Rational Design of Protein-Specific Folding Modifiers

Abstract

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a “xenonucleus”, which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

Potential Self-Peptide Inhibitors of the SARS-CoV-2 Main Protease

“…The overall order of helix packing (α1 structuring early followed by α4 with α5 forming last) is similar to the order of helix stabilities seen in NMR spectroscopy studies of SARS-1 M pro C . Thus, the folding of SARS-2 M pro C seen in simulations is similar to that of SARS-1 M pro C as seen in both simulations and experiments. , Specifically, both SARS-1 M pro C and SARS-2 M pro C fold cooperatively, and there is an order to the α-helix formation potentially due to a difference in the helix stabilities. , It has previously been shown that a self-peptide containing the folding nucleus (residues that gain structure early during folding) of ubiquitin can modulate its folding by replacing ubiquitin’s own nucleus . Since SARS-2 M pro C has localized early structure formation (Figure E), akin to a polarized folding nucleus, , we tested if self-peptides of SARS-2 M pro C could insert into the whole protein during folding similar to ubiquitin.…”

“…A larger number of contacts in a specific region also makes that region fold early and nucleate folding. It was shown previously that the ubiquitin self-peptide containing the folding nucleus can act as an alternate xenonucleus during folding . In fact, some of the earliest forming contacts in SARS-2 M pro C are present between α1 and loop4 (Figure E).…”

“…Since the loop between helices α4 and α5 (loop4) is a long loop, it was included as a separate peptide. As in previous work, the potential energy function of the peptide is a subset of the potential energy function of M pro C (V in eq ) and includes bond, angle, dihedral and contact terms for all peptide Cα beads derived from its folded structure within M pro C. Additionally, if a Cα bead, i , from the segment corresponding to the peptide makes a contact with a Cα bead, j , in any part of M pro C, then the same Cα bead in the peptide, i pep , can form an identical contact (with the same interaction function) with the protein Cα bead j . Prefolded or stabilized self-peptides are modeled using intrapeptide contacts whose interaction strength is scaled up by a factor of 5.…”

“…Since the same self-peptide had no inhibitory effect on folded DHFR, it was concluded that the self-peptide affected DHFR folding possibly by competing with the corresponding native peptide during folding. Recently, it was shown that a self-peptide derived from the folding nucleus of ubiquitin, termed the “xenonucleus”, could function as an external seed and allow ubiquitin to fold onto it . Since the folding of several proteins is guided by the formation of specific early forming contacts (the folding nucleus), − it may be easy to rationally design self-peptides that modulate the folding of many proteins. , Here, using self-peptides spanning the entire protein, we test whether folding nucleus-derived self-peptides are more likely than other self-peptides to affect folding by replacing the corresponding native segment in the native fold.…”

Computational design of stapled peptide inhibitor against SARS‐CoV‐2 receptor binding domain

“…By altering the length, attachment point, and chemical nature of the stapling agents, the design of stapled peptides can be tuned to achieve desirable affinity and target specificity. [ 18 , 24 , 25 ] Few experimental and computational reports have checked the stability of stapled ACE2 and their binding with RBD. [ 21 , 26 , 27 ] However, a rationale to combine stapling agents chemical nature and length with their binding affinity is still lacking which is crucial for the future design of stapled peptides.…”