1. 1 Refined Solution Structure of HIV-1 Nef Stephen Grzesiek, Ad Bax, Jin-Shan Hu, Joshua Kaufman, Ira Palmer, Stephen J Stahl, Nico Tjandra and Paul T. Wingfield. Protein Science (1997), 6:1248-1263

2. 2  Length : 206 amino acid  Importance: expressed at high level in the early stages of HIV infection and key protein in viral replication.  Residues 2-39 ( N-terminus ) are unstructured so these residues were deleted.  Three deletion mutant (i) NEF δ2-39, (ii ) NEF δ2-39, δ159-153 and (iii) NEF δ2-39, δ159-153 in complex with the SH3 domain of tyrosine protein Kinase were studied. About the protein:

3. 3 Flow Chart for 3D structure determination using NMR

4. 4 Assignment methods that use heteronuclear shift correlation  For larger proteins (>10-15 kD), assignment methods based on 2D homonuclear 1H-1H correlation methods (COSY/TOCSY/NOESY) don’t work very well because of overlapping resonances and broad linewidths.  An alternative (which is now used even for small proteins in most cases) is to use heteronuclear shift correlation experiments on 13C, 15N labelled samples.  In these experiments, magnetization is transferred from 1H to 13C and/or 15N through large one-bond scalar couplings.

5. 5 Hetero-nuclear multi-dimensional NMR experiments for resonance assignment

6. 6 Some Triple Resonance experiments

7. 7 Frequencies Observed with correlation

8. 8 Assignment Strategy for HIV-1 NEF:  Identify a cross peak in HNCO spectra.  For corresponding 1 H and 15 N identify CA and CB resonance in HNCA and HNCACB. ( intraresidue connections)  HA and HB can be identified from NOESY and HOHAHA.  Using CBCA(CO)NH and HBHA(CO)NH inter-residue connections can be established.  Repeating the procedure with all HNCO peaks can lead to long fragments of protein sequence.  This fragments can be assigned to probable amino acid type using characteristic CA and CB frequencies.

9. 9

10. 10 Assignment strategy : An example

11. 11 Structural information:  Not much from chemical shift so….  Inter-proton distances from NOESY.  J-coupling experiments.

12. 12 NOE informations

13. 13 Assigned HSQC shows random coil region Random coil region corresponding to Residue 159-173

14. 14 Structural information from T2 relaxation and NOE relaxation data

15. 15 Final Structure and Statistics

16. 16