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1.
J Mol Biol ; : 168665, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38878854

ABSTRACT

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

2.
Handb Exp Pharmacol ; 283: 319-360, 2024.
Article in English | MEDLINE | ID: mdl-37947907

ABSTRACT

Solute carrier family 26 (SLC26) is a family of functionally diverse anion transporters found in all kingdoms of life. Anions transported by SLC26 proteins include chloride, bicarbonate, and sulfate, but also small organic dicarboxylates such as fumarate and oxalate. The human genome encodes ten functional homologs, several of which are causally associated with severe human diseases, highlighting their physiological importance. Here, we review novel insights into the structure and function of SLC26 proteins and summarize the physiological relevance of human members.


Subject(s)
Anion Transport Proteins , Humans , Sulfate Transporters/metabolism , Anion Transport Proteins/genetics , Anion Transport Proteins/chemistry , Anion Transport Proteins/metabolism , Anions/metabolism , Biological Transport
3.
Biol Chem ; 404(7): 715-725, 2023 06 27.
Article in English | MEDLINE | ID: mdl-36916166

ABSTRACT

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na+-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.


Subject(s)
Carrier Proteins , Membrane Proteins , Humans , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Membrane Transport Proteins/metabolism , Biological Transport
4.
J Proteome Res ; 21(6): 1408-1417, 2022 06 03.
Article in English | MEDLINE | ID: mdl-35561006

ABSTRACT

Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. The FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa), stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q)-peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R)-peptides that relate its abundance to a single protein standard (bovine serum albumin, BSA). FastCAT not only alleviates the need to purify CP or use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but also improves the accuracy, precision, and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of the target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in human cerebrospinal fluid at the low ng/mL level.


Subject(s)
Proteins , Proteomics , Electrophoresis, Polyacrylamide Gel , Humans , Peptides/metabolism , Proteomics/methods , Reference Standards
5.
J Proteome Res ; 21(1): 132-141, 2022 01 07.
Article in English | MEDLINE | ID: mdl-34807614

ABSTRACT

By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.


Subject(s)
Peptides , Proteome , Calibration , Isotope Labeling/methods , Peptides/chemistry , Reference Standards
6.
J Mol Biol ; 434(2): 167393, 2022 01 30.
Article in English | MEDLINE | ID: mdl-34896363

ABSTRACT

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.


Subject(s)
Biological Transport/physiology , Ion Transport , Membrane Transport Proteins/chemistry , Protons , Animals , Ascorbic Acid/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Catalytic Domain , Escherichia coli , Humans , Membrane Transport Proteins/metabolism , Nucleobase Transport Proteins/chemistry , Nucleobase Transport Proteins/metabolism , Pentosyltransferases/chemistry , Pentosyltransferases/metabolism , Sodium/metabolism , Symporters , Uracil/metabolism
7.
Biochem Soc Trans ; 48(3): 1047-1055, 2020 06 30.
Article in English | MEDLINE | ID: mdl-32573703

ABSTRACT

The activity of enzymes is subject to regulation at multiple levels. Cooperativity, the interconnected behavior of active sites within a protein complex, directly affects protein activity. Cooperativity is a mode of regulation that requires neither extrinsic factors nor protein modifications. Instead, it allows enzymes themselves to modulate reaction rates. Cooperativity is an important regulatory mechanism in soluble proteins, but also examples of cooperative membrane proteins have been described. In this review, we summarize the current knowledge on interprotomer cooperativity in elevator-type proteins, a class of membrane transporters characterized by large rigid-body movements perpendicular to the membrane, and highlight well-studied examples and experimental approaches.


Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Bacteria/metabolism , Binding Sites , Catalytic Domain , Escherichia coli/metabolism , Humans , Kinetics , Ligands , Membrane Transport Proteins/metabolism , Protein Binding , Protein Multimerization , Solubility
8.
Nat Protoc ; 15(5): 1707-1741, 2020 05.
Article in English | MEDLINE | ID: mdl-32269381

ABSTRACT

Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.


Subject(s)
Chemistry Techniques, Synthetic/methods , Single-Domain Antibodies/chemistry , Bacteriophages/chemistry , Ribosomes/chemistry
9.
Methods Mol Biol ; 2127: 151-165, 2020.
Article in English | MEDLINE | ID: mdl-32112321

ABSTRACT

The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.


Subject(s)
Avidin/metabolism , Biotin/metabolism , Biotinylation/methods , Membrane Proteins/metabolism , Single-Domain Antibodies/isolation & purification , Amino Acid Sequence , Avidin/chemistry , Biotin/chemistry , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/metabolism , Cell Surface Display Techniques/methods , Cloning, Molecular/methods , Escherichia coli , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Klebsiella pneumoniae , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Protein Binding , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Streptavidin/chemistry , Streptavidin/metabolism
10.
Ann N Y Acad Sci ; 1459(1): 38-68, 2020 01.
Article in English | MEDLINE | ID: mdl-31588569

ABSTRACT

Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.


Subject(s)
Anti-Bacterial Agents/metabolism , Drug Resistance, Multiple, Bacterial/physiology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Humans , Protein Structure, Secondary , Protein Structure, Tertiary
11.
Nature ; 571(7766): 580-583, 2019 07.
Article in English | MEDLINE | ID: mdl-31316210

ABSTRACT

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/metabolism , Cryoelectron Microscopy , Thermus thermophilus/chemistry , ATP-Binding Cassette Transporters/ultrastructure , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Hydrolysis , Kinetics , Models, Molecular , Mutation , Protein Conformation , Protein Multimerization , Substrate Specificity , Thermus thermophilus/ultrastructure , Vanadates/metabolism
12.
Nat Commun ; 10(1): 2032, 2019 05 02.
Article in English | MEDLINE | ID: mdl-31048734

ABSTRACT

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.


Subject(s)
Bacterial Proteins/metabolism , Molecular Dynamics Simulation , Protein Multimerization , Protein Structure, Quaternary , Sulfate Transporters/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Deinococcus , Electron Spin Resonance Spectroscopy , Mutagenesis, Site-Directed , Organic Anion Transporters, Sodium-Dependent/chemistry , Organic Anion Transporters, Sodium-Dependent/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SLC4A Proteins/chemistry , SLC4A Proteins/metabolism , Sulfate Transporters/chemistry , Sulfate Transporters/genetics , Sulfate Transporters/isolation & purification
13.
Elife ; 72018 05 24.
Article in English | MEDLINE | ID: mdl-29792401

ABSTRACT

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.


Subject(s)
ATP-Binding Cassette Transporters/isolation & purification , Equilibrative Nucleoside Transporter 1/isolation & purification , Glycine Plasma Membrane Transport Proteins/isolation & purification , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/immunology , ATP-Binding Cassette Transporters/metabolism , Cell Surface Display Techniques , Equilibrative Nucleoside Transporter 1/chemistry , Equilibrative Nucleoside Transporter 1/immunology , Equilibrative Nucleoside Transporter 1/metabolism , Glycine Plasma Membrane Transport Proteins/chemistry , Glycine Plasma Membrane Transport Proteins/immunology , Glycine Plasma Membrane Transport Proteins/metabolism , Humans , Protein Binding , Protein Conformation , Protein Stability , Single-Domain Antibodies/genetics
14.
Methods Enzymol ; 594: 139-164, 2017.
Article in English | MEDLINE | ID: mdl-28779839

ABSTRACT

Escherichia coli is one of the most widely used expression hosts for membrane proteins. However, establishing conditions for its recombinant production of membrane proteins remains difficult. Attempts to produce membrane proteins frequently result in either no expression or expression as misfolded aggregates. We developed an efficient pipeline for improving membrane protein overexpression in E. coli that is based on two approaches. The first involves transcriptional fusions, small additional RNA sequences upstream of the target open reading frame, to overcome no or poor overall expression levels. The other is based on a tunable promoter in combination with a fusion to green fluorescent protein serving as a reporter for the folding state of the target membrane protein. The latter combination allows adjusting the membrane protein expression rate to the downstream folding capacity, in order to decrease the formation of protein aggregates. This pipeline has proven successful for the efficient and parallel optimization of a diverse set of membrane proteins.


Subject(s)
Escherichia coli/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Engineering/methods , Recombinant Fusion Proteins/metabolism , Cloning, Molecular/methods , Genetic Vectors , Green Fluorescent Proteins/genetics , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics
15.
Sci Rep ; 7(1): 8390, 2017 08 21.
Article in English | MEDLINE | ID: mdl-28827559

ABSTRACT

Broadly neutralizing antibodies (bnAbs) against HIV-1 protect from infection and reduce viral load upon therapeutic applications. However no vaccine was able so far to induce bnAbs demanding their expensive biotechnological production. For clinical applications, nanobodies (VHH) derived from heavy chain only antibodies from Camelidae, may be better suited due to their small size, high solubility/stability and extensive homology to human VH3 genes. Here we selected broadly neutralizing nanobodies by phage display after immunization of dromedaries with different soluble trimeric envelope proteins derived from HIV-1 subtype C. We identified 25 distinct VHH families binding trimeric Env, of which 6 neutralized heterologous primary isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The complementary neutralization pattern of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of epidemiologically relevant HIV-1 subtypes. The CD4 binding site was preferentially targeted by the broadly neutralizing VHHs as determined by competition ELISAs and 3D models of VHH-Env complexes derived from negative stain electron microscopy. The nanobodies identified here are excellent candidates for further preclinical/clinical development for prophylactic and therapeutic applications due to their potency and their complementary neutralization patterns covering the majority of epidemiologically relevant HIV-1 subtypes.


Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV-1/immunology , Single-Domain Antibodies/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/isolation & purification , Camelus , Cell Surface Display Techniques , Genotype , HIV Antibodies/chemistry , HIV Antibodies/isolation & purification , HIV Infections/immunology , HIV Infections/virology , HIV-1/classification , HIV-1/drug effects , HIV-1/genetics , Humans , Protein Binding , Protein Conformation , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/isolation & purification , env Gene Products, Human Immunodeficiency Virus/chemistry
16.
Biochemistry ; 56(30): 3962-3971, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28731329

ABSTRACT

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.


Subject(s)
Central Nervous System Depressants/pharmacology , Cerebral Cortex/drug effects , Membrane Transport Modulators/pharmacology , Models, Molecular , Nerve Tissue Proteins/antagonists & inhibitors , Neurons/drug effects , Single-Domain Antibodies/pharmacology , Vesicular Glutamate Transport Protein 1/antagonists & inhibitors , Animals , Biological Transport/drug effects , Camelids, New World , Cells, Cultured , Central Nervous System Depressants/chemistry , Central Nervous System Depressants/metabolism , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Embryo, Mammalian/cytology , Glutamic Acid/metabolism , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Membrane Transport Modulators/chemistry , Membrane Transport Modulators/metabolism , Mice , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Peptide Library , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Synaptic Transmission/drug effects , Synaptic Vesicles/drug effects , Synaptic Vesicles/metabolism , Vesicular Glutamate Transport Protein 1/chemistry , Vesicular Glutamate Transport Protein 1/genetics , Vesicular Glutamate Transport Protein 1/metabolism
17.
Bio Protoc ; 7(3): e2116, 2017 Feb 05.
Article in English | MEDLINE | ID: mdl-34458442

ABSTRACT

The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure ( Geertsma et al., 2015 ). The procedure may be adapted to purify and crystallize other membrane protein complexes.

18.
Biol Chem ; 398(2): 165-174, 2017 02 01.
Article in English | MEDLINE | ID: mdl-27865089

ABSTRACT

Solute carriers from the SLC4, SLC23, and SLC26 families are involved in pH regulation, vitamin C transport and ion homeostasis. While these families do not share any obvious sequence relationship, they are united by their unique and novel architecture. Each member of this structural class is organized into two structurally related halves of seven transmembrane segments each. These halves span the membrane with opposite orientations and form an intricately intertwined structure of two inverted repeats. This review highlights the general design principles of this fold and reveals the diversity between the different families. We discuss their domain architecture, structural framework and transport mode and detail an initial transport mechanism for this fold inferred from the recently solved structures of different members.


Subject(s)
Cell Membrane/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Repetitive Sequences, Amino Acid , Animals , Biological Transport , Humans , Protein Domains , Protein Multimerization
20.
Nat Struct Mol Biol ; 22(10): 803-8, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26367249

ABSTRACT

The SLC26 family of membrane proteins combines a variety of functions within a conserved molecular scaffold. Its members, besides coupled anion transporters and channels, include the motor protein Prestin, which confers electromotility to cochlear outer hair cells. To gain insight into the architecture of this protein family, we characterized the structure and function of SLC26Dg, a facilitator of proton-coupled fumarate symport, from the bacterium Deinococcus geothermalis. Its modular structure combines a transmembrane unit and a cytoplasmic STAS domain. The membrane-inserted domain consists of two intertwined inverted repeats of seven transmembrane segments each and resembles the fold of the unrelated transporter UraA. It shows an inward-facing, ligand-free conformation with a potential substrate-binding site at the interface between two helix termini at the center of the membrane. This structure defines the common framework for the diverse functional behavior of the SLC26 family.


Subject(s)
Anion Transport Proteins/chemistry , Deinococcus/genetics , Fumarates/metabolism , Models, Molecular , Multigene Family/genetics , Anion Transport Proteins/metabolism , Base Sequence , Chromatography, Gel , Cloning, Molecular , Crystallography, X-Ray , DNA Primers/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Fluorescence , Molecular Sequence Data , Open Reading Frames/genetics , Polymerase Chain Reaction , Protein Conformation , Selenomethionine , Sequence Analysis, DNA
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