Zhou, Y. Guan, L. Kaback, H. R. Opening and closing of the periplasmic gate in lactose permease.
Proc Natl Acad Sci U S A
2008;
in press.
Guan, L. Kaback, H. R. Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins.
Nat Protoc.
2007;
2( 8):
2012-7.
Kaback, H. R. Structure and mechanism of the lactose permease.
C R Biol.
2005;
328( 6):
557-67.
Kaback, H. R The Passion of the Permease.
Biophysical and Structural Aspects of Bioenergetics
2005;
359-373.
Nie, Y. Ermolova, N. Kaback, H. R. Site-directed Alkylation of LacY: Effect of the Proton Electrochemical Gradient.
J Mol Biol.
2007;
374( 2):
356-64.
Smirnova, I. Kasho, V. Choe, J. Y. Altenbach, C. Hubbell, W. L. Kaback, H. R. Sugar binding induces an outward facing conformation of LacY.
Proc Natl Acad Sci U S A.
2007;
104:
16504-16509.
Guan, L. Mirza, O. Verner, G. Iwata, S. Kaback, H. R. Structural determination of wild-type lactose permease.
Proc Natl Acad Sci U S A.
2007;
104( 39):
15294-8.
Majumdar, D. S. Smirnova, I. Kasho, V. Nir, E. Kong, X. Weiss, S. Kaback, H. R. Single-molecule FRET reveals sugar-induced conformational dynamics in LacY.
Proc Natl Acad Sci U S A.
2007;
104( 31):
12640-12645.
Shimohata, N. Nagamori, S. Akiyama, Y. Kaback, H. R. Ito, K. SecY alterations that impair membrane protein folding and generate a membrane stress.
J Cell Biol.
2007;
176( 3):
307-17.
Smirnova, I. N. Kasho, V. N. Kaback, H. R. Direct Sugar Binding to LacY Measured by Resonance Energy Transfer.
Biochemistry.
2006;
45( 51):
15279-87.
Kaback, H. R. Dunten, R. Frillingos, S. Venkatesan, P. Kwaw, I. Zhang, W. Ermolova, N. Site-directed alkylation and the alternating access model for LacY.
Proc Natl Acad Sci U S A.
2006;
.
Nie, Y. Smirnova, I. Kasho, V. Kaback, H. R. Energetics of Ligand-induced Conformational Flexibility in the Lactose Permease of Escherichia coli.
J Biol Chem.
2006;
281( 47):
35779-84.
Vadyvaloo, V. Smirnova, I. N. Kasho, V. N. Kaback, H. Ronald Conservation of residues involved in sugar/H(+) symport by the sucrose permease of Escherichia coli relative to lactose permease.
J Mol Biol.
2006;
358( 4):
1051-9.
Kasho, V. N. Smirnova, I. N. Kaback, H. R. Sequence alignment and homology threading reveals prokaryotic and eukaryotic proteins similar to lactose permease.
J Mol Biol.
2006;
358( 4):
1060-70.
Ermolova, N. Madhvani, R. V. Kaback, H. R. Site-directed alkylation of cysteine replacements in the lactose permease of Escherichia coli: helices I, III, VI, and XI.
Biochemistry.
2006;
45( 13):
4182-9.
Mirza, O. 2006)200251183Guan, L. Verner, G. Iwata, S. Kaback, H. R. Structural evidence for induced fit and a mechanism for sugar/H(+) symport in LacY.
Embo J.
2006;
25:
1177-1183.
Guan, L. Smirnova, I. N. Verner, G. Nagamoni, S. Kaback, H. R. Manipulating phospholipids for crystallization of a membrane transport protein.
Proc Natl Acad Sci U S A.
2006;
103( 6):
1723-6.
Ermolova, N. V. Smirnova, I. N. Kasho, V. N. Kaback, H. R. Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli.
Biochemistry.
2005;
44( 21):
7669-77.
van Bloois, E. Nagamori, S. Koningstein, G. Ullers, R. S. Preuss, M. Oudega, B. Harms, N. Kaback, H. R. Herrmann, J. M. Luirink, J. The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential.
J Biol Chem.
2005;
280( 13):
12996-3003.
Weinglass, A. B. Soskine, M. Vazquez-Ibar, J. L. Whitelegge, J. P. Faull, K. F. Kaback, H. R. Schuldiner, S. Exploring the role of a unique carboxyl residue in EmrE by mass spectrometry.
J Biol Chem.
2005;
280( 9):
7487-92.
Sun, J. Savva, C. G. Deaton, J. Kaback, H. R. Svrakic, M. Young, R. Holzenburg, A. Asymmetric binding of membrane proteins to GroEL.
Arch Biochem Biophys.
2005;
434( 2):
352-7.
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The lactose permease of Escherichia coli (LacY), a particularly well-studied representative of the the Major Facilitator Superfamily (MFS), is solely responsible for all translocation reactions catalyzed by the galactoside transport system in E. coli. Like many members of the MFS, LacY couples the free energy released from downhill translocation of protons in response to a proton electrochemical gradient to drive the energetically uphill stoichiometric accumulation of D-galactopyranosides.
The x-ray structure of LacY mutant C154G in an inward-facing conformation was solved at a resolution of 3.5 U confirming many conclusions derived from biochemical and biophysical studies carried out over the past 20 years. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the molecule. A large internal hydrophilic cavity is exposed to the cytoplasm, and ligand is bound at the two-fold axis of symmetry at the apex of the hydrophilic cavity and in the approximate middle of the molecule. By combining a large body of experimental data derived from systematic studies of site-directed mutants, residues involved in substrate binding and proton translocation have been identified, and based on the functional properties of the mutants and the x-ray structure, a working model for the mechanism has been postulated.
Clearly, an alternative, outward-facing conformation open to the periplasmic side is absolutely required for substrate transport across the membrane. A simulation of the outward-facing conformation has been constructed on the basis of structurally flexibility, ligand-induced increase in the reactivity of certain Cys-replacement mutants in the periplasmic region of LacY with N-ethylmaleimide (NEM) and a discrepancy between distances in the crystal structure and distances approximated from thiol-cross-linking across the hydrophilic cavity facing the cytoplasm. Based on these considerations as a whole, it is postulated that LacY contains a single binding site with alternating access to either side of membrane during turnover. Supporting evidence for the hypothesis has been presented.
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