An isotope-edited FT-IR study of a symporter, the lactose permease.
The lactose permease of Escherichia coli transports protons and lactose across the plasma membrane and uses a transmembrane ion gradient as the energy source to drive the uphill accumulation of 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 bacteriorhodopsin contributions were subtracted. In previous work, positive bands in the 1765-1730 cm(-1) region of the reaction-induced FT-IR spectrum were attributed to the perturbation of carboxylic acid residues in the permease [Patzlaff, J. S., Brooker, R. J., and Barry, B. A. (2000) J. Biol. Chem. 275, 28695-28700]. In this study, we have globally labeled the permease with (13)C or (15)N. Isotopic labeling demonstrates that features in the reaction-induced FT-IR spectrum arise from permease carboxylic acid, amide I, and amide II vibrational modes. In addition, isotope labeling leads to a tentative assignment of spectral features to lysine, arginine, histidine, glutamine, and/or asparagine in the permease. These results indicate that the electrochemical gradient causes changes in the environment or protonation state of carboxylic acid residues in the permease and suggest an interaction between these carboxylic acid side chains and nitrogen-containing amino acid side chains. Evidence for a change in secondary structure, corresponding to an interconversion of secondary structural elements, a change in the hydrogen-bonding strength, or coupling of peptide vibrational modes, is also presented. These experiments demonstrate the usefulness of reaction-induced spectroscopy in the study of transmembrane transport.
Patzlaff
Jason S
JS
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, MN 55108, USA.
Zhang
Jingyan
J
Brooker
Robert J
RJ
Barry
Bridgette A
BA
D001436:Bacteriorhodopsins / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism*; D004563:Electrochemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes / Q000378:metabolism*; D010782:Photolysis; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D027981:Symporters; D001436:Bacteriorhodopsins; C021499:lactose permease