https://doi.org/10.69107/koomesh-149931

Computational redesign of thermostable DARPin G3 antibody mimetic to facilitate production, storage, and administration management

authors:

avatar Maryam Ehsasatvatan ORCID 1 , avatar Bahram Baghban Kohnehrouz 1 , *

Department of Plant Breeding & Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
Koomesh: Vol.26, issue 5; e149931
published online: January 22, 2025
article type: Research Article

How To Cite? Ehsasatvatan M, Baghban Kohnehrouz B. Computational redesign of thermostable DARPin G3 antibody mimetic to facilitate production, storage, and administration management. koomesh. 2024;26(5):e149931. https://doi.org/10.69107/koomesh-149931.

Abstract

Introduction: Designed ankyrin repeat proteins (DARPins) are relatively small non-immunoglobulin scaffolds with high affinity for specific targets. G3 is a DARPin designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. This study aimed to evaluate the thermostability of DARPin G3 using in silico analyses and amino acid substitutions in the sequences of its N- and C-capping repeats. Bioinformatic tools were used to compare the structural changes and dynamic stability of the designed thermostable proteins with those of the original DARPin G3.
Materials and methods: Computational analysis of the primary and secondary structures of the redesigned DARPin G3 was conducted to predict certain properties. The 3D model of the redesigned proteins was created using the Robetta server, and molecular dynamic simulations were conducted using GROMACS for 50 ns. Molecular docking between the redesigned proteins and the extracellular domain of the HER2 receptor was conducted using the ZDOCK server, and amino acid interactions were plotted using LigPlot.
Results: Molecular dynamic simulations of the structures revealed that the redesigned thermostable DARPin G3 at 350 K was more stable than the original DARPin G3. Molecular docking confirmed the binding of the redesigned thermostable DARPin G3 to the HER2 receptor, with a binding energy comparable to that of the original DARPin G3. The amino acid substitution in the capping repeats of DARPin G3 resulted in an increase of 16–35 °C in the melting temperature of this protein, as well as an improvement in its dynamic stability when compared to the original DARPin G3.
Conclusion: The design and production of thermostable proteins enhance their durability and stability at high temperatures, thereby addressing the need for costly cold storage and simplifying the administration management and transportation of these proteins.