Title: How to determine the solution structure of murine epidermal growth factor by NMR Spectroscopy Hong Liu.

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Presentation transcript:

Title: How to determine the solution structure of murine epidermal growth factor by NMR Spectroscopy Hong Liu

General protocols 1 COSY, TOCSY, DQFCOSY Spin System identification; 3JNH-αH 3JαH-βH; Dihedral angles. 2 NOESY Sequential resonance assignments; Distance between different close nuclear pairs 3 Structure constraints table 4 Calculation (Submit NMR data to a specific program) 5 Refinement

Spin system identification-COSY αH-βH cross peak 2DF-COSY spectrum

COSY 2DF-COSY spectrum Relayed-COSY spectrum NH-βH cross-peak

1H Chemical Shifts for murine epidermal growth factor at 28oC and PH 3

Sequential resonance assignments-NOESY Containing the intra-residue NH-αH and sequential αH –NH cross-peaks Containing the sequential NH –NH cross-peaks Survey of the sequential connectivities used in the sequence-specific 1H NMR assignment NOESY spectrum

Stereospecific Cβ H2 assignment for mEGF at 28oC and PH3.1 14AA(degenerate) 53 AA 2prolines 41AA(Cβ H2) 10AA(not estimate) 27AA 6AA(3JαH-βH inconsistent with any single staggered rotamer state for χ1)# 25AA 15AA 9AA(consistent) # To assume that the protein side chains adopt one of the three staggered conformations with χ1=+60o,180o,-60o

Stereospecific Cβ H2 assignment for mEGF

Stereospecific assignment of side-chain amide protons

Other assignments Stereospecific assignment of Valine isopropyl Methyl groups Based on NOE: Val-34, Val19

Experimental conformational constraints 1 2 3 4 5

Survey of NOE-derived distance constraints for mEGF in a diagonal plot

Structural calculation and refinement Submit the NMR data to the DISMAN program. Structures refined by restrained energy minimization.

Conclusion 1 Advantage: Test samples in solutions, not need crystals, so convenient. 2 Disadvantage: Molecular-weight can not be big.