Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design
We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all...
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doaj-2e6e476b430f40a0b20ce665437be6092020-11-24T22:55:27ZengHindawi LimitedBioMed Research International2314-61332314-61412013-01-01201310.1155/2013/752514752514Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided DesignPavel Mikulecký0Jiří Černý1Lada Biedermannová2Hana Petroková3Milan Kuchař4Jiří Vondrášek5Petr Malý6Peter Šebo7Bohdan Schneider8Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicInstitute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech RepublicWe describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.http://dx.doi.org/10.1155/2013/752514 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Pavel Mikulecký Jiří Černý Lada Biedermannová Hana Petroková Milan Kuchař Jiří Vondrášek Petr Malý Peter Šebo Bohdan Schneider |
spellingShingle |
Pavel Mikulecký Jiří Černý Lada Biedermannová Hana Petroková Milan Kuchař Jiří Vondrášek Petr Malý Peter Šebo Bohdan Schneider Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design BioMed Research International |
author_facet |
Pavel Mikulecký Jiří Černý Lada Biedermannová Hana Petroková Milan Kuchař Jiří Vondrášek Petr Malý Peter Šebo Bohdan Schneider |
author_sort |
Pavel Mikulecký |
title |
Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design |
title_short |
Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design |
title_full |
Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design |
title_fullStr |
Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design |
title_full_unstemmed |
Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design |
title_sort |
increasing affinity of interferon-γ receptor 1 to interferon-γ by computer-aided design |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2013-01-01 |
description |
We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor. |
url |
http://dx.doi.org/10.1155/2013/752514 |
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