Transcript Slide 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
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About the protein:
 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) NEFδ2-39 , (ii ) NEFδ2-39, δ159-153 and
(iii) NEFδ2-39, δ159-153 in complex with the SH3 domain of tyrosine protein
Kinase were studied.
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Flow Chart for 3D structure determination using NMR
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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.
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Hetero-nuclear multi-dimensional NMR experiments for
resonance assignment
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Some Triple Resonance experiments
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Frequencies Observed with correlation
Experiment
Assignment
Comment
HNCO
H(i), N(i), C’(i-1)
<20 kD, above use H labeling
HNCA
H(i), N(i), C(i),C(i-1)
<20 kD, above use H labeling
HN(CO)CA
H(i), N(i), C(i-1)
<20 kD, above use H labeling
HN(CA)CO
H(i), N(i), C’(i)
<20 kD, above use H labeling
CBCA(CO)NH
H(i), N(i), C(i-1), C(i-1)
<20 kD, above use H labeling
HBHA(CO)NH
H(i), N(i), H(i-1), H(i-1)
<20 kD, above use H labeling
CBCANH,
HNCACB
H(i), N(i), C(i), C(i),
C(i-1),C(i-1)
<15 kD, above use H labeling
(H)CC(CO)NHTOCSY
H(i), N(i), Caliph.(i-1)
<15-20 kD, above use H labeling
H(CC)(CO)NHTOCSY
HCCH-TOCSY
H(i), N(i), Haliph.(i-1)
<15-20 kD, above use H labeling
Haliph., Caliph.
<25 kD, - sensitive, but tedious to analyze,
combine with HCCONH type experiments
Relative
S/N [%]
2
100
2
50/15
2
71
2
13/4
2
13/9 
2
13/9 
2
4/1.7 (i)
1.3/0.5
i-1)
2
2
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Assignment Strategy for HIV-1 NEF:
 Identify a cross peak in HNCO spectra.
 For corresponding 1H and 15N 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.
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Assignment strategy : An example
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Structural information:
 Not much from chemical shift so….
 Inter-proton distances from NOESY.
 J-coupling experiments.
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NOE informations
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Assigned HSQC shows random coil region
Random coil
region corresponding to
Residue 159-173
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Structural information from T2 relaxation and NOE relaxation data
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Final Structure and Statistics
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