An investigation into the native‐like properties of de novo designed cavitand‐based four‐helix bundle proteins

Abstract

We investigated the hydrophobic packing of two previously designed caviteins, LG2 and LG3, which differ by one Gly in the linker regions between the peptide sequence and the cavitand scaffold. We sought to diminish the putative native-like properties of LG2 and LG3, and see if we could diagnose a change in the conformational specificity of the hydrophobic core. We replaced the leucine residues with norleucine residues at the hydrophobic positions in LG2 and LG3, to create NG2 and NG3, respectively. LG2 exhibited more dispersion, but less sharp signals than LG3 in the amide region of its (1)H NMR spectrum. NG3 and NG2 were found to be slightly less helical and significantly less stable toward guanidine hydrochloride compared with their reference caviteins. The (1)H NMR spectrum of NG2 was very similar to that of LG2, whereas there was a noticeable loss in the number and sharpness of the amide signals of NG3 compared with LG3. These data suggest that LG3 is very well packed; a loss in conformational specificity resulted from replacement of the leucine residues with norleucine residues. In contrast, the packing and dynamics of the hydrophobic core in LG2 were similar to those in NG2 (both more modest than LG3), as their (1)H NMR spectra were virtually indistinguishable. Overall, substitution of leucine by norleucine provided an efficient, convenient, and informative probe of the packing and dynamics of our caviteins' hydrophobic cores.

X-ray Crystal Analysis of a TASP: Structural Insights of a Cavitein Dimer

“…The calculated value of Δ G ° H 2 O for cavitein Q4 is −9.6 ± 0.6 kcal mol −1 . This is within error of the previously reported values for N1GG by Mezo and Huttunen (Table ). , …”

“…This is within error of the previously reported values for N1GG by Mezo and Huttunen (Table 1). 34,35 Concentration-dependent GuHCl unfolding experiments (high, 200 µM; low, 4 µM) were carried out to examine the aggregation state of Q4. 61 The high and low concentration studies showed indistinguishable denaturation curves, indicating that Q4 denatures cooperatively as a monomeric species (Figure 11).…”

“…Our group’s TASPs often use a cavitand as the template, as they are rigid and can be readily linked to peptides (Scheme ). − We have named these model systems “caviteins” from the combination of cavi tand + pro tein . Caviteins are typically helical bundles that owe their high thermostability to the template-driven saving of entropic cost of bundle organization.…”

Systematic Moiety Variations of Ultrashort Peptides Produce Profound Effects on Self-Assembly, Nanostructure Formation, Hydrogelation, and Phase Transition

“…The methyl shifts do not change the “hydrophobic content” of the peptide, but they can affect intermolecular peptide packing. As noted before, there is a difference in packing between Leu and Nle, in which Leu can pack more intimately than Nle 72 . As our crystal structure illustrates, the isobutyl side chains of Ile can interdigitate with each other to maximize hydrophobic interaction, a packing conformation inaccessible to Nle.…”