title: Stabilization of hyperactive dihydrofolate reductase by cyanocysteine-mediated backbone cyclization.
abstract: Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine- and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteine-mediated cyclization method is complementary to the intein-mediated cyclization method in stabilizing a protein without affecting its activity.
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Munehito
authors.initials: M
authors.lastname: Arai
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Takenawa
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sota
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Qui Hong
authors.initials: QH
authors.lastname: Xie
authors.affiliation: Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address: masa-iwakura@aist.go.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Iwakura
mesh_terms: D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000502:physiology; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology; D006801:Humans; D008024:Ligands; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000737:chemistry*; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008024:Ligands; D010456:Peptides, Cyclic; D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine