To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.

Peptide “mimics” (mimotopes) of linear protein epitopes and carbohydrate epitopes have been successfully used as immunogens to elicit cross-reactive antibodies against their cognate epitopes; however, immunogenic mimicry has been difficult to achieve for discontinuous protein epitopes. To explore this, we developed from phage-displayed peptide libraries optimized peptide mimics for three well-characterized discontinuous epitopes on hen egg lysozyme and horse cytochrome C. The peptides competed with their cognate antigens for antibody binding, displayed affinities in the nM range, and shared critical binding residues with their native epitopes. Yet, while immunogenic, none of the peptides elicited antibodies that cross-reacted with their cognate antigens. We analyzed the 3-D structure of the site within each discontinuous epitope that shared critical binding residues with its peptide mimic, and observed that in each case it formed a ridge-like patch on the epitope; in no case did it cover most or all of the epitope. Thus, the peptides’ lack of immunogenic mimicry could be attributed to their inability to recapitulate the topological features of their cognate epitopes. Our results suggest that direct peptide immunizations are not a practical strategy for generating targeted antibody responses against discontinuous epitopes.

: The combination of IM silicon tetrachloride and 3M phenol serves as an efficient N a -t-butoxycarbonyl deblocking agent in solid phase peptide synthesis, the duration of the cleavage being 10 rain. This is demonstrated by the synthesis of the naturally occurring g-receptor selective opioid heptapeptide, dermorphin. Copyright © 1996 Elsevier Science Ltd t-Butoxycarbonyl (Boc) group is extensively used for temporary protection of the amino group in solid phase peptide synthesis (SPPS). Its subsequent removal is effected by acidolysis. A major concern of peptide chemists is the repetitive acidolysis that becomes mandatory in this approach and the consequent side reactions that result. In recent years, instead of acidolysis, some organo-silicon reagents have been tried. Lott et al. t have observed that iodotrimethylsilane can bring about the cleavage of Boc group from several protected amino acids and peptides. The cleavage was followed by NMR. However, its application for the synthesis of any specific peptide was not reported by them. We have earlier described the utility of iodotrichlorosilane, prepared from SiCI4 and NaI, for Boc deprotection during SPPS of oxytocin 2. Merrifield and co-workers 3'4 observed that 1M chlorotrimethylsilane and 3M phenol can be employed for Boc group deprotection in SPPS. This communication outlines the use of the combination of 1M SiCI4 and 3M phenol in CH2C12 for efficient Boc group deprotection in SPPS. The reagent is inexpensive and is easily prepared by mixing freshly distilled SiCI4 and phenol in CHIC12. The cleavage of Boc group from Boc-Phe-OCH2-C6H4-resin was effected by combinations of 1M SiCI4 with 1M phenol, 2M phenol and 3M phenol and the duration of cleavage in these cases was found to be 45 rain, 30 min and 10 min respectively as estimated by the picrate method s. Subsequently further deprotection experiments were carried out employing 20 mmoles of SiCh and 60 mmoles of phenol in CH2C12 (20 ml) per gram of protected aminoacyl resin with an esterification level of about 0.5 mmole/g.The stability of different amino and carboxyl protecting groups to 1M SiCI4-3M phenol has been studied by the addition of the reagent to the amino acid derivative and stirring at room temperature. The reaction was monitored by thin layer chromatography. It was observed that Boc group was cleaved completely in 10 min, while the N~-9-fluorenylmethoxycarbonyl, l~-benzyloxycarbonyl, benzyl ester and benzyl ether groups were not cleaved to any noticeable extent even after 18 hr. The reagent does not also affect the side chain protecting groups such as nitro group of nitroarginine, ethyl and methyl carboxylic esters after continuous treatment for 18 hr. 5989

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