An Isotope-Edited FT-IR Study of a Symporter, the Lactose Permease

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• 作者：Jason S. Patzlaff ; Jingyan Zhang ; Robert J. Brooker ; and Bridgette A. Barry
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：June 11, 2002
• 年：2002
• 卷：41
• 期：23
• 页码：7366 - 7372
• 全文大小：78K
• 年卷期：v.41,no.23(June 11, 2002)
• ISSN：1520-4995

文摘

The lactose permease of Escherichia coli transports protons and lactose across the plasmamembrane and uses a transmembrane ion gradient as the energy source to drive the uphill accumulationof lactose. In this report, the effect of the electrochemical gradient on the permease has been studied.Bacteriorhodopsin was co-reconstituted with the lactose permease to provide a light-triggered electrochemical gradient. Reaction-induced Fourier transform infrared spectra were acquired, and bacteriorhodopsincontributions were subtracted. In previous work, positive bands in the 1765-1730 cm^-1 region of thereaction-induced FT-IR spectrum were attributed to the perturbation of carboxylic acid residues in thepermease [Patzlaff, J. S., Brooker, R. J., and Barry, B. A. (2000) J. Biol. Chem. 275, 28695-28700]. Inthis study, we have globally labeled the permease with ¹³C or ¹⁵N. Isotopic labeling demonstrates thatfeatures in the reaction-induced FT-IR spectrum arise from permease carboxylic acid, amide I, and amideII vibrational modes. In addition, isotope labeling leads to a tentative assignment of spectral features tolysine, arginine, histidine, glutamine, and/or asparagine in the permease. These results indicate that theelectrochemical gradient causes changes in the environment or protonation state of carboxylic acid residuesin the permease and suggest an interaction between these carboxylic acid side chains and nitrogen-containingamino acid side chains. Evidence for a change in secondary structure, corresponding to an interconversionof secondary structural elements, a change in the hydrogen-bonding strength, or coupling of peptidevibrational modes, is also presented. These experiments demonstrate the usefulness of reaction-inducedspectroscopy in the study of transmembrane transport.