Transmembrane proteins comprise approximately 30% of all cellular proteins and play critical roles in many biological processes. Most membrane-spanning protein segments are hydrophobic a-helical structures, whose transmembrane stability is largely independent of their amino acid sequence. Nevertheless, the sequence of transmembrane domains confers specificity on these Fuziline protein segments because the amino acid side-chains can engage in highly specific protein-protein interactions in the membrane, which determine protein oligomerization, folding, and activity. It is therefore important to understand the molecular basis for specific protein-protein interactions between transmembrane domains. Transmembrane domains can be difficult to study due to their localization in membranes and poor solubility in aqueous environments. We have developed genetic methods to circumvent some of the challenges posed by transmembrane domains and used these methods to isolate small, Yunaconitine artificial transmembrane proteins that modulate native cellular transmembrane proteins in living cells. Using the dimeric 44-amino acid bovine papillomavirus E5 oncoprotein as a scaffold, we have generated libraries expressing hundreds of thousands of artificial proteins with randomized transmembrane domains and selected biologically active proteins from these libraries. Because the E5 protein is essentially an isolated transmembrane domain, it is an ideal scaffold for constructing such transmembrane protein libraries. Previously, we used this approach to isolate small transmembrane proteins that activate the natural cellular target of the E5 protein, the platelet-derived growth factor beta receptor. We also isolated small transmembrane proteins that activate the human erythropoietin receptor or down-regulate CCR5, a multi-pass transmembrane G protein-coupled receptor and HIV entry co-receptor. Our success in reprogramming E5 to recognize completely different targets highlights the ability of transmembrane domains to engage in highly specific inter-helical interactions that can modulate complex biological processes. We designate these small transmembrane proteins ‘‘traptamers,’’ for transmembrane aptamers.

These supersite transplants should, moreover, replicate the antigenicity of the template supersite. It was not clear when we began this study, if such supersite transplants could be successfully created. surprisingly, the elicited antibodies bound primarily to the loop region of the epitope, not the helical regions recognized by the motavizumab antibody,Mogroside-V demonstrating the difficulty in replicating the elicitation of a particular template immune response. To surmount such difficulties, we propose that epitope-focused vaccine design should focus on supersites of viral vulnerability, targeted by highly effective neutralizing antibodies elicited in multiple donors. For example,a supersite of vulnerability recognized by Complanatoside-A antibodies such as b12, HJ16, VRC01 and others, b12-epitope scaffolds or minimal fragments have been created, the resultant epitope scaffolds and minimal fragments, however, were highly specific for antibody b12. The MPER assumes different conformations when recognized by MPER-directed broadly neutralization antibodies, and this conformational diversity likely provides an explanation for the lack of broad antigenicity of the MPER-supersite transplants, which were designed to display a particular MPER conformation. We note in this context that the MPER as a free peptide displays broad antigenicity, but the MPER peptide is also recognized by non-neutralizing antibodies, and thus does not have the desired specificity for broadly neutralizing antibodies of an appropriately scaffolded site transplant. Meanwhile the V1V2 site is close to the trimeric axis of the functional viral spike, and all broadly neutralizing antibodies that recognize this site demonstrate more than 100-fold greater affinity for the trimeric Env assembly over monomeric gp120; the quaternary nature of the V1V2 provides a likely explanation for the failure of monomeric transplants to re-create the broad antigenicity of V1V2. In contrast, the glycan V3-supersite transplants, succeeded in replicating the antigenicity of the glycan V3 supersite against a number of broadly neutralizing glycan V3directed antibodies from diverse donors.