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Study of cavitand-based de novo four-helix bundle proteins Mezo, Adam Robert


The protein folding problem is studied by designing, synthesizing and characterizing the structures of de novo four-helix bundles. Each de novo protein (e.g., 27) consists of four designed peptides (e.g., 26) attached to a rigid cavitand macrocycle (e.g., 3) in order to overcome large entropic barriers to folding. We have named these hybrid de novo proteins "caviteins" as a result of their constituent parts (cavitand + protein). Firstly, a number of different cavitand macrocycles were synthesized bearing reactive thiol or benzylbromide moieties at the cavitand's "rim" position to allow for peptide attachment. In addition, cavitands bearing methyl or propyl-phosphates moieties at the "foot" position were synthesized in order to study the effect of the cavitand foot on cavitein structure. As a synthetic model for cavitein synthesis, A-activated phenylalanine ethyl ester derivatives were coupled to methyl-footed tetrathiol cavitand 3 in 34-76% yield. Interestingly, the hydrogen bonding characteristics of their amide NHs vary considerably: only the cavitand-phenylalanine hybrid bearing a single methylene linker displays significant hydrogen bonding. This behaviour is attributed to an NH hydrogen bond to a cavitand "bridge" oxygen and the nearby sulfur atom. The design of each cavitein consists of an JV-activated amphiphilic amino acid sequence (e.g., 26) and a cavitand macrocycle (e.g., 3) bearing sulfur moieties at its rims. The coupling of four activated peptides to each cavitand proceeded efficiently in varying yields (9-62%). Their structures are highly helical and stable towards guanidine hydrochloride in comparison to peptide 28, much a result of the cavitand template. We observe that the cavitand-peptide linker has a profound effect on the structure and oligomeric state of the cavitein. In general, we find that the glycine linker variants possess native-like structural characteristics while the methylene linker variants possess molten globule-like structural characteristics. We attribute the enhanced structural characteristics of the glycine variants to the effect of their added hydrogen bond donors and acceptors to the ends of each helix. The caviteins presented herein represent simple model systems that demonstrate the complexities and subtleties of forces involved in protein folding and allow for further study of the protein folding problem. [Figure.]

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