A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Abstract

In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions. This process occurs in all cells, but the microscopic mechanism is not understood. Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters. To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples. The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient. Positive bands in the 1765-1730 cm ؊1 region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK a , protonation state, or environment. This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level. Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.In secondary active transport, proteins employ a transmembrane electrochemical potential to translocate solutes across the membrane. This process plays a central metabolic role in all living cells. The molecular mechanism by which a transporter transduces a protonmotive force into a solute gradient is the focus of our study. The lactose permease is a transmembrane protein that is responsible for the uptake of lactose into the bacterial cytoplasm (1). The permease is a member of the major facilitator superfamily; members of the major facilitator superfamily catalyze uniport, symport, or antiport (reviewed in Ref.2). The lactose permease is a symporter that can harness the free energy stored in a proton electrochemical gradient to drive the accumulation of lactose against a concentration gradient (1). The permease transports protons and lactose with a one-to-one stoichiometry. A number of mutagenesis studies have led to models suggesting that a few ionizable residues along transmembrane segments play key roles in the mechanism of H ϩ /lactose symport (3, 4). However, direct evidence regarding the mechanism of chemiosmotic coupling in the lactose permease and related transporters has not been available. The goal of this study was to obtain dynamic information about structural changes in the permease associated with the imposition of an electrochemical gradient. To carry out this experiment, we have employed co-reconstitution (5-7) of the permease with a light-driven proton pump, bacteriorhodopsin, and reaction-induced FT-IR 1 spectroscopy (8). Reaction-induced FT-IR spectroscopy requires that a single sample be modulated between two structural states of interest. Co-reconstituted samples provide such a light-activatable and reversible experimental system. When a laser flash is given, bacteriorhodopsin pumps protons across the membrane and creates a H ϩ...

Evidence for Spontaneous Structural Changes in a Dark-Adapted State of Photosystem II

“…Therefore, we assessed the water content of our samples by calculating the ratio of the absorbance at 3290 cm ÿ1 (water plus protein contributions) to the absorbance at 1545 cm ÿ1 (protein contributions). The ratio was derived from each FT-IR absorption spectrum (Patzlaff et al, 2000). The average ratios for the 2-h, 1-h, and 20-min data sets were indistinguishable and were 2.15 1 0.35, 2.22 1 0.40, and 2.13 1 0.39, respectively.…”

Reaction-Induced FT-IR Spectroscopic Studies of Biological Energy Conversion in Oxygenic Photosynthesis and Transport

“…The bacteriorhodopsin contributions have been subtracted out using an optical measurement of the bacteriorhodopsin and total protein concentrations. 37 The lactose permease spectrum decays in the 5 min following saturating laser flashes (data not shown), presumably with the decay of a component of the proton motive force. A positive vibrational band attributable to the permease is observed at 1734 cm -1 (Figure 15A); this band is tentatively assigned to the CdO stretch of carboxylic acid residues in the permease.…”

“…130 Bacteriorhodopsin-containing purple membrane was isolated and fused with these liposomes by a freeze-thaw technique. 37 Control experiments on the coreconstituted vesicles demonstrated a stimulation of lactose efflux under illumination and suggested that each permeasecontaining liposome also contained one or more bacteriorhodopsin molecules. 37 These experiments also showed that bacteriorhodopsin was preferentially oriented to pump protons into the liposome (Figure 14).…”

“…37 Control experiments on the coreconstituted vesicles demonstrated a stimulation of lactose efflux under illumination and suggested that each permeasecontaining liposome also contained one or more bacteriorhodopsin molecules. 37 These experiments also showed that bacteriorhodopsin was preferentially oriented to pump protons into the liposome (Figure 14). This preferential orientation of the reconstituted form of bacteriorhodopsin has been observed previously.…”

“…1,2,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] Recently, we have also successfully applied reaction-induced FT-IR spectroscopy to a transport protein, the lactose permease, which is not directly photoactivatable. 37 To monitor the effect of an electrochemical gradient on the permease, the protein was incorporated into a liposome containing bacteriorhodopsin, which acted as the light-inducible trigger for the electrochemical gradient. This approach will ultimately give new information about membrane transport.…”