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bioRxiv2021

Structure Insight into Photosystem I Octamer from Cyanobacteria

Diversity of photosystem oligomers is essential to understand how photosynthetic organisms adopted to light conditions. Given by the structural and physiological significance, the assemblies of PSI supercomplex is of great interest in both chloroplast and cyanobacteria recently. In this study, two novel photosystem I supercomplexes were isolated for the first time from the low light incubated culture of filamentous cyanobacterium Anabaena sp. PCC 7120. These complexes were defined as PSI hexamers and octamers through biochemical and biophysical characterization. Their 77K emission spectra indicated that the red forms of chlorophylls seemed not to be affected during oligomerization. By cryo-EM single particle analysis, a near-atomic (7.0 [A]) resolution structure of PSI octamer was resolved, and the molecular assemblies of stable PSI octamer was revealed.

biophysics↗

bioRxiv2021

A Trypanosoma brucei orphan kinesin employs a convergent microtubule organization strategy to complete cytokinesis

Many single-celled eukaryotes have complex cell morphologies defined by cytoskeletal elements comprising microtubules arranged into higher-order structures. Trypanosoma brucei (T. brucei) cell polarity is mediated by a parallel array of microtubules that underlie the plasma membrane and define the auger-like shape of the parasite. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the division plane and is essential for the completion of cytokinesis. To gain mechanistic insight into how this novel kinesin functions to complete cleavage furrow ingression, we characterized the biophysical properties of the KLIF motor domain in vitro. We found that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked in an antiparallel orientation are translocated relative to one another by KLIF, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we found that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new posterior, which is a unique requirement for the completion of cytokinesis in T. brucei.

biophysics↗

bioRxiv2021

Self-organization of kinetochore-fibers in human mitotic spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

biophysics↗

bioRxiv2021

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.

The pathogenic fungus Candida albicans causes severe invasive candidiasis. C. albicans infection requires the action of the virulence factor Candidalysin (CL), which damages the plasma membrane of the target human cells. However, the molecular mechanism that CL uses to permeabilize membranes is poorly understood. We employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism. Unexpectedly, it was observed that CL readily assembles into linear polymers in solution. The basic structural unit in polymer formation is a CL 8-mer, which is sequentially added into a string configuration. Finally, the linear polymers can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in different synthetic lipid membranes systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed the co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL molecules assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that might stabilize pore formation to achieve more robust cellular damage. Taken together, the data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

biophysics↗

bioRxiv2021

Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. However, interactions with ubiquitin or multivalent K48-linked chains eliminate LLPS. Here, we found that, although some polyubiquitin chain types (K11-Ub4 and K48-Ub4) did generally inhibit UBQLN2 LLPS, others (K63-Ub4, M1-Ub4 and a designed tetrameric ubiquitin construct) significantly enhanced LLPS. Using nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in chain conformations, but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin binding surface significantly promoted UBQLN2 LLPS by enabling a switch between homotypically to partially heterotypically-driven phase separation. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic phase separation with ubiquitin-binding shuttles and adaptors. HighlightsO_LIUbiquitin or short polyubiquitin chains bind to phase separation-driving stickers on UBQLN2 and inhibit its phase separation whereas longer chains provide the multivalency needed to enhance UBQLN2 phase separation. C_LIO_LIPhase separation of UBQLN2 is promoted over a wide range of Ub:UBQLN2 ratios in the presence of extended M1- and K63-linked Ub4 chains, but not compact K11- and K48-linked Ub4 chains. C_LIO_LIChain conformation and accessibility of the Ub interacting surface is a driving factor of UBQLN2/polyUb co-phase separation. C_LIO_LIUBQLN2 condensates assemble during in vitro enzymatic assembly of K63-linked polyUb chains as free ubiquitin is reduced. C_LI

biophysics↗

bioRxiv2021

Key Factors Governing Initial Stages of Lipid Droplet Formation

Lipid droplets (LDs) are neutral lipid storage organelles surrounded by a phospholipid (PL) monolayer. LD biogenesis from the endoplasmic reticulum (ER) is driven by phase separation of neutral lipids, overcoming surface tension and membrane deformation. However, the core biophysics of the initial steps of LD formation remain relatively poorly understood. Here, we use a tunable, phenomenological coarse-grained (CG) model to study triacylglycerol (TG) nucleation in a bilayer membrane. We show that PL rigidity has a strong influence on TG lensing and membrane remodeling: When membrane rigidity increases, TG clusters remain more planar with high anisotropy but a minor degree of phase nucleation. This finding is confirmed by free energy sampling simulations that calculate the potential of mean force (PMF) as a function of the degree of nucleation and anisotropy. We also show that asymmetric tension, controlled by the number of PLs on each membrane leaflet, determines the budding direction. A TG lens buds in the direction of the monolayer containing excess PLs to allow for better PL coverage of TG, consistent with reported experiments. Finally, two governing mechanisms of the LD growth, Ostwald ripening and merging, are observed. Taken together, this study characterizes the interplay between two thermodynamic quantities during the initial LD phases, the TG bulk free energy and membrane remodeling energy.

biophysics↗

bioRxiv2021

Contingency and selection in mitochondrial genome dynamics

Eukaryotic cells contain numerous copies of mitochondrial DNA (mtDNA), allowing for the coexistence of mutant and wild-type mtDNA in individual cells. The fate of mutant mtDNA depends on their relative replicative fitness within cells and the resulting cellular fitness within populations of cells. Yet the dynamics of the generation of mutant mtDNA and features that inform their fitness remain unaddressed. Here we utilize long read single-molecule sequencing to track mtDNA mutational trajectories in Saccharomyces cerevisiae. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that may be applicable in more complex organisms.

biophysics↗

bioRxiv2021

Systematic Interrogation of Protein Refolding Under Cellular-Like Conditions

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chains intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting proteins safe passage to their native states; however, the complexity of this medium has generally precluded biophysical techniques from interrogating protein folding under cellular-like conditions for single proteins, let alone entire proteomes. Here, we develop a limited-proteolysis mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding E. coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data, and is particularly important for restoring acidic proteins and proteins with three to five domains, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than indirect measures like binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones both unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. The data support a model in which chaperone-nonrefolders have evolved to fold efficiently once and only once, co-translationally, and remain kinetically trapped in their native conformations.

biophysics↗

bioRxiv2021

Laser Ablation and Fluid Flows Show a Single Force Mechanism Governs Spindle Positioning

Few techniques are available for elucidating the nature of forces that drive subcellular behaviors. Here we develop two complementary ones: 1) femtosecond stereotactic laser ablation (FESLA), which rapidly creates complex cuts of subcellular structures, thereby allowing precise dissection of when, where, and in what direction forces are generated; and 2) assessment of subcellular fluid flows, by comparing direct flow measurements, using microinjected fluorescent nanodiamonds, to large-scale fluid-structure simulations of different models of force transduction. We apply these to study centrosomes in Caenorhabditis elegans early embryos, and use the data to construct a biophysically-based model of centrosome dynamics. Taken together, we demonstrate that cortical pulling forces provide a general explanation for many behaviors mediated by centrosomes, including pronuclear migration/centration and rotation, metaphase spindle positioning, asymmetric spindle elongation and spindle oscillations. In sum, this work establishes new methodologies for disentangling the forces responsible for cell biological phenomena.

biophysics↗

bioRxiv2021

Computational Compensatory Mutation Discovery Approach: Predicting a PARP1 Variant Rescue Mutation

The prediction of protein mutations that affect function may be exploited for multiple uses. In the context of disease variants, the prediction of compensatory mutations that reestablish functional phenotypes could aid in the development of genetic therapies. In this work, we present an integrated approach that combines coevolutionary analysis and molecular dynamics (MD) simulations to discover functional compensatory mutations. This approach is employed to investigate possible rescue mutations of a poly(ADP-ribose) polymerase 1 (PARP1) variant, PARP1 V762A, associated with lung cancer and follicular lymphoma. MD simulations show PARP1 V762A exhibits noticeable changes in structural and dynamical behavior compared with wild type PARP1. Our integrated approach predicts A755E as a possible compensatory mutation based on coevolutionary information, and molecular simulations indicate that the PARP1 A755E/V762A double mutant exhibits similar structural and dynamical behavior to WT PARP1. Our methodology can be broadly applied to a large number of systems where single nucleotide polymorphisms (SNPs) have been identified as connected to disease and can shed light on the biophysical effects of such changes as well as provide a way to discover potential mutants that could restore wild type-like functionality. This can in turn be further utilized in the design of molecular therapeutics that aim to mimic such compensatory effect. Significance StatementDiscovering protein mutations with desired phenotypes can be challenging due to its combinatorial nature. Herein we employ a methodology combining gene SNP association to disease, direct coupling analysis and molecular dynamics simulations to systematically predict rescue mutations. Our workflow identifies A755E as a potential rescue for the PARP1 V762A mutation, which has been associated with cancer. This methodology is general and can be applied broadly.

biophysics↗

bioRxiv2021

Sampling the conformational landscapes of transporters and receptors with AlphaFold2

Equilibrium fluctuations and triggered conformational changes often underlie the functional cycles of membrane proteins. For example, transporters mediate the passage of molecules across cell membranes by alternating between inward-facing (IF) and outward-facing (OF) states, while receptors undergo intracellular structural rearrangements that initiate signaling cascades. Although the conformational plasticity of these proteins has historically posed a challenge for traditional de novo protein structure prediction pipelines, the recent success of AlphaFold2 (AF2) in CASP14 culminated in the modeling of a transporter in multiple conformations to high accuracy. Given that AF2 was designed to predict static structures of proteins, it remains unclear if this result represents an underexplored capability to accurately predict multiple conformations and/or structural heterogeneity. Here, we present an approach to drive AF2 to sample alternative conformations of topologically diverse transporters and G-protein coupled receptors (GPCRs) that are absent from the AF2 training set. Whereas models generated using the default AF2 pipeline are conformationally homogeneous and nearly identical to one another, reducing the depth of the input multiple sequence alignments (MSAs) by stochastic subsampling led to the generation of accurate models in multiple conformations. In our benchmark, these conformations spanned the range between two experimental structures of interest, with models at the extremes of these conformational distributions observed to be among the most accurate (average template modeling (TM)-score of 0.94). These results suggest a straightforward approach to identifying native-like alternative states, while also highlighting the need for the next generation of deep learning algorithms to be designed to predict ensembles of biophysically relevant states.

biophysics↗

bioRxiv2021

Influenza A virus hemagglutinin prevents extensive membrane damage upon dehydration

While the molecular mechanisms of virus infectivity are rather well known, the detailed consequences of environmental factors on virus biophysical properties are poorly understood. Seasonal influenza outbreaks are usually connected to the low winter temperature, but also to the low relative air humidity. Indeed, transmission rates increase in cold regions during winter. While low temperature must slow degradation processes, the role of low humidity is not clear. We studied the effect of relative humidity on a model of influenza A H1N1 virus envelope, a supported lipid bilayer containing the surface glycoprotein hemagglutinin (HA), which is present in the viral envelope in very high density. For complete cycles of hydration, dehydration and rehydration, we evaluate the membrane properties in terms of structure and dynamics, which we assess by combining confocal fluorescence microscopy, raster image correlation spectroscopy, line-scan fluorescence correlation spectroscopy and atomic force microscopy. Our findings indicate that the presence of HA prevents macroscopic membrane damage after dehydration. Without HA, fast membrane disruption is followed by irreversible loss of lipid and protein mobility. Although our model is principally limited by the membrane composition, the macroscopic effects of HA under dehydration stress reveal new insights on the stability of the virus at low relative humidity.

biophysics↗

bioRxiv2021

Single virus assay reveals membrane determinants and mechanistic features of Sendai virus binding

Sendai virus (SeV, formally murine respirovirus) is a membrane-enveloped, negative sense RNA virus in the Paramyxoviridae family, and is closely related to human parainfluenza viruses. SeV has long been utilized as a model paramyxovirus and has recently gained attention as a viral vector candidate for both laboratory and clinical applications. To infect host cells, SeV must first bind to sialic-acid glycolipid or glycoprotein receptors on the host cell surface via its hemagglutinin-neuraminidase (HN) protein. Receptor binding induces a conformational change in HN, which allosterically triggers the viral fusion (F) protein to catalyze membrane fusion. While it is known that SeV binds to 2,3-linked sialic acid receptors, and there has been some study into the chemical requirements of those receptors, key mechanistic features of SeV binding remain unknown, in part because traditional approaches often convolve binding and fusion. Here, we develop and employ a fluorescence microscopy-based assay to observe SeV binding to supported lipid bilayers (SLBs) at the single particle level, which easily disentangles binding from fusion. Using this assay, we investigate mechanistic questions of SeV binding. We identify chemical structural features of ganglioside receptors that influence viral binding and demonstrate that binding is cooperative with respect to receptor density. We measure the characteristic decay time of unbinding and provide evidence supporting a "rolling" mechanism of viral mobility following receptor binding. We also study the dependence of binding on target cholesterol concentration. Interestingly, we find that while SeV binding shows striking parallels in cooperative binding with a prior report of Influenza A virus, it does not demonstrate a similar sensitivity to cholesterol concentration and receptor nano-cluster formation. STATEMENT OF SIGNIFICANCEParamyxoviruses are a family of membrane-enveloped viruses with many notable human and animal pathogens. In this study, we develop and use an assay to observe the initial step of infection - virus binding to the host membrane - for Sendai virus, the prototypical paramyxovirus, at the single virus level. This assay uses cell membrane mimics - supported lipid bilayers - as targets for virus binding to enable easy control of the membrane components with which the virus interacts. Using our assay, we gain insight into basic biophysical questions about Sendai virus binding, including the chemical characteristics of the receptor, the cooperative nature of binding, the influence of cholesterol, and the mechanism of viral mobility following binding.

biophysics↗

bioRxiv2021

Aquaglyceroporin AQP7's affinity for its substrate glycerol

AQP7 is one of the four human aquaglyceroporins that facilitate glycerol transport across the cell membrane, a biophysical process that is essential in human physiology. Therefore, it is interesting to compute AQP7s affinity for its substrate (glycerol) with reasonable certainty to compare with the experimental data suggesting high affinity in contrast with most computational studies predicting low affinity. In this study aimed at computing the AQP7-glycerol affinity with high confidence, we implemented a direct computation of the affinity from unbiased equilibrium molecular dynamics (MD) simulations of three all-atom systems constituted with 0.16M, 4.32M, and 10.23M atoms, respectively. These three sets of simulations manifested a fundamental physics law that the intrinsic fluctuations of pressure in a system are inversely proportional to the system size (the number of atoms in it). These simulations showed that the computed values of glycerol-AQP7 affinity are dependent upon the system size (the inverse affinity estimations were, respectively, 47.3 mM, 1.6 mM, and 0.92 mM for the three model systems). In this, we obtained a lower bound for the AQP7-glycerol affinity (an upper bound for the dissociation constant). Namely, the AQP7-glycerol affinity is stronger than 1087/M (the dissociation constant is less than 0.92 mM). Additionally, we conducted hyper steered MD (hSMD) simulations to map out the Gibbs free-energy profile. From the free-energy profile, we produced an independent computation of the AQP7-glycerol dissociation constant being approximately 0.18 mM. Table of contents entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/469753v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@420c90org.highwire.dtl.DTLVardef@46d27borg.highwire.dtl.DTLVardef@1a4b269org.highwire.dtl.DTLVardef@1bd3979_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

On the reproductive mechanisms of Gram-positive protocells

Prokaryotes are hypothesized to have evolved from more primitive protocells. Unlike present-day cells, protocells are thought to have been devoid of complex molecular biological processes. They are believed to have mediated reproduction entirely by biophysical forces under favorable environmental conditions. Despite this proposition, little is known about the actual mechanism of their reproduction. To understand the reproduction process of protocells in their native habitat, here we used a top-down approach to transform bacterial cells into a primitive lipid vesicle-like state. Given that environmental conditions are thought to have played an essential role in mediating protocell reproduction, we then studied these cells under the presumed environmental conditions of Archaean Eon Earth. Even in the absence of functioning biological processes, cells in our study reproduced in a defined sequence of steps, always leading to the formation of viable daughter cells. Their reproduction mechanism can be explained by the interaction between intracellular metabolism, physicochemical properties of cell constituents, and, most importantly, environmental conditions. Given the simplicity of this reproduction mechanism and its suitability to environmental conditions of early Earth, we propose that protocells reproduced by this process. Moreover, this method of reproduction is also in tune with the earlier theoretical propositions on protocells, the results of the top- down approach of building a minimal cell, and the paleontological record of the Achaean Eon. Our study is the first to bridge the gap between non-living systems like lipid vesicles, living cells, and the paleontology of the Archaean Eon.

biophysics↗

bioRxiv2021

ABC Transporters are billion-year-old Maxwell Demons

ABC transporters are a broad family of biological machines, found in most prokaryotic and eukaryotic cells, performing the crucial import or export of substrates through both plasma and organellar membranes, and maintaining a steady concentration gradient driven by ATP hydrolysis. Building upon the present biophysical and biochemical characterization of ABC transporters, we propose here a model whose solution reveals that these machines are an exact molecular realization of the Maxwell Demon, a century-old abstract device that uses an energy source to drive systems away from thermodynamic equilibrium. In particular, the Maxwell Demon does not perform any direct mechanical work on the system, but simply selects which spontaneous processes to allow and which ones to forbid based on information that it collects and processes. In the molecular model introduced here, the different information-processing steps that characterize Maxwell Demons (measurement, feedback and resetting) are features that emerge from the biochemical and structural properties of ABC transporters, allowing us to develop an explicit bridge between the molecular level description and the higher-level language of information theory.

biophysics↗

bioRxiv2021

The Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614 Mutant: Conformational Plasticity and Frustration-Driven Allostery as Energetic Drivers of Highly Transmissible Spike Variant

The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern(VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model which can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the inter-protomer hinge modulate the inter-domain interactions, global mobility change and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the inter-protomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.

biophysics↗

bioRxiv2021

The Prion Protein Octarepeat Domain Forms Transient β-sheet Structures Upon Residue-Specific Cu(II) and Zn(II) Binding

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in the protein misfolding, and metal imbalance may be part of TSE pathologies. PrPC is a combined Cu(II) and Zn(II) metal binding protein, where the main metal binding site is located in the octarepeat (OR) region. Here, we used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Upon metal binding, the OR region seems to adopt a transient antiparallel {beta}-sheet hairpin structure. Fluorescence spectroscopy data indicates that under neutral conditions, the OR region can bind both Cu(II) and Zn(II) ions, whereas under acidic conditions it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of both metal ions to the OR region results in formation of {beta}-hairpin structures. As formation of {beta}-sheet structures is a first step towards amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSEs.

biophysics↗