Ligand-Induced Movement of Helix X in the Lactose Permease fromEscherichia coli:A Fluorescence Quenching Study

Abstract

Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues (Trp-less permease). Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity. The intrinsic fluorescence emission of each single-Trp mutant does not change significantly after addition of beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside (TDG), indicating that ligand induces little change in the microenvironment of the Trp residues. However, fluorescence quenching studies with the brominated detergent 7,8-dibromododecyl beta,d-maltoside (BrDM) demonstrate that a Trp residue in place of Val315, Val326, or Val331 becomes less accessible to BrDM in the presence of TDG, while a Trp residue in place of Leu318 or Leu329 becomes more accessible. Acrylamide quenching studies with Leu318-->Trp and Val331-->Trp permeases or 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS)-labeled Thr320-->Cys and Glu325-->Cys permeases indicate that positions 318 and 325 also become more accessible to a hydrophobic environment in the presence of TDG, while positions 320 and 331 become less accessible. The findings are consistent with a recently proposed mechanism for energy coupling in lactose permease [Kaback, H. R. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5539-5543] in which substrate binding causes a conformational change resulting in movement of Glu325 to a nonpolar environment with a dramatic increase in pKa.

Locating ligand binding and activation of a single antiporter

“…The peak at 90–100 pN suggested that a certain fraction of the molecules (30–35%) still possessed strong molecular interactions within helix V. The specificity of Na + ions strongly suggests that they have an important role in establishing the molecular interactions at the ligand‐binding site of helix V. Together with the observed pH‐dependent formation of these interactions, it may be suggested that the accessibility of the binding site to Na + ions is governed by pH. It is assumed that this ion‐binding capability may be altered with small changes of the side‐chain orientation (Wang et al , 1997), which is promoted by minute spatial rearrangements of the NhaA helices. Thus, we conclude that the antiporter is activated by intramolecular interactions, which are established only at neutral pH, and simultaneously occurring ligand binding.…”

“…Ligand binding and activation of NhaA A. Kedrov et al membrane proteins that ligand binding during the functional cycle can alter the protein conformation (Wang et al, 1997;Ferguson et al, 2002;le Coutre et al, 2002), causing changes in molecular interactions. As the observed change in interactions is localized in the direct proximity of the ligand-binding site of NhaA, we applied force spectroscopy to probe the effect of Na þ ions on this site.…”

Spectroscopic studies of the interaction of Ca2+‐ATPase‐peptides with dodecyl maltoside and its brominated analog

“…This shows that the detergent efficiently covers the hydrophobic area in contact with the lipid alkyl chain in the native state, as predicted more than 20 years ago [39,40] and subsequently confirmed in threedimensional crystals of membrane protein-detergent complexes [41,42]. Br 2DodMal has recently proved to be a useful tool for the study of protein-detergent interactions during the different steps of SR membrane solubilization [38] and as an aid to follow the change of structure (movement of helix X) associated with ligand binding to lactose permease [43]. Here, we use Br 2 Dod-Mal to analyze the propensity of the peptides to form TM spanning segments from their interaction with detergent micelles and to investigate the position of specific Trp residues within detergent micelles and hence, by analogy, their location within the native membrane.…”

Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter

“…SDFQS is a highly sensitive method that has been successfully applied to reveal conformational rearrangements in other membrane proteins, such as the ␤ 2 -adrenergic receptor and the lactose permease of E. coli (41)(42)(43)(44)(45). Note that LeuT contains no endogenous cysteines, which enables straightforward site-selective incorporation of thiol-reactive fluorophores into targeted cysteine mutants (Fig.…”