Project/Area Number |
12480204
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Research Category |
Grant-in-Aid for Scientific Research (B)
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Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
Biophysics
|
Research Institution | National Institute of Genetics |
Principal Investigator |
NISHIKAWA Ken National Institute of Genetics, Center for Information Biology and DNA Data Bank of Japan, Professor, 生命情報・DDBJ研究センター, 教授 (10093288)
|
Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Kaoru (FUKAMI Kaoru) National Institute of Genetics, Center for Information Biology and DNA Data Bank of Japan, Assistant Professor, 生命情報・DDBJ研究センター, 助手 (20225494)
SHIBA Kiyotaka Japanese Foundation for Cancer Research, Cancer Institute, Department of Protein Engineering, Research Director, 癌研究所・蛋白創製研究部, 部長 (40196415)
|
Project Period (FY) |
2000 – 2002
|
Project Status |
Completed (Fiscal Year 2002)
|
Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2002: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥9,100,000 (Direct Cost: ¥9,100,000)
|
Keywords | periplasmic binding protein / molecular evolution / design of artificial protein / evolutionary protein engineering / folding pattern / chimera protein / assembly PCR method / protein three-dimensional structure / アセンフーリーPCR法 |
Research Abstract |
Periplasmic binding proteins (PBPs) are classified into type 1 and type 2 groups according to the folding pattern of their three-dimensional structure. It has been inferred that the change from type 1 into type 2 occurred only once in the evolution of PBPs by exchanging beta-strands in the core structure. To find out the trigger of the change, we attempted to create a chimera protein that have type 1 function and type 2 folding, by artificially causing the strand exchange. Based on the comparison of the three-dimensional structures, an artificial chimera protein was made out of two pieces of peptides from E.coli type 1 protein (MglB or AraF) and two pieces of peptides from type 2 protein (ArgT). Although partial formation of the secondary structures was observed in the CD spectroscopic analysis, the chimera protein showed substantially weaker ligand binding than the wild type proteins in the equilibrium dialysis. We tried to recover the ligand binding using the phage display method, which was obstructed by non-specific adsorption. We thus investigate the folding pathways of the two types to re-design the artificial protein. We characterized the folding pathways of MglB and ArgT by using urea gradient gel electophoresis, fast protein size-exclusion liquid chromatography and hydrophobic dye ANS binding assay, and found that ArgT has more complicated folding pathway than MglB (J. Biochem133 : 371). We then constructed chimera proteins where only one region was replaced with the corresponding region of another type. Cooperative folding was shown in some of the proteins where a structurally conserved region was replaced with another type (in preparation). We are now trying to modify them using in vitro evolution system and combine them to build chimera proteins where two regions are replaced with another type.
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