Computational design of cyclic peptides for the customized oriented immobilization of globular proteins

Abstract

The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of β2-microglobulin (β2m). Designed sequences were screened by explicit solvent molecular dynamics simulations (MD) followed by experimental validation. Five candidates gave dose-response surface plasmon resonance signals with dissociation constants in the micromolar range. One of them was further analyzed by means of isothermal titration calorimetry, nuclear magnetic resonance, and 250 ns of MD. Atomic-force microscopy imaging showed that this peptide is able to immobilize β2m on a gold surface. In short, we have shown by a variety of experimental techniques that it is possible to capture a protein through an epitope of choice by computational design.

Assessing the capability ofin silicomutation protocols for predicting the finite temperature conformation of amino acids

“…Mutational protocols have been used successfully for predicting structures of proteins that were not resolved experimentally, 1 for determining the interfaces in protein-protein interactions, 2,3 and for designing de novo peptides. [4][5][6] One widespread application is the computational scanning of alanine or glycine residues, in order to identify hot spots and key amino acids responsible of the protein-protein stabilizing interactions. 7,8 The placement of the mutated residue is crucial to understand the potential effects of the mutation.…”

Proteomimetics of Natural Regulators of JAK–STAT Pathway: Novel Therapeutic Perspectives

“…In this context, peptides and peptidomimetics are elective compounds ( Scognamiglio et al, 2013 ; Carotenuto et al, 2014 ; La Manna et al, 2018a ): they act as proteomimetics able to selectively recognize their target protein in biophysical assays and to mimic, totally or partially, the cellular function of native proteins ( Lonardo et al, 2010 ; Causa et al, 2013 ; Sawyer et al, 2017 ). A prominent strategy, mostly lying on the knowledge of the structure of protein complex, consists in the identification of isolated protein fragments mainly responsible for the formation of the protein complex and successively on their conformational stabilization through covalent restraints to improve the affinity and stabilities of unstructured peptides for target proteins ( Russo et al, 2015 ; Soler et al, 2017 ; Adedeji Olulana et al, 2021 ; La Manna et al, 2021 ). In this scenario, several structure-based inhibitors demonstrated discrete success.…”

Homology Modeling-Based in Silico Affinity Maturation Improves the Affinity of a Nanobody

“…The main point is the requirement of three-dimensional structural data of ADAPT-involved components. As a priori knowledge of these crystallographic structures is often not fully available, homologous modeling can be used to get the initial structure data for computational screening [31,32,33].…”