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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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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.

About the protein:

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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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Experiment Assignment Comment RelativeS/N [%]

HNCO H(i), N(i), C’(i-1) <20 kD, above use 2H labeling 100

HNCA H(i), N(i), C(i),C(i-1) <20 kD, above use 2H labeling 50/15

HN(CO)CA H(i), N(i), C(i-1) <20 kD, above use 2H labeling 71

HN(CA)CO H(i), N(i), C’(i) <20 kD, above use 2H labeling 13/4

CBCA(CO)NH H(i), N(i), C(i-1), C(i-1) <20 kD, above use 2H labeling 13/9

HBHA(CO)NH H(i), N(i), H(i-1), H(i-1) <20 kD, above use 2H labeling 13/9

CBCANH,HNCACB

H(i), N(i), C(i), C(i),

C(i-1),C(i-1)<15 kD, above use 2H labeling 4/1.7 (i)

1.3/0.5i-1)

(H)CC(CO)NH-TOCSY

H(i), N(i), Caliph.(i-1) <15-20 kD, above use 2H labeling

H(CC)(CO)NH-TOCSY

H(i), N(i), Haliph.(i-1) <15-20 kD, above use 2H labeling

HCCH-TOCSY Haliph., Caliph. <25 kD, - sensitive, but tedious to analyze,combine with HCCONH type experiments

Frequencies Observed with correlation

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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 toResidue 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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