Rational thermostabilisation of four-helix bundle dimeric de novo proteins. 2021

Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
Department of Science and Technology, Graduate School of Medicine, Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.

The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

UI MeSH Term Description Entries
D011489 Protein Denaturation Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein. Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D000072756 Protein Conformation, alpha-Helical A secondary structure of proteins that is a right-handed helix or coil, where each amino (N-H) group of the peptide backbone contributes a hydrogen bond to the carbonyl(C alpha-Helical Conformation, Protein,alpha-Helical Protein Conformation,alpha-Helical Structures,alpha-Helices,alpha-Helix,Conformation, Protein alpha-Helical,Conformation, alpha-Helical Protein,Conformations, Protein alpha-Helical,Conformations, alpha-Helical Protein,Protein Conformation, alpha Helical,Protein Conformations, alpha-Helical,alpha Helical Conformation, Protein,alpha Helical Protein Conformation,alpha Helical Structures,alpha Helices,alpha Helix,alpha-Helical Conformations, Protein,alpha-Helical Protein Conformations,alpha-Helical Structure
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D015202 Protein Engineering Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes. Genetic Engineering of Proteins,Genetic Engineering, Protein,Proteins, Genetic Engineering,Engineering, Protein,Engineering, Protein Genetic,Protein Genetic Engineering
D016297 Mutagenesis, Site-Directed Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion. Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses
D017433 Protein Structure, Secondary The level of protein structure in which regular hydrogen-bond interactions within contiguous stretches of polypeptide chain give rise to ALPHA-HELICES; BETA-STRANDS (which align to form BETA-SHEETS), or other types of coils. This is the first folding level of protein conformation. Secondary Protein Structure,Protein Structures, Secondary,Secondary Protein Structures,Structure, Secondary Protein,Structures, Secondary Protein
D055550 Protein Stability The ability of a protein to retain its structural conformation or its activity when subjected to physical or chemical manipulations. Protein Stabilities,Stabilities, Protein,Stability, Protein
D056004 Molecular Dynamics Simulation A computer simulation developed to study the motion of molecules over a period of time. Molecular Dynamics Simulations,Molecular Dynamics,Dynamic, Molecular,Dynamics Simulation, Molecular,Dynamics Simulations, Molecular,Dynamics, Molecular,Molecular Dynamic,Simulation, Molecular Dynamics,Simulations, Molecular Dynamics

Related Publications

Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
June 2012, The journal of physical chemistry. B,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
December 2000, Biochemistry,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
March 2001, Journal of the American Chemical Society,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
May 2011, Protein science : a publication of the Protein Society,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
December 2014, Science (New York, N.Y.),
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
April 2015, Protein science : a publication of the Protein Society,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
May 2002, The Journal of biological chemistry,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
May 2019, Accounts of chemical research,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
July 2015, European journal of inorganic chemistry,
Shin Irumagawa, and Kaito Kobayashi, and Yutaka Saito, and Takeshi Miyata, and Mitsuo Umetsu, and Tomoshi Kameda, and Ryoichi Arai
January 2008, Biopolymers,
Copied contents to your clipboard!