Archive by category: Advances in the Field

Active MPSDC participation in the Biophysical Society 60th Annual Meeting in 2016

MPSDC Awards: Francisco Bezanilla, Past President presenting an award to Robert Nakamoto.
(Click to enlarge)

The Membrane Protein Structural Dynamics Consortium is always very well represented at the annual Biophysical Society meeting, as can be seen by Klaus Schulten‘s National Lecture in 2015 as well as at the level of participants in a remarkably large number of workshops, symposia, and presentations. This is also reflected in Consortium members being actively involved at the leadership level, including Francisco Bezanilla‘s tenure as Biophysical Society President from 2012 to 2014 and Robert Nakamoto and Olga Boudker‘s service in the current Biophysical Society leadership council.

On August 31, 2015 the Biophysical Society announced that MPSDC Chair Eduardo Perozo was elected as a 2016 Society Fellow. This award honors the Society’s distinguished members who have demonstrated excellence in science, contributed to the expansion of the field of biophysics, and supported the Biophysical Society. The Fellows will be honored at the Awards Ceremony during the Biophysical Society’s 60th Annual Meeting on Monday February 29, 2016 at the Los Angeles Convention Center in Los Angeles, California. Perozo was elected for his leadership and fundamental contributions in ion channel biophysics.

At next year’s meeting in Los Angeles, MPSDC members will participate in a number of specialty symposia and workshops organized by the Biophysical society (more information on the nature of these symposia and workshops can be found on the Biophysical Society meeting website here). We would like to highlight the following in particular (though there are and will certainly be more ways in which Consortium members are involved with the meeting):

  • Francisco Bezanilla (University of Chicago) is participating in a symposium on Voltage Sensing and Gating.
  • Olga Boudker (Weill Cornell Medical College) is receiving the Michael and Kate Bárány Award during the meeting’s award symposium. During this session, award recipients are recognized and each give a short talk about the work for which they are being recognized. Congratulations, Olga!
  • Yifan Cheng (University of California, San Francisco) is co-chairing the Cryo-EM Subgroup 2016 Symposium
  • Claudio Grosman(UIUC) is participating in a symposium on Pentameric Ligand-gated Ion Channels: New Insights from Structure and Function.
  • Benoît Roux (University of Chicago) is participating in a workshop about Computational Methods for Ion Permeation and Selectivity
  • Emad Tajkhorshid (UIUC) will once again be chairing the Permeation & Transport Subgroup 2015 Symposium. He chaired the same subgroup last year.

Last year, MPSDC member and one of our keynote speakers at Frontiers in Membrane Protein Structural Dynamics 2014 Klaus Schulten (UIUC) gave the prestigious National Lecture. The National Lecturer is the highest award given each year by the Biophysical Society. Dr. Schulten’s 2015 National Lecture lecture can be viewed in full here.

See also: interviews from the 59th Biophysical Society Meeting with 2015 National Lecturer Klaus Schulten and Harel Weinstein.

See also: interviews from the 58th Biophysical Society Meeting with Francisco Bezanilla, Past President and Robert Nakamoto, Chair of the 2014 BPS Program Committee.

Solving a Major Piece of a Cellular Mystery about Nuclear Pore Complexes

Drs. Shohei Koide and Anthony Kossiakoff recently worked together with a team to determines the architecture of a second subcomplex of the nuclear pore complex, in so doing solving a major quandary of answering how a nuclear pore complex (NPC) can be such an effective gatekeeper, preventing much from entering the nucleus while helping to shuttle certain molecules across the nuclear envelope.

The scientific team was featured in multiple publications including Caltech and the Argonne National Laboratory Science Highlights (reproduced in part below). The research was supported in part by the Consortium, and culminated in the following publication:

Architecture of the fungal nuclear pore inner ring complex

Stuwe T, Bley CJ, Thierbach K, Petrovic S, Schilbach S, Mayo DJ, Perriches T, Rundlet EJ, Jeon YE, Collins LN, Huber FM, Lin DH, Paduch M, Koide A, Lu V, Fischer J, Hurt E, Koide S, Kossiakoff AA, Hoelz A. Science. 2015 Oct 2;350(6256):56-64. PMID: 26316600.

Learn more »

Solving a Major Piece of a Cellular Mystery

This article by Argonne reproduced in part can be read here

Not just anything is allowed to enter the nucleus, the heart of eukaryotic cells where, among other things, genetic information is stored. A double membrane, called the “nuclear envelope,” serves as a wall, protecting the contents of the nucleus. Any molecules trying to enter or exit the nucleus must do so via a cellular gatekeeper known as the nuclear pore complex (NPC), or pore, which exists within the envelope.

How can the NPC be such an effective gatekeeper — preventing much from entering the nucleus while helping to shuttle certain molecules across the nuclear envelope? Scientists have been trying to figure that out for decades, at least in part because the NPC is targeted by a number of diseases, including some aggressive forms of leukemia and nervous system disorders such as a hereditary form of Lou Gehrig’s disease. Now a team of researchers from Caltech, The University of Chicago, and the Biochemistry Center of Heidelberg University (Germany), led by André Hoelz and working at three U.S. Department of Energy synchrotron light sources including the Advanced Photon Source (APS) at Argonne, has solved a crucial piece of the puzzle.

In February of this 2015, the team published a paper describing the atomic structure of the NPC’s coat nucleoporin complex, a subcomplex that forms what they now call the outer rings (see the figure). Building on that work, the team has now solved the architecture of the pore’s inner ring, a subcomplex that is central to the NPC’s ability to serve as a barrier and transport facilitator. In order to determine that architecture, which determines how the ring’s proteins interact with each other, the biochemists built up the complex in a test tube and then systematically dissected it to understand the individual interactions between components. Then they validated that this is actually how it works in vivo, in a species of fungus.

Read more »

Researchers discover structure of fluoride-specific ion channel

Dr. Shohei Koide‘s collaborations in a scientific team seeking to discover the structure of a fluoride-specific ion channel was recently featured in Brandeis NOW, a scientific research publication published by Brandeis University. The work focuses on Dr. Christopher Miller’s lab, based at Brandeis University. The research was supported in part by the Consortium, and culminated in the following publication:

Crystal structures of a double-barrelled fluoride ion channel

Stockbridge RB, Kolmakova-Partensky L, Shane T, Koide A, Koide S, Miller C, Newstead S. Nature. 2015 Sep 24;525(7570):548-51. PMID: 26344196.

Learn more »

Researchers discover structure of fluoride-specific ion channel

Creation of ‘atomic blueprint’ is a biological novelty

The original Brandeis NOW press release by Kimm Fesenmaier can be read here

Fluoride protects our teeth against cavity-causing bacteria by making our teeth stronger. But what if we could find a way to trap fluoride ions (the negatively charged form of the chemical element fluorine) inside bacteria? At the right concentration, fluoride ions are highly toxic to bacteria, wreaking havoc on their proteins and disrupting critical cellular functions. Bacteria, however, can fight back, exporting the toxic fluoride ions out using specialized proteins called fluoride-specific ion channels.

How these proteins remove fluoride ions from the cell is poorly understand. To glimpse into the inner workings of these proteins, researchers in Christopher Miller’s lab at Brandeis University in collaboration with Simon Newstead at the University of Oxford have determined the structure of one such fluoride-specific ion channel from the Bordetella pertussis bacteria called Bpe.

In a paper published by Nature on Sept. 7, lead author Randy Stockbridge, a post-doctorate fellow in Miller’s lab, and colleagues used a technique called X-ray crystallography to obtain an “atomic blueprint” illustrating the arrangement of the amino acids that make up Bpe. The details provide important insight into how Bpe exports fluoride out of the cell. Intriguingly, the schematics also point out potential weaknesses in Bpe that could be exploited to trap fluoride inside bacteria.

Based on the blueprint, the Bpe fluoride-specific ion channel resembles an hourglass and is actually composed of two Bpe protein molecules. At the center where the hourglass constricts is a sodium ion that may act like a pin that fastens the two Bpe proteins together. However, rather than having a central pore like an hourglass, the arrangement of the two Bpe molecules forms two parallel tunnels through which fluoride ions could flow. The blueprint also helps explain why Bpe only exports fluoride; the tunnels are just the right size for fluoride ions, but are too narrow for the biologically abundant chloride ion, fluoride’s larger but chemically similar cousin.

Notably, the researchers observed two unidentifiable “hazy shadows” in each tunnel, which they concluded were fluoride ions. First, many of the amino acids along the walls of each channel that protrude toward the shadows are chemically attracted to fluoride ions. Consistently, when the researchers mutated some of these amino acids to change their chemical properties, Bpe’s ability to export fluoride ions out of the bacteria was dramatically reduced. The researchers also studied the structure of a related fluoride-specific ion channel, Ec2, and found similar hazy shadows in its tunnels.

The researchers also noted a peculiar feature of Bpe that has implications for how the protein moves fluoride out. At one point in each of the tunnels, the side chain, or chemical appendage, of a particular amino acid protrudes inward, contacts the fluoride ion, and impedes its path to the exit. The side chain can swivel though, so it may be that the fluoride ion grasps it as though it were a turnstile and is then pulled to the other side when the side chain rotates.

Armed with Bpe’s blueprints, researchers can exploit structural weaknesses and develop strategies that kill bacteria by preventing fluoride from being moved out. Chemical compounds could be designed that pull the sodium ion pin and dismantle Bpe or that lock the turnstile causing a fluoride traffic jam that backs up into the cell.

In addition to Stockbridge, Miller and Newstead, the paper’s other authors are Ludmila Kolmakova-Partensky, Tania Shane, Akiko Koide and Shohei Koide.

The research was supported in part by a Wellcome Trust Investigator Award and grants from the National Institutes of Health (NIH) (RO1-GM107023 and U54-GM087519). Stockbridge also was supported by an NIH grant (K99-GM-111767). Miller is a Howard Hughes Medical Investigator.

Highlights and overview of selected recent Consortium-sponsored publications

Biomolecular DNP- supported NMR spectroscopy using site directed spin labeling

Authors: Elwin A.W. van der Cruijsen, Eline J. Koers, Claire Sauvee, Raymond E. Hulse, Markus Weingarth, Olivier Ouari, Eduardo Perozo, Paul Tordo, and Marc Baldus.

In the press, Journal of the American Chemical Society. (Consortium project: Dynamics of Ion Permeation and Conformation Coupling in KcsA)

Molecular probes that combine the benefits of enhanced spectroscopic sensitivity with site-specific localization have significantly expanded our ability to track molecular structure and function in applications ranging from cell biology to material science . In the field of magnetic resonance, dynamic nuclear polarization (DNP) has become a widely usable method to significantly enhance overall spectroscopic sensitivity in NMR and MRI. Here, the authors show that DNP can be established by creating local spin clusters via site-directed spin labeling using mono- or biradicals. Applied to a membrane-embedded potassium channel, we show that this approach can significantly enhance NMR sensitivity while preserving the intrinsic spectroscopic properties of (bi)radicals as paramagnetic relaxation enhancers.

The results suggest that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of PRE-based NMR approaches. Our results are consistent with the idea that the magnitude in DNP enhancement are highly dependent on the nearest neighbor electron-electron distances.

13C NMR detects conformational change in the 100-kD membrane transporter Cl-C-ec1

Authors: Sherwin J. Abraham, Ricky C. Cheng, Thomas A. Chew, Chandra M. Khantwal, Corey W. Liu, Shimei Gong, Robert K. Nakamoto, and Merritt Maduke.

J Biomol NMR. 2015 Jan 29. (Consortium project: Conformational Dynamics in the CLC Channel Transporter Family. Consortium core: Membrane Protein Production)

Members of the Cl-C (‘‘Chloride-Channel’’) family play central roles in cardiovascular, neuronal, bone, and epithelial function. Cl-C transporters catalyze the exchange of Cl--for H+ across cellular membranes. To do so, they must couple Cl-- and H+ binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, the authors used solution-state 13Cmethyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H+) dependent conformational change outside the restraints of crystallization. They show that methyl labels in several regions report H+-dependent spectral changes. They  identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl-–permeation pathway, to the extracellular solution. The H+-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein.

Together, the results suggest that H+ binding is mechanistically coupled to Cl-osing of the intracellular access-pathway for Cl--. These studies set the stage for investigating the structural details and dynamics of this change.

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Authors: Virginia Meyer, Michael A. Swanson, Laura J. Clouston, Przemyslaw J. Boratynski, Richard A. Stein, Hassane S. Mchaourab, Andrzej Rajca, Sandra S. Eaton, and Gareth R. Eaton.

Biophys J. 2015 Mar 10;108(5):1213-9. (Consortium Core: Membrane Protein Production)

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ~80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ~4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl) methanethio-sulfonate label.

The leap from 80 K DEER measurements to room temperature is an important step toward DEER measurement in physiological environments. The current fundamental requirement for DEER of protein immobilization provides additional avenues toward improvement of the technique.

Architecture of the nuclear pore complex coat

Authors: Tobias Stuwe, Ana R. Correia, Daniel H. Lin, Marcin Paduch, Vincent T. Lu, Anthony A. Kossiakoff, and André Hoelz.

Science. 2015 Mar 6;347(6226):1148-52. (Consortium core: Synthetic Antigen Binder (SAB) Generation and Crystallography)

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. In this research report, the authors present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central “triskelion” of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass.

Multilevel Summation Method for Electrostatic Force Evaluation

Authors: David J. Hardy, Zhe Wu, James C. Phillips, John E. Stone, Robert D. Skeel, and Klaus Schulten.

J Chem Theory Comput. 2015 Feb 10;11(2):766-779. PMCID: PMC4325600. (Consortium core: Computational Modeling Core)

Significant long-range electrostatic interactions arise in many biomolecular systems, such as negatively charged DNA and RNA, polar or charged membranes, ion channels, and electrostatic steering of protein−protein and enzyme−substrate association. Accordingly, electrostatic interactions need to be accurately represented in molecular modeling calculations. The computational cost increases in principle as N2, where N is the number of charged partiCl-es in the system.

The multilevel summation method (MSM) offers an efficient algorithm utilizing convolution for evaluating long-range forces arising in molecular dynamics simulations. Shifting the balance of computation and communication, MSM provides key advantages over the ubiquitous partiCl-e−mesh Ewald (PME) method, offering better scaling on parallel computers and permitting more modeling flexibility, with support for periodic systems as does PME but also for semiperiodic and nonperiodic systems. The version of MSM available in the simulation program NAMD is described, and its performance and accuracy are compared with the PME method. The accuracy feasible for MSM in practical applications reproduces PME results for water property calculations of density, diffusion constant, dielectric constant, surface tension, radial distribution function, and distance dependent Kirkwood factor, even though the numerical accuracy of PME is higher than that of MSM. Excellent agreement between MSM and PME is found also for interface potentials of air−water and membrane−water interfaces, where long-range Coulombic interactions are crucial. Applications demonstrate also the suitability of MSM for systems with semiperiodic and nonperiodic boundaries. For this purpose, simulations have been performed with periodic boundaries along directions parallel to a membrane surface but not along the surface normal, yielding membrane pore formation induced by an imbalance of charge across the membrane. Using a similar semiperiodic boundary condition, ion conduction through a graphene nanopore driven by an ion gradient has been simulated. Furthermore, proteins have been simulated inside a single spherical water droplet. Finally, parallel scalability results show the ability of MSM to outperform PME when scaling a system of modest size (less than 100 K atoms) to over a thousand processors, demonstrating the suitability of MSM for large-scale parallel simulation.

Ongoing is the development of improved interpolation for MSM to provide higher accuracy for a given polynomial degree p without increasing the computational cost. Future work inCl-udes also the calculation of dispersion forces without truncation with MSM-based NAMD; these forces, in particular, their long-range contribution, are considered to be important for membrane properties. With support in NAMD also for long-range dispersion forces, the present CHARMM-prescribed 12 Å cutoff/splitting distance can be used as a true control for MSM accuracy. High performance simulations will then be able to achieve practical accuracy with a reduced splitting distance, where a splitting distance of between 8 and 9 Å is expected to double the overall simulation performance.

Potential Application of Alchemical Free Energy Simulations to Discriminate GPCR Ligand Efficacy

Authors: Hui Sun Lee, Chaok Seok, and Wonpil Im.

J. Chem. Theory Comput. 2015 Feb 10;11:1255-1266. (Consortium core: Computational Modeling Core)

G protein-coupled receptors (GPCRs) constitute the largest protein superfamily in the human genome with almost 1,000  members. They play key functional roles as major contributors of information flow from the outside to the inside of the cell, making them one of the most important protein families. As a result of their broad influence on human physiology and behavior, GPCRs are arguably the most promising targets for development of new and more effective therapeutic agents.

Based on the fact that GPCR-mediated signaling is modulated in a ligand-specific manner such as agonist, inverse agonist, and neutral antagonist (termed ligand efficacy), quantitative characterization of the ligand efficacy is essential for rational design of selective modulators for GPCR targets. As experimental approaches for this purpose are time-, cost-, and labor-intensive, computational tools that can systematically predict GPCR ligand efficacy can have a big impact on GPCR drug design. Here, the authors have performed free energy perturbation molecular dynamics simulations to calculate absolute binding free energy of an inverse agonist, a neutral antagonist, and an agonist to β2-adrenergic receptor (β2-AR) active and inactive states, respectively, in explicit lipid bilayers. Relatively short alchemical free energy calculations reveal that both the time series of the total binding free energy and decomposed energy contributions can be used as relevant physical properties to discriminate β2-AR ligand efficacy. This study illustrates a merit of the current approach over simple, fast docking calculations or highly expensive millisecond-time scale simulations.

It is the authors’ hope that their computational approach improves research and development efficiency in designing novel lead compounds targeting various GPCRs for the treatment of various human diseases.

Recent Consortium advances in the study of neurotransmitter transporters point the way to future research initiatives

Cartoon model of LeuT transport derived from EPR data, click to enlarge (Publication source)

Using the recently determined crystal structure of a prokaryotic leucine transporter (LeuT), the Transport Cycle in Neurotransmitter Uptake Project explores conformational changes and dynamic properties relevant to function in Neurotransmitter transporters translocation cycle using a combination of computational, functional, and spectroscopic approaches.

The conceptual design, scope and integration of this Project exemplifies the consortium approach to discovery of mechanistic principles of secondary active transport, including the conformational dynamics that govern alternating access in transporters and the allosteric interplay of substrate sites that transduce the energy stored in the electrochemical Na+ gradient into transport work. One of the most recent collaborative publications from members of this Project (lab of Harel Weinstein) and the Consortium at large (labs of Consortium member Olga Boudker and associate member Scott Blanchard) describes such conformational dynamics for a complex transporter system, illustrating the power of the combined experimental / computational approaches we have implemented for the study of these membrane proteins in the possible next phase of the grant:

Akyuz N, Georgieva ER, Zhou Z, Stolzenberg S, Cuendet MA, Khelashvili G, Altman RB, Terry DS, Freed JH, Weinstein H, Boudker O, Blanchard SC.
Transport domain unlocking sets the uptake rate of an aspartate transporter
Nature. 2015 Feb 5;518(7537):68-73. PMID: 25652997. PMCID: PMC4351760.

Crystal structure of the H276,395-GltPh.

Publication abstract: Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these ‘elevator-like’ transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the ‘humanizing’ mutations favour structurally ‘unlocked’ intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

In a second publication from this Project, Matthias Quick (who is working with Project PI Jonathan Javitch), and Computational Modeling Core participant Lei Shi (from Harel Weinstein’s group), published findings that reveal the existence of two substrate sites in vSGLT, a member of the solute-sodium symporter (SSS) family of secondary transporters, as well as in PutP, another member of the SSS family. This publication follows a number of Consortium-sponsored interactions between Quick and Shi as well as Javitch and Weinstein. An earlier 2013 research initiative examined the aggregation dynamics and detergent binding to the second substrate site binding site of LeuT, and in the same year, their team published a key paper on the chloride binding site of neurotransmitter sodium transporters. The very first paper published in the name of the Project, was also the product of collaboration between this team of scientists, and showed how experimental conditions can block the second high-affinity site of LeuT. The important discovery of the present publication suggests that our findings in LeuT are generalizable to a different family of secondary transporters. These findings are the basis for forthcoming scientific collaborations between Quick and Shi, further demonstrating the broadening of the impact of the consortium and the synergy that the “glue” of the consortium has engendered.

Li Z, Lee AS, Bracher S, Jung H, Paz A, Kumar JP, Abramson J, Quick M, Shi L.
Identification of a second substrate-binding site in solute-sodium symporters
J Biol Chem., 2015 Jan 2;290(1):127-41. PMID: 25398883. PMCID: PMC4281715.

Crystallographically identified substrate-binding site of vSGLT is located more extracellularly than that of LeuT.

Publication abstract: The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.

Recent Posts
Recent Comments
Recent Photos