RiSLnet: Rapid identification of smart mutant libraries using protein structure network. Application to thermal stability enhancement

Abstract

A key point of protein stability engineering is to identify specific target residues whose mutations can stabilize the protein structure without negatively affecting the function or activity of the protein. Here, we propose a method called RiSLnet (Rapid identification of Smart mutant Library using residue network) to identify such residues by combining network analysis for protein residue interactions, identification of conserved residues, and evaluation of relative solvent accessibility. To validate its performance, the method was applied to four proteins, that is, T4 lysozyme, ribonuclease H, barnase, and cold shock protein B. Our method predicted beneficial mutations in thermal stability with~62% average accuracy when the thermal stability of the mutants was compared with the ones in the Protherm database. It was further applied to lysine decarboxylase (CadA) to experimentally confirm its accuracy and effectiveness. RiSLnet identified mutations increasing the thermal stability of CadA with the accuracy of~60% and significantly reduced the number of candidate residues (~99%) for mutation. Finally, combinatorial mutations designed by RiSLnet and in silico saturation mutagenesis yielded a thermally stable triple mutant with the half-life (T 1/2 ) of 114.9 min at 58°C, which is approximately twofold higher than that of the wild-type.

Enhancing the Thermo-Stability and Anti-Bacterium Activity of Lysozyme by Immobilization on Chitosan Nanoparticles

“…Kawaguchi et al prepared a lysozyme mutant with glycosylation at proper sites by yeast expression system, improving the thermodynamic stabilization of lysozyme but sacrificing the activity [23]. Upadhyay et al applied a rapid identification of smart mutant libraries into protein stabilization, retaining catalytic activity, but the drawback is the absolute requirement of 3D protein structures [24]. Immobilization of enzymes on surfaces can possibly increase the stability of the enzyme and protect it from heat or chemical attack and prolong the time of use [25,26].…”

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing)

“…To meet this high demand, hybrid approaches of protein engineering, which combine computational prediction of potential target residues and rapid experimental validation of these target residues, become an effective alternative. Such attempts have demonstrated great improvements in protein stability, activity, or regio- and stereo-specificity. − Protein solubility could also be improved using such hybrid approaches, but not many studies have yet been reported since rapid protein solubility screening or quantitative evaluation methods are not often available . Therefore, we herein introduce a novel hybrid approach for protein solubility engineering that integrates sequence-based prediction of solubility hot spots, construction of a large, yet focused library for 13 candidate residues in one pot using Darwin assembly, and a fluorescence-based mutant screening with split GFP as a reporter system.…”

Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcus furiosus Phosphoglucose Isomerase

“…However, the screening of such large libraries is typically costly and time inefficient. Hence, the recently introduced ‘smart library design’ approach, which involves reductions in library size by choosing only a small set of promising amino acid candidates and their substitutions, is considered an appreciable step forward [1,2,3,4,5,6,7]. Smart library design requires the identification of key residues, often through functional predictions using bioinformatics followed by high-throughput experimental analyses.…”