Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli.
The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.
Jeong
Jae Kap
JK
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-district, 609-735, Busan, Republic of Korea.
Shin
Hae Ja
HJ
Kim
Jong Won
JW
Lee
Choon Hwan
CH
Kim
Han Do
HD
Lim
Woon Ki
WK
D000817:Anilino Naphthalenesulfonates; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005456:Fluorescent Dyes; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D021122:Protein Subunits; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D014367:Tryptophan Synthase