Using Cys-Scanning Analysis Data in the Study of Membrane Transport Proteins (original) (raw)
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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
Structural perspectives on secondary active transporters
Trends in pharmacological sciences, 2010
Secondary active transporters catalyze concentrative transport of substrates across lipid membranes by harnessing the energy of electrochemical ion gradients. These transporters bind their ligands on one side of the membrane, and undergo a global conformational change to release them on the other side of the membrane. Over the last few years, crystal structures have captured several bacterial secondary transporters in different states along their transport cycle, providing insight into possible molecular mechanisms. In this review, we will summarize recent findings focusing on the emerging structural and mechanistic similarities between evolutionary diverse transporters. We will also discuss the structural basis of substrate binding, ion coupling and inhibition viewed from the perspective of these similarities.
Membrane transport proteins: implications of sequence comparisons
Current Opinion in Cell Biology, 1992
Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.
European Biophysics Journal, 2013
Secondary active transporters from several protein families share a core of two five-helix inverted repeats that has become known as the LeuT fold. The known highresolution protein structures with this fold were analyzed by structural superposition of the core transmembrane domains (TMDs). Three angle parameters derived from the mean TMD axes correlate with accessibility of the central binding site from the outside or inside. Structural transitions between distinct conformations were analyzed for four proteins in terms of changes in relative TMD arrangement and in internal conformation of TMDs. Collectively moving groups of TMDs were found to be correlated in the covariance matrix of elastic network models. The main features of the structural transitions can be reproduced with the 5 % slowest normal modes of anisotropic elastic network models. These results support the rocking bundle model for the major conformational change between the outward-and inwardfacing states of the protein and point to an important role for the independently moving last TMDs of each repeat in occluding access to the central binding site. Occlusion is also supported by flexing of some individual TMDs in the collectively moving bundle and hash motifs.
Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins
PLoS ONE, 2011
Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassiumchloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.
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...
Structure and function of amino acid and peptide transport proteins
2008
15 2.3 Introduction 15 2.4 Materials and Methods 16 2.5 Results 20 2.6 Discussion 24 2.7 Acknowledgments 26 2.8 References 3. High-Throughput Single Molecule Force Spectroscopy for Membrane Proteins 31 3.1 Abstract 3.2 Introduction 3.3 Materials and Methods 3.4 Results 3.5 Discussion 3.6 Summary and Perspectives 48 3.7 Acknowledgments 49 3.8 References 4. Functional and Structural Characterization of the first Prokaryotic Member of the LAT Family: a Model for APC Transporters 55 4.1 Abbreviations
The International Journal of Biochemistry & Cell Biology, 2010
The recent breakthrough discoveries of transport systems assigned with atypical functions provide evidence for complexity in membrane transport biochemistry. Some channels are far from being simple pores creating hydrophilic passages for solutes and can, unexpectedly, act as enzymes, or mediate highaffinity uptake, and some transporters are surprisingly able to function as sensors, channels or even enzymes. Furthermore, numerous transport studies have demonstrated complex multiphasic uptake kinetics for organic and mineral nutrients. The biphasic kinetics of glucose uptake in Saccharomyces cerevisiae, a result of several genetically distinct uptake systems operating simultaneously, is a classical example that is a subject of continuous debate. In contrast, some transporters display biphasic kinetics, being bona fidae dual-affinity transporters, their kinetic properties often modulated by post-translational regulation. Also, aquaporins have recently been reported to exhibit diverse transport properties and can behave as highly adapted, multifunctional channels, transporting solutes such as CO 2 , hydrogen peroxide, urea, ammonia, glycerol, polyols, carbamides, purines and pyrimidines, metalloids, glycine, and lactic acid, rather than being simple water pores. The present review provides an overview on some atypical functions displayed by transporter proteins and discusses how this novel knowledge on cellular uptake systems may be related to complex multiphasic uptake kinetics often seen in a wide variety of living organisms and the intriguing diffusive uptake of sugars and other solutes.