Solution Structure Determination by NMR and Distance Geometry Calculations

1. Pure Protein (0.3 mL, 0.5 - 1 mM; ~ 10 mg)2. Sequence-specific resonance assignments3. Sequence-specifice distance constraints

disulfide bondsspin-spin coupling constantsinternuclear distance constraints from NOEsresidual dipolar couplingshydrogen bonds standard bond lengths; bond angles

4. Distance Geometry Calculations5. Constrained Energy Minimization; Refinement

Basic Principles

Nuclear Spin and Magnetization

Magnetization and Coherence

Fourier Transform NMRErnst & AndersonRev. Scientific Instrumentation 37: 93 (1966)

Nuclear Relaxation

Spin-Spin Coupling

Chemical Shift with Spin-Spin Coupling

1D NMR Spectrum of a Protein

2D Correlated SpectroscopY

AV-COSY

Nuclear Overhauser Effect (NOE)

Def: The change in the intensity of resonance i upon irradiation of resonance j due to “cross relaxation” between spins i and j through space

2D NOESY Spectrum of a Protein

Some Useful 2D NMR Experiments

1H-13C Heteronuclear Correlation Spectrum

Solution Structure Determination by NMR and Distance Geometry Calculations

1. Pure Protein (0.3 mL, 0.5 - 1 mM; ~ 10 mg)2. Sequence-specific resonance assignments3. Sequence-specifice distance constraints

disulfide bondsspin-spin coupling constantsinternuclear distance constraints from NOEsresidual dipolar couplingshydrogen bonds standard bond lengths; bond angles

4. Distance Geometry Calculations5. Constrained Energy Minimization; Refinement

WR41

Protein NMR Structures

C-TmZip

ER14

MMP-1

IL13

FGF-2

WR90

WR64

LC8

ER115

N-TmZip

JR19

ZR18

OP3

WR33

ZR31 ER75Z-domain

IR24

ER75: Ecol YgdK (nmr)ER30: Ecol SufE (xray)IR24: Hinf IscU (nmr)SR17 Bsub IscU (nmr)

N N

C C

YgdKG. Liu et al. (Szyperski)

Gram neg IscU

IscU, SufE,YgdK Domain Families are Structurally

Similar

YgdK cf SufEA. Kuzin et al. (Hunt)

Ramelot et al, J. Mol. Biol. 2004Goldsmith et al., J. Mol. Biol. 2004Liu et al. submitted

T. Ramelot et al. (Kennedy)

Gram pos IscU

G. Kornhaber et al. (Montelione)