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

Abstract

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.

Evidence for Coupled Biogenesis of Yeast Gap1 Permease and Sphingolipids: Essential Role in Transport Activity and Normal Control by Ubiquitination

“…It has also recently been reported that the patchy distribution of some proteins normally present in the MCC is lost upon membrane depolarization (Grossmann et al, 2007). This depolarization might affect the conformation of the intrinsic membrane proteins (Patzlaff et al, 2002) and consequently their association with surrounding lipids and their ability to aggregate preferentially with other proteins.…”

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

“…For instance, illumination of bacteriorhodopsin was used to generate a proton gradient in the presence of co-reconstituted lactose permease (LacY), a proton coupled sugar transporter, and the resulting spectral changes were measured by IR difference spectroscopy. 764 With universal 13 C and 15 N labeling of LacY, it was possible to obtain double-difference spectra free from spectral contributions from BR, containing information about changes in LacY induced by the proton gradient.…”

Isotope-edited IR spectroscopy for the study of membrane proteins

“…Excitonic model simulations and comparisons with the measured spectra enabled the authors to show conclusively that the peptide forms an anti-parallel b-sheet in the membrane, and that the spectra are modulated by the vibrational couplings both in individual strands and between them. Barry and co-workers [13 ], on the other hand, have analyzed a significantly larger protein: the lactose permease (LacY), which contains 417 amino acid residues. The authors performed a 'reaction-induced difference FTIR', in which a reaction is initiated by changing the sample conditions and the resulting difference in the FTIR spectra is recorded.…”

“…spectra of the influenza A M2 transmembrane domain (25 amino acids) reconstituted in lipid bilayers[23]. The spectra shown are of samples with no label (gray line), a single13 C 16 O label (dashed line) or a single 13 C 18 O label (black line). The location of the unlabeled and labeled peaks are shown.…”