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

2. 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

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

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

5. 1H Chemical Shifts for murine epidermal growth factor at 28oC and PH 3

6. 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

7. 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

8. Stereospecific Cβ H2 assignment for mEGF

9. Stereospecific assignment of side-chain amide protons

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

11. Experimental conformational constraints1 2 3 4 5

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

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

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