Fixation of transporters in the active or inactive state (original) (raw)
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Functional Role for Transporter Isoforms in Optimizing Membrane Transport
Biophysical Journal, 2011
Quantitative analysis of carrier parameters demonstrates that with decreasing substrate concentration the optimal strength of substrate-carrier interaction which maximizes the flux across the membrane increases and requires less fine-tuning than at higher concentrations of the substrate.
2021
Plasmalemmal solute carriers (SLCs) gauge and control solute abundance across cellular membranes. By virtue of this action, they play an important role in numerous physiological processes. Mutations in genes encoding the SLCs alter amino acid sequence that often leads to impaired protein function and onset of monogenic disorders. To understand how these altered proteins cause disease, it is necessary to undertake relevant functional assays. These experiments reveal descriptors of SLC function such as the maximal transport velocity (Vmax), the Michaelis constant for solute uptake (KM), potencies for inhibition of transporter function (IC50/EC50), and many more. In several instances, the mutated versions of different SLC transporters differ from their wild-type counterparts in the value of these descriptors. While determination of these experimental parameters can provide conjecture as to how the mutation gives rise to disease, they seldom provide any definitive insights on how a vari...
Structures and Models of Transporter Proteins
Journal of Pharmacology and Experimental Therapeutics, 2004
Transporter proteins in biological membranes may be divided into channels and carriers. Channels function as selective pores that open in response to a chemical or electrophysiological stimulus, allowing movement of a solute down an electrochemical gradient. Active carrier proteins use an energy producing process to translocate a substrate against a concentration gradient. Secondary active transporters use the movement of a solute down a concentration gradient to drive the translocation of another substrate across a membrane. ATP-binding cassette (ABC) transporters couple hydrolysis of adenosine triphosphate (ATP) to the translocation of various substrates across cell membranes. High-resolution three-dimensional structures have now been reported from X-ray crystallographic studies of six different transporters, including two ATP-binding cassette (ABC) transporters. These structures have explained the results
Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators
Journal of Biological Chemistry, 2003
Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala 122 3 Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (⌬ H ؉) markedly increases the rate of reaction between Cys 122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala 122 3 Cys mutation is combined with a mutation (Cys 154 3 Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and ⌬ H ؉ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala 122 in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.
General Principles of Secondary Active Transporter Function
arXiv: Biomolecules, 2019
Transport of ions and small molecules across the cell membrane against electrochemical gradients is catalyzed by integral membrane proteins that use a source of free energy to drive the energetically uphill flux of the transported substrate. Secondary active transporters couple the spontaneous influx of a "driving" ion such as Na+ or H+ to the flux of the substrate. The thermodynamics of such cyclical non-equilibrium systems are well understood and recent work has focused on the molecular mechanism of secondary active transport. The fact that these transporters change their conformation between an inward-facing and outward-facing conformation in a cyclical fashion, called the alternating access model, is broadly recognized as the molecular framework in which to describe transporter function. However, only with the advent of high resolution crystal structures and detailed computer simulations has it become possible to recognize common molecular-level principles between disp...
Simple models for the analysis of binding protein-dependent transport systems
Protein Science, 1995
Mathematical modeling was used to evaluate experimental data for bacterial binding protein-dependent transport systems. Two simple models were considered in which ligand-free periplasmic binding protein interacts with the membrane-bound components of transport. In one, this interaction was viewed as a competition with the ligandbound binding protein, whereas in the other, it was considered to be a consequence of the complexes formed during the transport process itself. Two sets of kinetic parameters were derived for each model that fit the available experimental results for the maltose system. By contrast, a model that omitted the interaction of ligand-free binding protein did not fit the experimental data. Some applications of the successful models for the interpretation of existing mutant data are illustrated, as well as the possibilities of using mutant data to test the original models and sets of kinetic parameters. Practical suggestions are given for further experimental design. Keywords: bacterial transport; computer simulation; maltose transport; mutant proteins; periplasmic binding proteins monella typhimurium. J Biol Chem 26923931-8936. Hor LI, Shuman HA. 1993. Genetic analysis of periplasmic binding protein dependent transport in E. coli: Each lobe of maltose-binding protein interacts with a different subunit of the MalFGK2 membrane transport complex. J Mol BioI233:659-670. Jacobson BL, He JJ, Vermersch PS, Lemon DD, Quiocho FA. 1991. Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. J Biol Chem 2665220-5225. Kehres DG. 1992. A kinetic model for binding protein-mediated arabinose Kehres DG, Hogg RW. 1992. Escherichia coli K12 arabinose-binding pro-transport. Protein Sci 1:1661-1665. Maddock JR, Shapiro L. 1993. Polar location of the chemoreceptor com-tein mutants with altered transport kinetics. Protein Sci 1:1652-1660. Manson MD, Boos W, Bassford PJ Jr, Rasmussen BA. 1985. Dependence plex in the Escherichia coli cell. Science 259: 17 17-1723. of maltose transport and chemotaxis on the amount of maltose-binding protein. J Biol Chem 260:9727-9733. Miller DM 111, Olson JS, Pflugrath JW, Quiocho FA. 1983. Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis. J Biol Chem 258:13665-13672. Mowbray SL. 1992. The ribose and glucose/galactose receptors: Competi-Olah GA, Trakhanov S, Trewhella J, Quiocho FA. 1993. Leucine/isoleu-tors in bacterial chemotaxis. J Mol Biol227:418-440. 269:16241-16247. cinehaline binding protein contracts upon binding of ligand. JBiol Chem Oliver DB. 1987. Periplasm and protein secretion. In: Neidhardt FC, Ingra-Escherichia coli and Salmonella typhimurium: Cellular and molecular ham JL, Low KB, Magasanick B, Schaechter M, Umbarger HE, eds. biology. Washington, D.C.: American Society for Microbiology. pp Prossnitz E, Gee A, Ames GFL. 1989. Reconstitution of the histidine peri-56-69. teraction between the binding protein and the membrane complex. J Biol plasmic transport system in membrane vesicles. Energy coupling and in-Chem 2645006-5014. Richarme G, Kepes A. 1983. Study of binding protein-ligand interaction by 172:83-94.
Frontiers in pharmacology, 2014
Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structur...
A facile approach for the in vitro assembly of multimeric membrane transport proteins
eLife, 2018
Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using Glt, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric Glt transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na-coupled succinate tra...