| Budget Amount *help |
¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000)
Fiscal Year 2014: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2013: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2012: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000)
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| Outline of Final Research Achievements |
High-pressure stopped-flow absorbance / fluorescence spectrometer was set upped to our laboratory. The absorbance and fluorescence spectra of fluorescein were sufficiently measured but the fluorescence spectrum of intrinsic tryptophan residues of a protein was not detected by this apparatus. We measured pressure dependences of the enzymatic kinetics parameters for the wild-type and D27E mutant dihydrofolate reductases from Escherichia coli (ecDHFR) and that from deep-sea bacterium Moritella profunda (mpDHFR). The obtained Km values for ecDHFR D27E mutant and mpDHFR were less pressure-dependent than that of the wild-type ecDHFR, suggesting that the hydration around active site is important for the adaptation of DHFR to deep-sea. The results of this study clarify the molecular adaptation mechanisms of proteins from deep-sea organisms to the high-pressure environment, and give a novel guideline for the alteration of enzymes to their utilization purposes such as high-pressure condition.
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