리보스 결합 단백질의 Folding 기작에 관한 연구

학위논문(박사) - 한국과학기술원 : 생명과학과, 1993.8, [ ix, 140 p. ]Three tyrosine residues but no trytophan exist in the ribose-binding protein (RBP) of Escherichia coli. In order to assess the role of each tyrosine in fluorescence emission, mutants were constructed by oligonucleotide-directed mutagenesis that creates phenylalanines in replace of tyrosines at 32, 115, and 261. The mutant proteins were functional as confirmed by their ability to exert in vivo tactic response on minimal swarm plate containing ribose and by their ability to bind to ribose, dissociation constant, kd. The fluorescence of the native proteins purified from various tyrosine mutants was measured from an emission scan with a peak at 303 nm. The tyrosines, at positions 32, 115, and 261, have 10.0 ($\pm$2.4), 69.6 ($\pm$5.2), and 23.4\% ($\pm$5.6) contributions, respectively, to the total intensity of fluorescence. In completely unfolded state, these tyrosines have almost the same intensities of fluorescence, implying that the fluorescences from 32 and 261 tyrosines are considerably quenched in the wild-type protein. The magnitudes of their fluorescences in native proteins are even less than those of unfolded states. Molecular modeling using the coordinate of RBP revealed that a phenolic proton of the tyrosine 32 neighbors with the O$\varepsilon$2 of aspartate 249 residue at 2.05A distance, consistent with the previous observation that the tyrosine fluorscence was quenched by a nearby electron acceptor attracting the phenolic proton of tyrosine. In the tyrosine 261, the phenolic protons are more distantly located, 4.97A from the O$\varepsilon$1 of glutamine 258 and 6.12A from the O$\varepsilon$1 of glutamate 246. Increase of fluirescence emission was monitored in both residues during guanidine hydrochloride induced unfolding in which Tyr 32 is greater than Tyr 261. The increase of fluroescence in these residues was also detected in temperature-induced unfolding. In a quenching experiment with sodium iodide, the tyrosine 115 was the most affected with a St...한국과학기술원 : 생명과학과