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bioRxiv2018

BAR scaffolds drive membrane fission by crowding disordered domains

Cylindrical protein scaffolds are thought to stabilize membrane tubules, preventing membrane fission. In contrast, Snead et al. find that when scaffold proteins assemble, bulky disordered domains within them become acutely concentrated, generating steric pressure that destabilizes tubules, driving fission.\n\nAbstractCellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysinrvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of BAR domains in isolation, the current paradigm is that they polymerize into cylindrical scaffolds that stabilize lipid tubules, preventing membrane fission. But in nature BAR domains are often part of multi-domain proteins that contain large intrinsically-disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data challenge the idea that cellular membranes adopt the curvature of BAR scaffolds, suggesting instead that the ability to concentrate disordered domains is the key requirement for membrane remodeling and fission by BAR domain-containing proteins.

biophysics↗

bioRxiv2018

Monomeric streptavidin: a versatile regenerative handle for force spectroscopy

Most avidin-based handles in force spectroscopy are tetravalent biotin binders. Tetravalency presents two issues: multiple pulling geometries as well as multiple targets bound simultaneously. Additionally, such tetravalent handles require elaborate purification protocols in order to reassemble. A stoichiometric, monomeric variant of streptavidin (mcSA2) had been engineered previously. It is readily expressed and purified, and it binds biotin with a nanomolar KD. For atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), we fused the monomeric streptavidin with a small protein domain as an experimental fingerprint and to improve solubility. A ybbR-tag was additionally included for covalent site-specific tethering. Rupture forces of the mcSA2:biotin complex were found to be in a comparable range above 150 pN at force loading rates of 1E4 pN/s as for previously published, tetravalent streptavidin:biotin systems. Additionally, when tethering mcSA2 from its C-terminus, rupture forces were found to be slightly higher than when tethered N-terminally. Due to its monomeric nature, mcSA2 could also be chemically denatured and subsequently refolded - and thus regenerated during an experiment, in case the handle gets misfolded or clogged. We show that mcSA2 features a straightforward expression and purification with flexible tags, high stability, regeneration possibilities and an unambiguous pulling geometry. Combined, these properties establish mcSA2 as a reliable handle for single-molecule force spectroscopy.

biophysics↗

bioRxiv2018

Dual roles of electrostatic-steering and conformational dynamics in the binding of calcineurin’s intrinsically-disordered recognition domain to calmodulin

calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The CaN regulatory domain (RD) is responsible for regulating the enzymes phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorderto-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating CaM binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder cofacilitate or hinder CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.

biophysics↗

bioRxiv2018

Effect of Ca2+ on the promiscuous target-protein binding mechanism of calmodulin

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling path- ways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question.\n\nHere, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity.\n\nThis work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.\n\nAuthor summaryCalmodulin is a protein involved in the regulation of a variety of cell signaling pathways. It acts by making usually calcium-insensitive proteins sensitive to changes in the calcium concentration inside the cell. Its two lobes bind calcium and allow the energetically unfavorable exposure of hydrophobic residues to the aqueous environment which can then bind target proteins. The mechanisms behind the simultaneous specificity and variation of target protein binding is yet unknown but will aid understanding of the calcium-signaling and regulation that occur in many of our cellular processes.\n\nHere, we used molecular dynamics simulations and data analysis techniques to investigate what effect calcium has on the binding modes of calmodulin. The simulations and analyses allow us to observe and differentiate specific states. One domain of calmodulin is shown to be selective with binding involving short- distance interactions between hydrophobic residues, while the other binds target proteins through a more flexible mechanism involving long-distance electrostatic interactions.

biophysics↗

bioRxiv2018

LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional single microtubule imaging

Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be resolved with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-resolution 3D microscopy.\n\nOCIS codes(180.3170) Interference microscopy; (120.4570) Optical design of instruments (350.4855); Optical tweezers or optical manipulation.

biophysics↗

bioRxiv2018

Tuning molecular motor transport through cytoskeletal filament network organization

The interaction of motor proteins with intracellular filaments is required for transport processes and force generation in cells. Within a cell, crosslinking proteins organize cytoskeletal filaments both temporally and spatially to create dynamic, and structurally diverse networks. The architecture of these networks changes both the mechanics as well as the transport dynamics; however, the effects on transport are less well understood. Here, we compare the transport dynamics of myosin II motor proteins moving on model cytoskeletal networks created by common crosslinking proteins. We observe that motor dynamics change predictably based on the microstructure of the underlying networks and discuss how this can be utilized by cells to achieve specific transport goals.

biophysics↗

bioRxiv2018

Single-molecular and Ensemble-level Oscillations of Cyanobacterial Circadian Clock

When three cyanobacterial proteins, KaiA, KaiB, and KaiC, are incubated with ATP in vitro, the phosphorylation level of KaiC hexamers shows stable oscillation with approximately 24 h period. In order to understand this KaiABC clockwork, we need to analyze both the macroscopic synchronization of a large number of KaiC hexamers and the microscopic reactions and structural changes in individual KaiC molecules. In the present paper, we explain two coarse-grained theoretical models, the many-molecule (MM) model and the single-molecule (SM) model, to bridge the gap between macroscopic and microscopic understandings. In the simulation results with these models, ATP hydrolysis drives oscillation of individual KaiC hexamers and ATP hydrolysis is necessary for synchronizing oscillations of a large number of KaiC hexamers. Sensitive temperature dependence of the lifetime of the ADP bound state in the CI domain of KaiC hexamers makes the oscillation period temperature insensitive. ATPase activity is correlated to the frequency of phosphorylation oscillation in the single molecule of KaiC hexamer, which should be the origin of the observed ensemble-level correlation between the ATPase activity and the frequency of phosphorylation oscillation. Thus, the simulation results with the MM and SM models suggest that ATP hydrolysis randomly occurring in each CI domain of individual KaiC hexamers is a key process for oscillatory behaviors of the ensemble of many KaiC hexamers.\n\nSignificance StatementCyanobacterial proteins, KaiA, KaiB, and KaiC, can reconstitute a circadian clock when they are incubated with ATP in vitro. In order to understand this prototypical oscillator, we need to analyze both synchronization of a macroscopically large number of oscillating molecules and microscopic reactions in individual molecules. We introduced two theoretical models to unify macroscopic and microscopic viewpoints. Simulation results suggest that ATP hydrolysis is necessary for synchronization and temperature compensation and that ATPase activity is correlated to the oscillation frequency in individual molecules. Thus, ATP hydrolysis randomly occurring in individual molecules should determine important features of the ensemble-level oscillation.

biophysics↗

bioRxiv2018

Defining a canonical ligand-binding pocket in the orphan nuclear receptor Nurr1

Nuclear receptor related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor that is considered to function without a canonical ligand-binding pocket. A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other nuclear receptors with solvent accessible apo-protein ligand-binding pockets bind synthetic and natural ligands. Using solution NMR spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show here that the putative canonical ligand-binding pocket in the Nurr1 LBD is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystalized conformation to allow binding of unsaturated fatty acids. These findings should stimulate future studies to probe the ligandability and druggability of Nurr1 for both endogenous and synthetic ligands, which could lead to new therapeutics for Nurr1-related diseases, including Parkinsons disease and schizophrenia.

biophysics↗

bioRxiv2018

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from pre-formed, non-growing MTs, but rapidly forms MT asters in the presence of soluble tubulin. HSET binds soluble (non-polymer) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin \"clusters\" containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from non-growing MTs. We then show that excess soluble tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble tubulin.

biophysics↗

bioRxiv2018

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

RNA polymerase (RNAP) contains a mobile structural module, the \"clamp,\" that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including loading of promoter DNA into the RNAP active-center cleft, unwinding of promoter DNA, transcription elongation complex stability, transcription pausing, and transcription termination. Crystal structures and single-molecule FRET studies establish that the clamp can adopt open and closed conformational states; however, the occurrence, pathway, and kinetics of transitions between clamp states have been unclear. Using single-molecule FRET (smFRET) on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme exists in three distinct conformational states: the previously defined open state, the previously defined closed state, and a previously undefined partly closed state. smFRET time-traces show dynamic transitions between open, partly closed, and closed states on the 0.1-1 second time-scale. Similar analyses of transcription initiation complexes confirm that the RNAP clamp is closed in the catalytically competent transcription initiation complex and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, in contrast to in RNAP holoenzyme, the clamp does not interconvert between the closed state and other states. The stringent-response alarmone ppGpp selectively stabilizes the partly-closed-clamp state, inhibiting interconversion between the partly closed state and the open state. The methods of this report should allow elucidation of clamp conformation and dynamics during all phases of transcription.\n\nSIGNIFICANCE STATEMENTThe clamp forms a pincer of the RNA polymerase \"crab-claw\" structure, and adopts many conformations with poorly understood function and dynamics. By measuring distances within single surface-attached molecules, we observe directly the motions of the clamp and show that it adopts an open, a closed, and a partly closed state; the last state is stabilized by a sensor of bacterial starvation, linking the clamp conformation to the mechanisms used by bacteria to counteract stress. We also show that the clamp remains closed in many transcription steps, as well as in the presence of a specific antibiotic. Our approach can monitor clamp motions throughout transcription and offers insight on how antibiotics can stop pathogens by blocking their RNA polymerase movements.

biophysics↗

bioRxiv2018

Three-dimensional cell geometry controls excitable membrane signaling in Dictyotelium cells

Phosphatidylinositol (3,4,5)-trisphosphate (PtdInsP3) is known to propagate as waves on the plasma membrane and is related to the membrane protrusive activities in Dictyostelium and mammalian cells. While there have been a few attempts to study the three-dimensional dynamics of these processes, most studies have focused on the dynamics extracted from single focal planes. However, the relation between the dynamics and three-dimensional cell shape remains elusive, due to the lack of signaling information about the unobserved part of the membrane. Here we show that PtdInsP3 wave dynamics are directly regulated by the three-dimensional geometry - size and shape - of the plasma membrane. By introducing an analysis method that extracts the three-dimensional spatiotemporal activities on the entire cell membrane, we show that PtdInsP3 waves self-regulate their dynamics within the confined membrane area. This leads to changes in speed, orientation and pattern evolution, following the underlying excitability of the signal transduction system. Our findings emphasize the role of the plasma membrane topology in reaction-diffusion driven biological systems and indicate its importance in other mammalian systems.

biophysics↗

bioRxiv2018

Expansion stimulated emission depletion microscopy (ExSTED)

Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a [~]10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample [~]4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and [~]50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

biophysics↗

bioRxiv2018

Variational algorithms for analyzing noisy multi-state diffusion trajectories

Single particle tracking offers a non-invasive high-resolution probe of biomolecular reactions inside living cells. However, efficient data analysis methods that correctly account for various noise soures are needed to realize the full quantitative potential of the method. We report new algorithms for hidden Markov based analysis of single particle tracking data, which incorporate most sources of experimental noise, including heterogeneuous localization errors and missing positions. Compared to previous implementations, the algorithms offer significant speed-ups, support for a wider range of inference methods, and a simple user interface. This will enable more advanced and exploratory quantitative analysis of single particle tracking data.

biophysics↗

bioRxiv2018

Volvox barberi flocks, forming near-optimal, two-dimensional, polydisperse lattice packings

Volvox barberi is a multicellular green alga forming spherical colonies of 10000-50000 differentiated somatic and germ cells. Here, I show that these colonies actively self-organize over minutes into \"flocks\" that can contain more than 100 colonies moving and rotating collectively for hours. The colonies in flocks form two-dimensional, irregular, \"active crystals\", with lattice angles and colony diameters both following log-normal distributions. Comparison with a dynamical simulation of soft spheres with diameters matched to the Volvox samples, and a weak long-range attractive force, show that the Volvox flocks achieve optimal random close-packing. A dye tracer in the Volvox medium revealed large hydrodynamic vortices generated by colony and flock rotations, providing a likely source of the forces leading to flocking and optimal packing.

biophysics↗

bioRxiv2018

Conformational flexibility of collagen molecules: composition and environmental effects

The predominant structural protein in vertebrates is collagen, which plays a key role in extracellular matrix and connective tissue mechanics. Despite its prevalence and physical importance in biology, the mechanical properties of molecular collagen are far from established. The flexibility of its triple helix is unresolved, with descriptions from different experimental techniques ranging from flexible to semirigid. Furthermore, it is unknown how collagen type (homo-vs. heterotrimeric) and source (tissue-derived vs. recombinant) influence flexibility. Using SmarTrace, a chain tracing algorithm we devised, we performed statistical analysis of collagen conformations collected with atomic force microscopy (AFM) to determine the proteins mechanical properties. Our results show that types I, II and III collagens - the key fibrillar varieties - exhibit molecular flexibilities that are very similar. However, collagen conformations are strongly modulated by salt, transitioning from compact to extended as KCl concentration increases, in both neutral and acidic pH. While analysis with a standard worm-like chain model suggests that the persistence length of collagen can attain almost any value within the literature range, closer inspection reveals that this modulation of collagens conformational behaviour is not due to changes in flexibility, but rather arises from the induction of curvature (either intrinsic or induced by interactions with the mica surface). By modifying standard polymer theory to include innate curvature, we show that collagen behaves as an equilibrated curved worm-like chain (cWLC) in two dimensions. Analysis within the cWLC model shows that collagens curvature depends strongly on pH and salt, while its persistence length does not. Thus, we find that triple-helical collagen is well described as semiflexible, irrespective of source, type, pH and salt environment. These results demonstrate that collagen is more flexible than its conventional description as a rigid rod, which may have implications for its cellular processing and secretion.

biophysics↗

bioRxiv2018

Effect of 5-trans isomer of arachidonic acid on model liposomal membranes studied by a combined simulation and experimental approach

Unsaturated fatty acids are found in humans predominantly in the cis configuration. Fatty acids in the trans configuration are primarily the result of human processing (trans fats), but can also be formed endogenously by radical stress. The cis-trans isomerization of fatty acids by free radicals could be connected to several pathologies. Trans fats have been linked to an increased risk of coronary artery disease; however, the reasons for the resulting pathogenesis remain unclear. Here, we investigate the effect of a mono trans isomer of arachidonic acid (C20:4-5trans,8cis,11cis,14cis) produced by free radicals in physiological concentration on a model erythrocyte membrane using a combined experimental and theoretical approach. Molecular Dynamics (MD) simulations of two model lipid bilayers containing arachidonic acid and its 5-trans isomer in 3% mol. were carried out for this purpose. The 5-trans isomer formation in the phospholipids was catalyzed by HOCH2CH2S* radicals, generated from the corresponding thiol by {gamma}-irradiation, in multilamellar vesicles (MLVs) of SAPC. Large unilamellar vesicles were made by the extrusion method (LUVET) as a biomimetic model for cis-trans isomerization. Atomic Force Microscopy and Dynamic Light Scattering were used to measure the average size, morphology, and the z-potential of the liposomes. Both results from MD simulations and experiments are in agreement and indicate that the two model membranes display different physicochemical properties in that the bilayers containing the trans fatty acids were more ordered and more rigid than those containing solely the cis arachidonic acid. Correspondingly, the average size of the liposomes containing trans isomers was smaller than the ones without.

biophysics↗

bioRxiv2018

SESCA: Predicting the Circular Dichroism Spectra of Proteins from Molecular Structure

Circular dichroism spectroscopy is a highly sensitive, but low-resolution technique to study the structure of proteins. Combined with molecular modelling or other complementary techniques, CD spectroscopy can provide essential information at higher resolution. To this end, we introduce a new computational method to calculate the electronic circular dichroism spectra of proteins from a structural model or ensemble using the average secondary structure composition and a pre-calculated set of basis spectra. We compared the predictive power of our method to existing algorithms - namely DichroCalc and PDB2CD - and found that it predicts CD spectra more accurately, with a 50% smaller average deviation from the measured CD spectra. Our results indicate that the derived basis sets are robust to experimental errors in the reference spectra and to the choice of the secondary structure classification algorithm. For over 80% of the globular reference proteins, our basis sets accurately predict the experimental spectrum solely from their secondary structure composition. For the remaining 20%, correcting for intensity normalization considerably improves the prediction power. Additionally, we show that the predictions for short peptides and intrinsically disordered proteins strongly benefit from accounting for side-chain contributions and structural flexibility. Table Of Content Graphics O_TBL View this table: org.highwire.dtl.DTLVardef@1a1d73forg.highwire.dtl.DTLVardef@3aaa8forg.highwire.dtl.DTLVardef@193c090org.highwire.dtl.DTLVardef@1474f92org.highwire.dtl.DTLVardef@b920da_HPS_FORMAT_FIGEXP M_TBL C_TBL

biophysics↗

bioRxiv2018

Pollen Patterns Form from Modulated Phases

Pollen grains are known for their impressive variety of species-specific, microscale surface patterning. Despite having similar biological developmental steps, pollen grain surface features are remarkably geometrically varied. Previous work suggests that a physical process may drive this pattern formation and that the observed diversity of patterns can be explained by viewing pollen pattern development as a phase transition to a spatially modulated phase. Several studies have shown that the polysaccharide material of plant cell walls undergoes phase separation in the absence of cross-linking stabilizers of the mixed phase. Here we show experimental evidence that phase separation of the extracellular polysaccharide material (primexine) during pollen cell development leads to a spatially modulated phase. The spatial pattern of this phase-separated primexine is also mechanically coupled to the undulation of the pollen cell membrane. The resulting patterned pools of denser primexine form the negative template of the ultimate sites of sporopollenin deposition, leading to the final micropattern observed in the mature pollen. We then present a general physical model of pattern formation via modulated phases. Using analytical and numerical techniques, we find that most of the pollen micropatterns observed in biological evolution could result from a physical process of modulated phases. However, an analysis of the relative rates of transitions from states that are equilibrated to or from states that are not equilibrated suggests that while equilibrium states of this process have occurred throughout evolutionary history, there has been no particular evolutionary selection for symmetric, equilibrated states.

biophysics↗