Engineering Proteins for Thermostability with iRDP Web Server

Abstract

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

Identification of a hot-spot to enhanceCandida rugosalipase thermostability by rational design methods

“…27 Intermolecular interactions were analyzed with the iMutants program of the iRDP web server (http://irdp.ncl.res.in/index.html accessed 12 October 2017). 34 Default settings were used for analysis.…”

“…55,56 Intermolecular interactions were analyzed by the iMutants application of the iRDP web server. 34 The program is designed to identify molecular interactions, such as hydrogen bonds, hydrophobic interactions, and disulde bonds, which are well known to be the major structural factors that are responsible for protein thermal stability. The Asp457Phe model showed that the introduced phenylalanine created a putative aromatic-aromatic interaction network containing three residues (Trp119, Phe133, and Tyr458), thereby stabilizing the overall structure ( Fig.…”

Enhancing the Thermostability of Rhizomucor miehei Lipase with a Limited Screening Library by Rational-Design Point Mutations and Disulfide Bonds

“…5A and B). The change of interactions via mutation was predicted by iMutants (59), and the overall results are presented in Table S5 in the supplemental material. Mutant T18K might eliminate two side chain-main chain hydrogen bonds and form a new cation-pi interaction between Lys18 and Phe261.…”

Role of Q177A and K173A/Q177A substitutions in thermostability and activity of the ELBn12 lipase

“…7A). Although K173A and Q177A lipases exhibited higher thermostability than the native during thermal stability of proteins [7,8]. Based on the bioinformatics results and similar studies [36,37], we expected that the double mutation should increase the thermal stability of the lipase more than each single one.…”

“…The most well-known strategies include shortening of loops, increasing covalent and noncovalent bonding interactions, higher content of the charged residues and lower thermolabile ones, higher hydrophobicity in the core of proteins, and greater structural packing [7][8][9]. Furthermore, introducing proline residues stabilize protein structures by decreasing the entropy of unfolding [8][9][10]. Applied Biochemistry Directed evolution and rational design are two main strategies for improving protein properties [1,11,12].…”