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bioRxiv2017

Mechanical mismatch between Ras transformed and untransformed epithelial cells.

The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (RasV12) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer RasV12 cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.

biophysics↗

bioRxiv2017

A method to minimize condenser lens-induced hysteresis effects in a JEOL JEM-3200FSC microscope to enable stable cryoEM low-dose operations

Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection.\n\nHighlights O_LIEnables consistent horizontal beam position during low-dose mode switching\nC_LIO_LIFacilitates EM automated data collection for the JEOL JEM-3200FSC\nC_LIO_LIImproves beam stability in both TEM imaging and diffraction modes\nC_LI

biophysics↗

bioRxiv2017

Molecular chaperones maximize the native state yield per unit time by driving substrates out of equilibrium

Molecular chaperones have evolved to facilitate folding of proteins and RNA in vivo where spontaneous self-assembly is sometimes prohibited. Folding of Tetrahymena ribozyme, assisted by the RNA chaperone CYT-19, surprisingly shows that at physiological Mg2+ ion concentrations, increasing the chaperone concentration reduces the yield of native ribozymes. In contrast, the more extensively investigated protein chaperone GroEL works in exactly the opposite manner--the yield of native substrate increases with the increase in chaperone concentration. Thus, the puzzling observation on the assisted-ribozyme folding seems to contradict the expectation that a molecular chaperone acts as an efficient annealing machine. We suggest a resolution to this apparently paradoxical behavior by developing a minimal stochastic model that captures the essence of the Iterative Annealing Mechanism (IAM), providing a unified description of chaperone mediated-folding of proteins and RNA. Our theory provides a general relation involving the kinetic rates of the system, which quantitatively predicts how the yield of native state depends on chaperone concentration. By carefully analyzing a host of experimental data on Tetrahymena (and its mutants) as well as the protein Rubisco and Malate Dehydrogenase, we show that although the absolute yield of native states decreases in the ribozyme, the rate of native state production increases in both the cases. By utilizing energy from ATP hydrolysis, both CYT-19 and GroEL drive their substrate concentrations far out of equilibrium, in an endeavor to maximize the native yield in a short time. Our findings are consistent with the general expectation that proteins or RNA need to be folded by the cellular machinery on biologically relevant timescales, even if the final yield is lower than what equilibrium thermodynamics would dictate. Besides establishing the IAM as the basis for functions of RNA and protein chaperones, our work shows that cellular copy numbers have been adjusted to optimize the rate of native state production of the folded states of RNA and proteins under physiological conditions.\n\nSignificance statementMolecular chaperones have evolved to assist the folding of proteins and RNA, thus avoiding the deleterious consequences of misfolding. Thus, it is expected that increasing chaperone concentration should lead to an enhancement in native yield. While this has been observed in GroEL-mediated protein folding, experiments on Tetrahymena ribozyme folding assisted by CYT-19, surprisingly show the opposite trend. Here, we reconcile these divergent experimental observations by developing a unified stochastic model of chaperone assisted protein and RNA folding. We show that chaperones drive their substrates out of equilibrium, and in the process maximize the rate of native substrate production rather than the absolute yield or the folding rate. In vivo the number of chaperones is regulated to optimize their functions.

biophysics↗

bioRxiv2017

Fractional Field Theory Approach to Protein Folding Dynamics

Understanding biological complexity is one of the most important scientific challenges nowadays. Protein folding is a complex process involving many interactions between the molecules. Fractional calculus is an effective modeling tool for complex systems and processes. In this work we have proposed a new fractional field theoretical approach to protein folding.

biophysics↗

bioRxiv2017

Quantum Calculations Of A Large Section Of The Voltage Sensing Domain Of The Kv1.2 Channel Show That Proton Transfer, Not S4 Motion, Provides The Gating Current

Quantum calculations on much of the voltage sensing domain (VSD) of the Kv1.2 potassium channel (pdb: 3Lut) have been carried out on a 904 atom subset of the VSD, plus 24 water molecules (total, 976 atoms). Those side chains that point away from the center of the VSD were truncated; in all calculations, S1,S2,S3 end atoms were fixed; in some calculations, S4 end atoms were also fixed, while in other calculations they were free. After optimization at Hartree-Fock level, single point calculations of energy were carried out using DFT (B3LYP/6-31G**), allowing accurate energies of different cases to be compared. Open conformations (i.e., zero or positive membrane potentials) are consistent with the known X-ray structure of the open state when the salt bridges in the VSD are not ionized (H+ on the acid), whether S4 end atoms were fixed or free (closer fixed than free). Based on these calculations, the backbone of the S4 segment, free or not, moves no more than 2.5 [A] upon switching from positive to negative membrane potential, and the movement is in the wrong direction for closing the channel. This leaves H+ motion as the principal component of gating current. Groups of 3-5 side chains are important for proton transport, based on the calculations. Our calculations point to a pair of steps in which a proton transfers from a tyrosine, Y266, through arginine (R300), to a glutamate (E183); this would account for approximately 20-25% of the gating charge. The calculated charges on each arginine and glutamate are appreciably less than one. Groupings of five amino acids appear to exchange a proton; the group is bounded by the conserved aromatic F233. Dipole rotations appear to also contribute. Alternate interpretations of experiments usually understood in terms of the standard model are shown to be plausible.

biophysics↗

bioRxiv2017

Spatiotemporal relationships defining the adaptive gating of the bacterial mechanosensitive channel MscS

Adaptive desensitization and inactivation are common properties of most ion channels and receptors. The mechanosensitive channel of small conductance MscS, which serves as a low-threshold osmolyte release valve in most bacteria, is unusual because it slowly inactivates not from the open, but from the resting state under moderate tensions. The manifestation of this mechanism is the channels ability to discriminate the rate of tension application, i.e., to ignore slow tension ramps but fully respond to abruptly applied stimuli. In this work, we present a reconstruction of the landscape for tension-dependent MscS transitions based on patch current kinetics recorded under specially designed pressure protocols. The data are analyzed with a three-state continuous time Markov model of gating, where the tension-dependent transition rates are governed by Arrhenius-type relations. The analysis provides assignments to the intrinsic opening, closing, inactivation, and recovery rates as well as their tension dependencies. These parameters, which define the spatial (areal) distances between the energy wells and the positions of barriers, describe the tension-dependent distribution of the channel population between the three states and quantitatively predict the experimentally observed dynamic pulse and ramp responses. Our solution also provides an analytic expression for the area of the inactivated state in terms of two experimentally accessible parameters: the tension at which inactivation probability is maximized, {gamma}*, and the midpoint tension for activation, {gamma}0.5. The analysis initially performed on Escherichia coli MscS shows its applicability to the previously uncharacterized MscS homolog from Pseudomonas aeruginosa. MscS inactivation minimizes metabolic losses during osmotic permeability response and thus contributes to the environmental fitness of bacteria.

biophysics↗

bioRxiv2017

Coupling of replisome movement with nucleosome dynamics can contribute to the parent-daughter information transfer

Positioning of nucleosomes along the genomic DNA is crucial for many cellular processes that include gene regulation and higher order packaging of chromatin. The question of how nucleosome-positioning information from a parent chromatin gets transferred to the daughter chromatin is highly intriguing. Accounting for experimentally known coupling between replisome movement and nucleosome dynamics, we propose a model that can explain the inheritance of nucleosome positioning. Simulating nucleosome dynamics during replication we argue that short pausing of the replication fork, associated with nucleosome disassembly, can be the event crucial for communicating nucleosome positioning information from parent to daughter. We show that the interplay of timescales between nucleosome disassembly ({tau}p) at the replication fork and nucleosome sliding behind the fork ({tau}s) can give rise to a rich \"phase diagram\" having different inherited patterns of nucleosome organization. Our model predicts that only when{tau} p [≥]{tau} s the daughter chromatin can inherit the precise nucleosome positioning of the parent.

biophysics↗

bioRxiv2017

Distinct Solubility and Cytotoxicity Regimes of Paclitaxel-Loaded Cationic Liposomes at Low and High Drug Content Revealed by Kinetic Phase Behavior and Cancer Cell Viability Studies

Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new Synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for > 3 mol% PTXL (low), days for {approx} 3 mol% PTXL (moderate), and [≥] 20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CLPTXL nanoparticles (NPs) in the distinct CLPTXL solubility regimes reveal an unexpected nonmonotonic dependence of efficacy on PTXL content in NPs delivered at short time scales (hours) after liposome hydration, where we see two distinct high-efficacy regimes at low (< 2 mol%) and high (9 mol%) drug loading. These newly identified high-efficacy regimes flank the membrane solubility limit ({approx} 3 mol%, where efficacy declines), which has been the focus of most previous physicochemical studies (and clinical trials) of PTXL-loaded CLs. At longer times scales (days), CLPTXL NPs with [≥] 3 mol% PTXL lose efficacy while formulations with 1-2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CLPTXL NPs to efficacy.

biophysics↗

bioRxiv2017

Binding Thermodynamics and Kinetics Calculations Using Chemical Host and Guest: A Comprehensive Picture of Molecular Recognition

Understanding the fine balance between changes of entropy and enthalpy and the competition between a guest and water molecules in molecular binding is crucial in fundamental studies and practical applications. Experiments provide measurements. However, illustrating the binding/unbinding processes gives a complete picture of molecular recognition not directly available from experiments, and computational methods bridge the gaps. Here, we investigated guest association/dissociation with {beta}-cyclodextrin ({beta}-CD) by using microsecond-timescale molecular dynamics (MD) simulations, post-analysis and numerical calculations. We computed association and dissociation rate constants, enthalpy, and solvent and solute entropy of binding. All the computed values of kon, koff, {Delta}H, {Delta}S, and {Delta}G using GAFF-CD and q4MD-CD force fields for {beta}-CD could be compared with experimental data directly and agreed reasonably with experiment findings. Both force fields resulted in similar computed {Delta}G from independently computed kinetics rates, {Delta}G=-RTln(kon {middle dot} C{degrees} / k off), and thermodynamics properties, {Delta}G={Delta}H - T{Delta}S. The water entropy calculations show that entropy gain of desolvating water molecules are a major driving force, and both force fields have the same strength of non-polar attractions between solutes and {beta}-CD as well. Water molecules play a crucial role in guest binding to {beta}-CD. However, collective water/{beta}-CD motions could contribute to different computed kon and {Delta}H values by different force fields, mainly because the parameters of {beta}-CD provide different motions of {beta}-CD, hydrogen-bond networks of water molecules in the cavity of free {beta}-CD and the strength of desolvation penalty. As a result, q4MD-CD suggests that guest binding is mostly driven by enthalpy, while GAFF-CD shows that gaining entropy is the major driven force of binding. The study further interprets experiments, deepens our understanding of ligand binding, and suggests strategies for force field parameterization.

biophysics↗

bioRxiv2017

Adsorption and conformation of lysozyme and α-lactalbumin at water-octane interface

As they contain both hydrophobic and hydrophilic amino acids proteins will readily adsorb onto interfaces between water and hydrophobic fluids such as oil. This adsorption normally causes changes in protein structure, which can result in a loss of protein function and irreversible adsorption, leading to the formation of protein interfacial films. While this can be advantageous in some applications (e.g. food technology) in most cases it limits our ability to exploit protein functionality at interfaces. To understand and control protein interfacial adsorption and function it is necessary to understand the microscopic conformation of proteins at liquid interfaces. In this paper molecular dynamics simulations are used to investigate the adsorption and conformation of two similar proteins, lysozyme and -lactalbumin, at a water-octane interface. While they both adsorb onto the interface -lactalbumin does so in a specific orientation, mediated by two amphipathic helices, while lysozyme adsorbs in a non-specific manner. Using replica exchange simulations both proteins are found to possess a number of distinct interfacial conformations, with compact states similar to the solution conformation being most common for both proteins. Decomposing the different contributions to the protein energy at oil-water interfaces, suggests that conformational change for -lactalbumin, unlike lysozyme, is driven by favourable protein-oil interactions. Revealing these differences between the factors that govern conformational change at interfaces in otherwise similar proteins can give insight into the control of protein interfacial adsorption, aggregation, and function.

biophysics↗

bioRxiv2017

A trapped double bond-photoisomerization intermediate in a bacterial photoreceptor

Abbreviations\n\nSummaryThe GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) with an in vivo assembled phycocyanobilin (PCB) chromophore has been crystallized in parental state (1.8 [A]) and photoproduct state (1.86 [A]), identified by 15-Z and 15-E chromophore configuration. Comparison of both structures for the same protein allows precise determination of structural changes after photo-activation. The chromophore photoisomerization causes an outward movement and partial helix formation of a formerly unstructured loop. A tryptophan residue located in this loop, in {pi}-{pi} stacking distance to PCB in the dark state, moves away by 14 [A] opening the binding cleft for the entry of water molecules. Also the in vitro assembled protein (chromophore addition to apo-protein) has been crystallized (1.6 [A] resolution). Most importantly, an intermediate structure was solved (2.1 [A]) with the protein in photoproduct conformation and the chromophore already isomerized into the parental 15-Z configuration, thereby giving insight into chromophore-initiated conformational protein changes.\n\nImpact StatementThis manuscript presents crystal structures of a photochromic protein in both states, before (1.6 [A]) and after (1.9 [A]) the light induced photochemical event with sufficient resolution to allow detailed description of conformational changes of chromophore and protein. The light driven reaction, double bond photoisomerization of a covalently bound bilin chromophore is presented here for the first time. Our results allow determining the impact of the chromophore photochemistry on the protein conformation. In addition, we succeeded in trapping an intermediate carrying the chromophore already in isomerized state with the protein still in unchanged conformation. Absorption spectra of this intermediate clearly demonstrate a color change, thus allowing conclusion that the absorption of phytochromes is predominantly determined by the chromophore conformation alone with only moderate effect of the surrounding protein.\n\nAuthors ContributionsXX, KHZ, and WG designed the experiment. XX generated the protein. AH performed crystallization trials, collected the X-ray diffraction data and solved the structure. All authors contributed in preparing the manuscript.

biophysics↗

bioRxiv2017

Competition of Charge-Mediated and Specific Binding by Peptide-Tagged Cationic Liposome-DNA Nanoparticles In Vitro and In Vivo

Cationic liposome-nucleic acid (CL-NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly(ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL-DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.\n\nGraphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2017

48-spot single-molecule FRET setup with periodic acceptor excitation

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time.\n\nHere we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

biophysics↗

bioRxiv2017

Cofilin Drives Rapid Turnover and Fluidization of Entangled F-actin

The shape of most animal cells is controlled by the actin cortex, a thin, isotropic network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and turnover: Myosin-II molecular motors drive deformations required for cell division, migration, and tissue morphogenesis, while turnover of the molecular components of the actin cortex relax stress and facilitate network reorganization. While many aspects of F-actin network viscoelasticity are well-characterized in the presence and absence of motor activity, a mechanistic understanding of how non-equilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system wherein the steady-state length and turnover rate of F-actin in entangled solutions are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and nucleate filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. Microrheology measurements demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover, and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number in spite of sustained severing.\n\nSignificance StatementWhen an animal cell moves or divides, a disordered network of actin filaments (F-actin) plays a central role in controlling the resulting changes in cell shape. While it is known that continual turnover of F-actin by cofilin-mediated severing aids in reorganization of the cellular cytoskeleton, it is unclear how the turnover of structural elements alters the mechanical properties of the network. Here we show that severing of F-actin by cofilin results in a stress relaxation mechanism in entangled solutions characterized by a single-timescale set by the severing rate. Additionally, we identify ATP hydrolysis and nucleotide-dependent cofilin binding as sufficient ingredients to generate a non-equilibrium steady-state in which asymmetric F-actin disassembly preserves filament number in spite of sustained severing.

biophysics↗

bioRxiv2017

Diffusion as a ruler: modeling kinesin diffusion as a length sensor for intraflagellar transport

An important question in cell biology is whether cells are able to measure size, either whole cell size or organelle size. Perhaps cells have an internal chemical representation of size that can be used to precisely regulate growth, or perhaps size is just an accident that emerges due to constraint of nutrients. The eukaryotic flagellum is an ideal model for studying size sensing and control because its linear geometry makes it essentially one-dimensional, greatly simplifying mathematical modeling. The assembly of flagella is regulated by intraflagellar transport (IFT), in which kinesin motors carry cargo adaptors for flagellar proteins along the flagellum and then deposit them at the tip, lengthening the flagellum. The rate at which IFT motors are recruited to begin transport into the flagellum is anticorrelated with the flagellar length, implying some kind of communication between the base and the tip and possibly indicating that cells contain some mechanism for measuring flagellar length. Although it is possible to imagine many complex scenarios in which additional signaling molecules sense length and carry feedback signals to the cell body to control IFT, might the already-known components of the IFT system be sufficient to allow length dependence of IFT? Here, we investigate a model in which the anterograde kinesin motors unbind after cargo delivery, diffuse back to the base, and are subsequently reused to power entry of new IFT trains into the flagellum. By modeling such a system at three different levels of abstraction we are able to show that the diffusion time of the motors can in principle be sufficient to serve as a proxy for length measurement. In all three implementations, we found that the diffusion model can not only achieve a stable steady-state length without the addition of any other signaling molecules or pathways, but also is able to produce the anticorrelation between length and IFT recruitment rate that has been observed in quantitative imaging studies.

biophysics↗

bioRxiv2017

Partial Unwinding of Transmembrane Helices Facilitates Alternating Access in the Neurotransmitter:Sodium Symporter LeuT

The prokaryotic neurotransmitter:sodium symporter (NSS) LeuT from Aquifex aeolicus is an established structural model for mammalian NSS counterparts. Here, we investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the solution-phase dynamics associated with transition of the transporter between outward- and inwardfacing configurations in a Na+- as well as K+-dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drive LeuT from a substrate-bound, outward-facing occluded conformation towards an inward-facing open state. We thereby envisage that substrate release is facilitated by formation of two distinct solvent pathways, which mediate access to the Na+ and substrate binding sites.\n\nThe neurotransmitter:sodium symporter (NSS) family includes prokaryotic and eukaryotic integral membrane proteins that harness the energy stored in the Na+ concentration gradient to transport solutes across the cell membrane (1, 2). Mammalian NSS proteins play an important role in terminating the neurotransmission in the central nervous system by rapid uptake of neurotransmitters against their concentration gradient into the presynaptic neuron or neighboring glial cells (3-5). Numerous neuropsychiatric conditions are associated with altered function of NSS transporters or low neurotransmitter concentrations in the synaptic cleft (2). Many NSS proteins therefore represent favorable targets for therapeutic drugs that act as potent transport inhibitors to increase neurotransmitter levels at the synaptic junction (2, 6). Despite the importance of mammalian NSS proteins in neurobiology and pharmacology, the molecular mechanisms underlying the transport function of mammalian NSS family members have proven difficult to elucidate by traditional methods as the target proteins are unstable in detergent-solubilized form (7) and difficult to purify in required quantity and purity.\n\nThe prokaryotic NSS protein LeuT from Aquifex aeolicus has emerged as an important structural model to investigate the structure-function relationship of mammalian NSS counterparts (7-9). High-resolution crystal structures of LeuT in distinct functional states (10-13) have established a structural framework and given rise to mechanistic models depicting the substrate transport mechanism (14, 15). LeuT comprises 12 transmembrane segments (TMs) that are interconnected by relatively short extracellular (EL) and intracellular (IL) loops. The first TMs 1-5 share a similar structural repeat with the following TMs 6-10 but are inverted in the membrane plane (10). The specific arrangement of TM domains, also referred to as the 5+5 or LeuT fold (10), has been observed in other NSS proteins (16-18), but also in transporters without sequence homology to LeuT (19, 20), suggesting the possibility of a conserved structural scaffold for many secondary active transporters (8). The primary binding site for the substrate (S1) and residues involved in coordination of two sodium ions (Na1 and Na2) are located approximately halfway across the membrane bilayer in the core of the transporter (10). The characteristic unwound regions of TMs 1 and 6 (providing both TMs with an a- and a b-section) fulfill a dual role in coordinating the sodium ions and forming interactions with the substrate molecule. Individual amino acid residues of TMs 3 and 8 as well as the sodium occupancy in the Na1 site complete the S1 binding pocket.\n\nAccording to the widely embraced alternating access model (21, 22), secondary active transporters isomerize between distinct functional states in a substrate-dependent manner. That is, the substrate binding site is alternatively exposed to either the intracellular or extracellular aqueous environment. X-ray crystallography provided structures of LeuT in outward-facing open (11, 12), outward-facing occluded (10), and inwardfacing open (11) conformations and led to the identification of external and internal gating residues in LeuT and related transporters (23-26). Based on these structural snapshots, it has been hypothesized that local and large-scale structural rearrangements are required to regulate the molecular gates and the outward-to-inward transition of the transporter, respectively (11). Combined evidence from crystallographic, functional, and simulation studies suggest that the underlying allosteric couplings are essential for LeuT to function as a symporter (27). Several key aspects of the transport cycle, in particular the molecular mechanism related to the transition of LeuT to the inward-facing open state, have remained controversial and are the subject of extensive debate (8, 28-30).\n\nHere, we have studied the substrate translocation mechanism of LeuT by measuring the structural dynamics of the protein in solution as a function of time and substrate/ion composition (e.g. leucine, Na+, K+, and Cs+) by local hydrogen/deuterium exchange mass spectrometry (HDX-MS). The exchange of hydrogen to deuterium (HDX) of backbone amides in a protein is dependent on the presence and stability of hydrogen bonds and thus provides a sensitive probe for higher-order structure and dynamics of the target protein in solution (31, 32). HDX-MS is a non-perturbing technique that allows the collection of structural dynamics data along the entire protein backbone in a coherent manner without the need for sequence alterations or changes to the covalent structure of the protein for labeling (33). Briefly, the target protein is diluted into deuterated buffer and labeled for various time intervals. The isotopic exchange reaction is quenched by lowering pH and temperature to approximately 2.5 and 0 {degrees}C, respectively, and the protein subsequently digested using an acid-stable protease (e.g. pepsin). Chromatographic separation and mass analysis of these peptides, in turn, reveal the shift in mass over time (i.e., deuterium uptake) of individual regions of the target protein, which is commonly referred to as local HDX analysis. A more detailed background on the HDX-MS technique and its applications in protein science can be found in several reviews (34-37).\n\nOur HDX-MS measurements provide a detailed map of LeuT regions involved in conformational changes during substrate transport (TMs 1a/1b/2/5/6a/6b/7 and interconnecting loops IL1/EL2/EL3/EL4b) and allow for the first time an unperturbed global view on the structural dynamics associated with the outward-to-inward transition of the wild-type transporter in solution. Of special interest, our acquired HDX data suggest that several helices (TMs 1a/5/6/7 and EL4b) are partially unwound in the course of substrate transport and that these unfolding events are dynamically coupled between individual helices that form the substrate binding site and the cytoplasmic gate. Addition of Na+ or the combination of Na+ and leucine destabilized discrete structural motifs on the extracellular side, stabilized the inner gate of LeuT, and substantially reduced the rate of unfolding in individual TM helices relative to the K+-bound state. We envisage that partial unwinding of TM helices accompanies the outward-to-inward isomerization in LeuT and that the same concept might be relevant to related transporters bearing the LeuT fold, hence extending the general model of NSS transport mechanism. Moreover, we provide additional experimental evidence for a potential role of K+ in the transport cycle as K+ selectively shifted the conformational equilibrium of LeuT in a dose-dependent manner towards an inwardfacing state under physiologically relevant concentrations.

biophysics↗

bioRxiv2017

The role of traction in membrane curvature generation

Curvature of biological membranes can be generated by a variety of molecular mechanisms including protein scaffolding, compositional heterogeneity, and cytoskeletal forces. These mechanisms have the net effect of generating tractions (force per unit length) on the bilayer that are translated into distinct shapes of the membrane. Here, we demonstrate how the local shape of the membrane can be used to infer the traction acting locally on the membrane. We show that buds and tubes, two common membrane deformations studied in trafficking processes, have different traction distributions along the membrane and that these tractions are specific to the molecular mechanism used to generate these shapes. Furthermore, we show that the magnitude of an axial force applied to the membrane as well as that of an effective line tension can be calculated from these tractions. Finally, we consider the sensitivity of these quantities with respect to uncertainties in material properties and follow with a discussion on sources of uncertainty in membrane shape.

biophysics↗

bioRxiv2017

Pivot-and-bond model explains microtubule bundle formation

During mitosis, bundles of microtubules form a spindle, but the physical mechanism of bundle formation is still not known. Here we show that random angular movement of microtubules around the spindle pole and forces exerted by passive cross-linking proteins are sufficient for the formation of stable microtubule bundles. We test these predictions by experiments in wild-type and ase1{Delta} fission yeast cells. In conclusion, the angular motion drives the alignment of microtubules, which in turn allows the cross-linking proteins to connect the microtubules into a stable bundle.

biophysics↗