Evaluating β-turn mimics as β-sheet folding nucleators

Abstract

␤-Turns are common conformations that enable proteins to adopt globular structures, and their formation is often rate limiting for folding. ␤-Turn mimics, molecules that replace the i ؉ 1 and i ؉ 2 amino acid residues of a ␤-turn, are envisioned to act as folding nucleators by preorganizing the pendant polypeptide chains, thereby lowering the activation barrier for ␤-sheet formation. However, the crucial kinetic experiments to demonstrate that ␤-turn mimics can act as strong nucleators in the context of a cooperatively folding protein have not been reported. We have incorporated 6 ␤-turn mimics simulating varied ␤-turn types in place of 2 residues in an engineered ␤-turn 1 or ␤-bulge turn 1 of the Pin 1 WW domain, a three-stranded ␤-sheet protein. We present 2 lines of kinetic evidence that the inclusion of ␤-turn mimics alters ␤-sheet folding rates, enabling us to classify ␤-turn mimics into 3 categories: those that are weak nucleators but permit Pin WW folding, native-like nucleators, and strong nucleators. Strong nucleators accelerate folding relative to WW domains incorporating all ␣-amino acid sequences. A solution NMR structure reveals that the native Pin WW ␤-sheet structure is retained upon incorporating a strong E-olefin nucleator. These ␤-turn mimics can now be used to interrogate protein folding transition state structures and the 2 kinetic analyses presented can be used to assess the nucleation capacity of other ␤-turn mimics.beta-sheet nucleator ͉ kinetic assessment of turn mimics ͉ Pin WW domain ͉ protein folding

Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts

“…The four-, five-, and six-residue reverse turns comprising loop 1 of WW were converted to their corresponding enhanced aromatic sequons by replacing the amino acid at position 16 (Ser in all cases) with Phe, replacing the amino acid at position 19 (Asn, Asp, or Ser, respectively) with Asn (GlcNAc1), and replacing the amino acid at position 21 (Arg in all cases) with Thr (36,38). Note that we are using the same number to indicate amino acids in analogous positions in WW variants with different loop 1 lengths (36,37). Thus, the sequences of the enhanced aromatic sequons in the four-, five-, and six-residue reverse turns comprising loop 1 are Phe16-Asn(GlcNAc1)19-Gly20-Thr21, Phe16-Ala18-Asn(GlcNAc1)19-Gly20-Thr21, and Phe16-Arg17-Ser18--Asn(GlcNAc1)19-Gly20-Thr21, respectively (see Table 1).…”

“…To meet these goals, we sought a single protein into which several types of enhanced aromatic sequons and their corresponding reverse turn types could be inserted without changing the overall structure or the flanking sequences. The WW domain is ideal for these requirements: Many WW variants harboring different reverse turn types in loop 1 have been structurally (34,36,37) and biophysically (36)(37)(38)(39) characterized. Crystal structures exist for WW domains harboring a type II β-turn in a six-residue loop (Fig.…”

FTIR Assessment of the Effect of Ginkgo biloba Leave Extract (EGb 761) on Mammalian Retina

“…b-turns are the smallest type of protein secondary structure, joining other elements of secondary structure such as a-helix and b-sheets and abruptly change the direction of the polypeptide chain and may dictating the folding of longer polypeptide chains [22,23]. b-turns are common conformations that enable protein to adopt globular structures, and may serve as a nucleation site for folding/refolding of proteins [24]. Their formation is often rate limiting for folding where protein stability is intimately connected with protein folding.…”

Protein prosthesis: β‐peptides as reverse‐turn surrogates

“…These results encourage additional interrogations of natural structural elements with foldameric equivalents. [27][28][29][30] …”