology

Part II Part II

Journal ClubJournal Club

A zinc clasp structure tethers lck to T cell coreceptors CD4 and CD8.

P.W Kim, Z.J. Sun, S. C. Blacklow, G. Wagner, M. J. Eck

Science 301(19 Sept): 1725-1728 (2003)

(the PDF file can be downloaded from www.sciencemag.org)

The physical principleThe physical principle

B (magnetic field)

Energy

nuclear spin, I=½

+½

-½

= B/2

At B=11.7 T (tesla, 104 Gauss) the resonant frequency for 1H is 500 MHz

(earth magnetic field = 30-60 T; magnetic stirrer = 0.1 T)

In NMR, excitation is achieved through short (s) electromagnetic (radio-frequency) pulses.

The E is very small (10-2 cal) low population difference low sensitivity

The spectrometerThe spectrometer

The NMR spectrumThe NMR spectrum

In biomolecules (proteins, nucleic acids, peptides, oligosaccharides, ...) the information available is contained in the NMR-active nuclei and limited by their

natural abundance and sensitivity:

1H (99.98%)

12C (98.93%) not active

14N (99.63%) not detectable

16O (99.76%) not active

32S (94.93%) not active

31P (100%) limited use

• resonant frequency (chemical shift, ppm)

• signal intensity

• J-couplings (spin-spin interactions through covalent bonds)

• lineshape

The chemical shiftThe chemical shift

The chemical shift depends on:

• the atom type (NH, aliphatic CH, aromatic CH, ...)

• the amino acid type (Ala, Phe, ...)

• the chemical (spatial) environment

The resonant frequency of a certain atom is called chemical shift.

For convenience, the chemical shift is expressed as follows:

(sample) – (reference)

spectrometer frequency(ppm) = 106 •

Advantages:

• more compact annotations

• independent on the spectrometer field

In practice, the 1H chemical shifts are in the range 0-10 ppm

The 1D spectrum of a peptideThe 1D spectrum of a peptide

N

H

C

H

C N

O HCH2 8 7 6 5 4 3 2 ppm

The assignment problemThe assignment problem

Which resonance corresponds to which atom?

N

H

C

H

C N

O H

CH3

CH

H3Cthrough-bonds magnetization transfer (J-couplings)

through-space magnetization transfer (NOE)

The 2D spectrumThe 2D spectrum

The information contained in 1D spectra can be expanded in a second (frequency) dimension 2D NMR

In a 1D experiment a resonance (line) is identified by a single frequency:

NH(f1nh)

In 2D spectra, a resonance (cross-peak) is identified by two different frequencies:

NH (f1nh, f2ha)

NH (f1nh, f2ha)

Usually, the second frequency depends on how the NMR experiment is designed.

f1

f2

The 2D spectrum of a peptide (DQF-COSY)The 2D spectrum of a peptide (DQF-COSY)

Ac-GRGGFGGRG-NHAc-GRGGFGGRG-NH22

N

H

C

H

C N

O HCH2

Ph

The 2D spectrum of a peptide (TOCSY)The 2D spectrum of a peptide (TOCSY)

Ac-GAc-GRRGGFGGGGFGGRRG-NHG-NH22

N

H

C

H

C N

O H

CH2

CH2

CH2

~

The 2D spectrum of a peptide (ROESY) The 2D spectrum of a peptide (ROESY)

Ac-GRGGFGGRG-NHAc-GRGGFGGRG-NH22

+

N

H

C

H

C N

O

H

CH2

~

+

++

+ + + ++

The NMR experiment as a black box ?The NMR experiment as a black box ?

sample NMR spectrum?

Playing around with r.f. pulses

(and magnetization):

• power

• length (s)

• phase (x, -x, y, -y)

• receiver phase (x, -x, y, -y)

• time delays

p1 p2 p3 detection (s)

t1t2

exp-1 exp-2 exp-3 ....

The 2D spectrum of a protein (NOESY)The 2D spectrum of a protein (NOESY)

Practical applications of 2D homonuclear NMR are limited

by peak overlap.

From 1D to 2D and 3D NMRFrom 1D to 2D and 3D NMR

nuclear magnetic resonance in structural biology, part Inuclear magnetic resonance 1D

in structural biology, part I

nuclear magnetic resonance 2D

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The NMR spectrumThe NMR spectrum

In biomolecules (proteins, nucleic acids, peptides, oligosaccharides, ...) the information available is contained in the NMR-active nuclei and limited by their

natural abundance. We can “add” information to the system replacing inactive/undetectable nuclei with active ones. This is called “labeling”.

In fact, it should be called “isotopic enrichment” or “isotope abundance reversal”. These are naturally occurring isotopes!

1H (99.98%)

12C (98.93%) not active 13C

14N (99.63%) not detectable 15N

The additional active nucleus can be used to:

• “label” 1H atoms with the frequency of the attached heteroatom (1H- 15N; 1H- 13C)

• transfer magnetization through covalents bonds using heteronuclear J-couplings

A 2D A 2D 11H-H-1515N heteronuclear NMR spectrum (HSQC)N heteronuclear NMR spectrum (HSQC)

15N

1H

C

H

1H (ppm)

15N (ppm)

H (f1,f2)

A 3D NMR spectrumA 3D NMR spectrum

A 2D plane of a 3D NMR spectrum (NOESY)A 2D plane of a 3D NMR spectrum (NOESY)

Applications Applications

• small flexible molecules that cannot be crystallized (peptides, oligosaccharides, ...)

• 3D structure determination of proteins, nucleic acids, protein/DNA complexes, ...)

• dynamics (ps to s)

• electrostatics (pKa values)

• hydrogen bonding (NH temperature coefficients, H2O/D2O exchange)

• unfolded/partially folded states of proteins

• bound solvent

• protein/ligand interactions (also very weak)

• diffusion coefficients

• analysis of biomolecules in vivo

• membrane peptides and proteins (solid-state NMR)

Protein NMR: a practical approachProtein NMR: a practical approach

Sample preparationSample preparation

• 500 l, ~1 mM protein solution (10 kDa 10 mg/ml solution 5 mg 0.5 mol)

• highly efficient (> 10 mg/l), inducible expression system in

M9 medium for isotopic enrichment (15NH4Cl, 13C6-glucose

are expensive; rich labelled media are available)

• the protein must be:solublemonodispersedstable 20-40° C, pH 3-7 over 2-3 weeks

• compatible buffers:inorganic buffers (phosphate)low ionic strength (1-100 mM)

Time scheduleTime schedule

data acquisition: 1-3 weeks

backbone assignments: 1-4 weeks

side-chain assignments: 1-4 weeks

list of restraint/structure calculation: 1-3 months

SpectrometersSpectrometers

500 MHz 800 MHz depending on :

the size (number of residues, molecular weight)

isotopic enrichment (1H, 15N, 15N/13C, 15N/13C/2H)

LimitsLimits

Molecular weight limits for protein structure calculation (monomer):

5-15 kDa: routine

15-20 kDa: usually feasible

20-30 kDa: long term project

40-50 kDa: in the next future?

Molecular weight limits for peptide/protein, protein/protein interactions (MW of the AB complex, A < 10 kDa):

20-30 kDa: routine

30-50 kDa: feasible

50-100 kDa: in the next future

Protein structure calculation by NMRProtein structure calculation by NMR

cloning

expression (labeling)

purification

data acquisition

sequential assignments

side-chain assignments

NOE assignments

list of geometrical restraints

structure calculation

structure refinement

validation

structure/function relationships (electrostatic potentials, surface analysis, ligand binding sites, ...


Structure calculation is the determination of atoms position (x,y,z) in a coordinate system

Atom positions in the liquid state are averaged over space and time

Structure calculation in liquids must rely on internal coordinates; in other words, you must measure something that does not depend on

atoms position

HB HC

HA

Geometrical restraints that allow structure calculations:

• distances (NOE)

• angles (J-couplings)


HB HC

HA

HB HC

HA


HBHC

HA

Books:Books:

• NMR of proteins and nucleic acidsKurt Wühtrich, Wiley (1986)

• NMR of macromolecules: a practical approachedited by G. C. K. Roberts, IRL Press (1993)

• Protein NMR spectroscopy: principles and practiceJ. Cavanagh, W. Fairbrother, A. Palmer, N. SkeltonAcademic Press (1996)

NMR on the web:NMR on the web:

http://www-keeler.ch.cam.ac.uk/lectures/

Useful links:Useful links:

http://www.spincore.com/nmrinfo/

http://www.spectroscopynow.com/

http://www.bmrb.wisc.edu/

Nuclear Magnetic Resonance in Structural BiologyNuclear Magnetic Resonance in Structural BiologyPart IPart I

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Nuclear Magnetic Resonance in Structural Biology Part I the physical principle the physical principle the spectrometer the spectrometer the NMR spectrum