著作: Kanai Taku/Maeda Ayumi/Hanafusa Eiichiro/[佐藤 高則]/Fluorescence spectroscopic studies on the conformational changes of thermostabilized Inorganic Pyrophosphatase mutants./Fifth East Asian Biophysics Symposium & Forty-Fourth Annual Meeting of the Biophysical Society of Japan.
(英) Fluorescence spectroscopic studies on the conformational changes of thermostabilized Inorganic Pyrophosphatase mutants.
(英) Inorganic pyrophosphatases (PPase) from thermophile form hexamer and show high thermostability. In previous studies, we suggested that some substitutions might affect the thermostabilities and oligomeric structure of thermophilic PPase from Thermus thermophilus (Tth PPase) and Geobacillus stearothermophilus (Bst PPase). In Tth PPase, the enhanced thermostabilities were observed by the combination of thermostabilizing factors (Multi-mutants), or insertion of archaeal sequence to C-terminal region (chimeric mutants). As to Bst PPase, the thermostability had increased by substitution in the deduced intertrimer interface (R129H/T). However, it was unclear in detail whether these improvements on PPases might induce the conformational changes. Therefore, we investigated the conformational changes of these PPase mutants by means of fluorescence spectra using intrinsic Tryptophans and ANS as fluorescence probe, and KI as quencher. Firstly, we measured Trp-excited fluorescence spectra of wild type Tth PPase (WT) and its mutants after heating. Although WT aggregated after heating at 85°C, some mutants might maintain solubility and conformation. Furthermore, ANS would not be bound to these mutants at 85°C. On the basis of KI quenching experiment, it was suggested that Trp residues of multi-mutants might be slightly exposed relative to the WT, whereas those of chimeric mutants buried. Same experiments were performed on the Bst PPase mutants, and we will discuss the relationship between conformation and stability of thermophilic PPase mutants.
(英) Fifth East Asian Biophysics Symposium & Forty-Fourth Annual Meeting of the Biophysical Society of Japan.
|都市||必須||(英) Ginowan, Okinawa|
|年月日||必須||2006年 11月 14日|