A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

Debnath, Asim K.

doi:10.1007/pl00010718

Cited by 2 publications

(1 citation statement)

References 32 publications

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“…Unconjugated peptides from another region of gp41 (e.g., fusion peptide) and from gp120 (e.g., V3 loop peptide 27 ) were included as controls. As shown in the Figure 7, C34 inhibited C34-FITC interacting with N36 in a dose-dependant manner with an IC 50 about 0.36 µM.…”

Section: Specificity and Sensitivity Of Flisa-based Screening Assaymentioning

confidence: 99%

Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41

Liu

Boyer-Chatenet²,

Liang³

et al. 2003

SLAS Discovery

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The human immuno deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays animportant role in the virus entry. During the process of fusion between the viral and target cell membranes, the N-and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.

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Section: Specificity and Sensitivity Of Flisa-based Screening Assaymentioning

confidence: 99%

Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41

Liu

Boyer-Chatenet²,

Liang³

et al. 2003

SLAS Discovery

View full text Add to dashboard Cite

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Modeling of loops in protein structures

2000

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Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM-22 force field, statistical preferences for the main-chain and side-chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4-, 8-, and 12-residue loop predictions, respectively, had Ͻ2 Å RMSD error for the mainchain N, C a , C, and O atoms; the average accuracies were 0.59 6 0.05, 1.16 6 0.10, and 2.61 6 0.16 Å, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main-chain stem atoms is 2.5 Å, the average loop prediction error increased by 180, 25, and 3% for 4-, 8-, and 12-residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by~1! comparing it with one of the most successful previously described methods, and~2! describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.

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A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

Cited by 2 publications

References 32 publications

Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41

Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41

Modeling of loops in protein structures

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