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Protein Structures by NMR

Thomas VosegaardLaboratory for Biomolecular NMR Spectroscopy,

Center for Insoluble Protein Structures (inSPIN) andInterdisciplinary Nanoscience Center (iNANO)

Thomas Vosegaard

Protein Structure Determination by NMR• Proteins

•Primary, secondary, tertiary structure• 1H NMR

•COSY•TOCSY•NOESY

• Assignment•Spin systems•NOEs

• Structure calculation•Restrained MD

• Membrane proteins and fibrils•Solid-state NMR

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Thomas Vosegaard

Protein Structure Determination by NMR• Proteins

•Primary, secondary, tertiary structure• 1H NMR

•COSY•TOCSY•NOESY

• Assignment•Spin systems•NOEs

• Structure calculation•Restrained MD

• Membrane proteins and fibrils•Solid-state NMR

Thomas Vosegaard

Proteins

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Thomas Vosegaard

Protein Primary StructureProteins are made of amino acids

Alanine Ala ACysteine Cys CAspartic Acid Asp DGlutamic Acid Glu EPhenylalanine Phe FGlycine Gly GHistidine His HIsoleucine Ile ILysine Lys KLeucine Leu LMethionine Met MAsparagine Asn NProline Pro PGlutamine Gln QArginine Arg RSerine Ser SThreonine Thr TValine Val VTryptophan Trp WTyrosine Tyr Y

H-Ala – Ser – Glu – Phe-OH

A peptide/protein

H2NHN

O

CH3

NH

HN

O

OOH

O

OHO

OH

N-terminus C-terminus

Thomas Vosegaard

Secondary Structure

α-Helix β-Sheet

Stable structures with extensive H-bonding

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Thomas Vosegaard

Tertiary Structure

3D arrangementof secondary

structure elements

Thomas Vosegaard

Protein Structure Determination by NMR• Proteins

•Primary, secondary, tertiary structure• 1H NMR

•COSY•TOCSY•NOESY

• Assignment•Spin systems•NOEs

• Structure calculation•Restrained MD

• Membrane proteins and fibrils•Solid-state NMR

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Thomas Vosegaard

1H NMR of Proteins

1D spectrum is too crowdedto provide information

Thomas Vosegaard

2D COSY Experiment

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Thomas Vosegaard

Coupling Pattern is Not in Phase

[ ]

[ ]))cos(())cos((21)sin()sin(

))sin(())sin((21)cos()sin(

1121112111211

1121112111211

tJtJtJt

tJtJtJt

πππ

πππ

+Ω−−Ω=Ω

+Ω+−Ω=Ω

ω1

ω2

cossin

sincos

Thomas Vosegaard

COSY Spectra

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Thomas Vosegaard

COSY Spectrum of a Protein

Thomas Vosegaard

TOCSY (Total Correlation Spectroscopy) Experiments

Isotropic mixing

sequence

Cross peaks are in-phase – Allows several transfers

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Thomas Vosegaard

Total Correlation

– CH2 – CH2 – CH2 – CH2 –COSY

– CH2 – CH2 – CH2 – CH2 –TOCSY

ValineCOrrelationSpecroscopY

TOtal CorrelationSpectroscopY

2-3 bonds

spin systemdirectly coupled

Thomas Vosegaard

TOCSY Spectrum of a Proteintmix = 48 ms tmix = 83 ms tmix = 102 ms

H2N

NH O

H

Lys (K)

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Thomas Vosegaard

NOESY

COSY Through bond correlations (J)

TOCSY Do

NOESY Through space correlations (Dipole)

Thomas Vosegaard

NOESY Experiment

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Thomas Vosegaard

NOESY Spectraτm = 40 ms τm = 100 ms

Intra-residueInter-residue

Thomas Vosegaard

Protein Structure Determination by NMR• Proteins

•Primary, secondary, tertiary structure• 1H NMR

•COSY•TOCSY•NOESY

• Assignment•Spin systems•NOEs

• Structure calculation•Restrained MD

• Membrane proteins and fibrils•Solid-state NMR

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Thomas Vosegaard

Resonance AssignmentOverall philosophy:1. Acquire spectra2. Assign resonances3. Use distance

restraints from NOEs

Overall philosophy:1. Acquire spectra2. Assign resonances3. Use distance

restraints from NOEs

Start using NH

HNHα

Hβ

Hδ

Hγ

Hβ

Thomas Vosegaard

1H Chemical Shifts

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Thomas Vosegaard

ProblemsCOSY and TOCSY only correlate through 2JHH and 3JHH

NH O

H

Phe (F)

NH O

H

HN

Trp (W)

Hβ is not coupled to the aromatic protonsThe amino acids appear like two spin systems

Thomas Vosegaard

NOESY SpectraLys-Thr-Leu

NHi- NHi+1αHi - NHi+1βHi - NHi+1Are the strongest cross-peaks in NOESY spectra

Also good for intra-residue assignments

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Thomas Vosegaard

Protein Structure Determination by NMR• Proteins

•Primary, secondary, tertiary structure• 1H NMR

•COSY•TOCSY•NOESY

• Assignment•Spin systems•NOEs

• Structure calculation•Restrained MD

• Membrane proteins and fibrils•Solid-state NMR

Thomas Vosegaard

Long-Range NOEs Help Defining the Structure

Nomenclature:NHi - NHi+1: dNN (i,i+1)αHi - NHi+1: dαN (i,i+1)βHi - NHi+1: dαN (i,i+1)

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Thomas Vosegaard

Torsion Angles LikewiseMeasurement of 3JHH

Thomas Vosegaard

Secondary Structure Summary

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Thomas Vosegaard

Distance Constraints

NOE intensity → distance constraint

Classificationstrong < 2.8 Åmedium < 3.5 Åweak < 5.0 Å

Iij ~ 1/ <Dij6> , Dij < 5 Å

δi

δj

NOEs

Thomas Vosegaard

Distance Constraints

1017 distances +175 anglescalbindin D9k

1 HA 1 HB11 5.01 HA 1 HB12 2.81 HA 1 HB21 2.81 HA 1 HB22 3.51 HA 1 HD 3.51 HA 1 HE 5.01 HA 1 HN 3.51 HB11 1 HB12 2.81 HB11 1 HB22 3.51 HB11 1 HD 5.01 HB11 1 HN 3.51 HB11 2 HN 2.81 HB12 1 HB21 5.01 HB12 1 HB22 5.01 HB12 1 HD 3.51 HB21 1 HB22 2.81 HB21 1 HD 3.51 HB21 1 HN 5.01 HB22 1 HD 3.51 HB22 1 HN 5.01 HD 1 HN 5.01 HD 4 HO 5.01 HE 4 HO 5.01 HN 2 HN 5.01 HN 4 HA 5.01 HN 4 HN 3.51 HN 4 HO 3.5

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Thomas Vosegaard

Restrained Molecular DynamicsStimulated Annealing

E = Ebond + Enonbond + ENMR

Eij NMR = k (Dij - Uij )2 , Dij > Uij0 , Dij ≤ Uij

T k

t (ps)

RT

~1000 K

Thomas Vosegaard

3D Structures

33 structuresM. Bak et al. Eur J Biochem 267:188-99 (2000)

PP3calbindin D9k

15 structures

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Thomas Vosegaard

Perspectives

< 10 kD:

10 kD - 30 kD:

homonuclear (1H) techniques

triple-resonance (1H,13C,15N) techniques

Thomas Vosegaard

TROSY – For Larger Proteins11HH 800 800 kDkD

150 150 kDkD50 50 kDkD

M. Salzmann et al. J Biomol NMR. 14:85-8 (1999) K. Wüthrich http://www.nobel.se/chemistry/laureates/2002/illpres/index.html

30 kD - 1000kD:TROSY + selective deuteration