We prepared a designed protein containing the gp41 NHR and CHR segments www.selleckchem.com/products/Enzastaurin.html which mimics the six helix bundle post fusion conformation. This protein consists of the NHR linked to the CHR by a short linker, followed by a trimeric coiled coil segment from T4 fibritin to promote trimerization. 6 Helix Fd was purified from E. coli by standard procedures and found to be helical by circular dichro ism consistent with design. To explore conformational specificity of the antibody clones, we performed competitive ELISA assays in which binding to immobilized 5 Helix was inhibited by binding free 5 Helix or free 6 Helix Fd. The IC50 obtained by competition with free 5 Helix provides an estimate for binding activity.

Furthermore, the relative IC50 obtained by competition with 6 Helix Fd enables evaluation of preference for the extended intermediate conformation over the post fusion conformation. These results are sum marized in Table 4. We previously reported an IC50 of D5 for 5 Helix of 0. 1 nM, and here we determined an IC50 for 6 Helix Fd of 11 nM. Therefore, the D5 is able to discriminate the extended and post fusion confor mations of gp41 by 100 fold difference in apparent af finity. Selectants from D5 Lib II ranged in their apparent affinity for 5 Helix, some were similar to D5 but others had 10 or 100 fold higher IC50. However, most retained their ability to distin guish 6 Helix Fd from 5 Helix by 100 fold difference in apparent affinity. In one case, 25D8, specificity for 5 Helix over 6 Helix Fd was enhanced relative to D5.

We have previously shown that analysis of binding to 5 Helix in this format, with the antibody fragment displayed on phage, agrees well with results using the purified antibody fragment. To further validate this assumption, we purified the scFv for D5 and several of the clones for binding analysis. In general, the IC50 obtained for the purified scFv proteins were 10 fold higher than those observed on phage. However, the overall trends were consistent with results on phage for the clones examined. Positional preferences Diverse populations of phage selectants can be used to assess positional requirements for protein protein inter actions by determining the degree of conservation for a particular residue in a functional selection relative to a selection for protein display.

In some cases, these datasets have been used to infer energetic consequences of mutation provided certain assumptions are validated. We performed a se lection of D5 Lib II against the anti FLAG antibody M2 to obtain a reference dataset to quantify display biases. A FLAG epitope sequence was included at the N terminus of our scFv construct, therefore selection against M2 should provide readout of display GSK-3 bias. We compiled se quences for 179 clones from the 5 Helix selection that scored well in terms of specificity profile analysis.