Bulletin of the American Physical Society
APS March Meeting 2022
Volume 67, Number 3
Monday–Friday, March 14–18, 2022; Chicago
Session B06: Emergent Biomolecular AssemblyFocus Recordings Available
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Sponsoring Units: DBIO Chair: Andrei Gasic, Rice University Room: McCormick Place W-178B |
Monday, March 14, 2022 11:30AM - 12:06PM |
B06.00001: Quinary structure modulates protein sequence stability in vivo Invited Speaker: Caitlin Davis Relying on evolutionary history within thousands of homologous sequences to map important residues, consensus sequence design has emerged as an effective approach for designing biologically functional proteins with high stability. Intriguingly, sequence differences between consensus and extant homologs are predominantly located at weakly conserved surface residues. Surface mutations can have large effects on protein quinary structure, interactions that organize protein interactions inside cells, which are largely dependent on surface charges. Here we compare the sequence, stability, and kinetics of consensus PGK and four extant PGK sequences. We find that all five sequences are stabilized in mammalian cells and in E. coli compared to in vitro. Of the five sequences, the consensus sequence was most stable and showed a larger thermodynamic stability in cells compared to the naturally occurring homologs. Perhaps unsurprisingly, the thermodynamic stabilities of the four extant sequences are near the temperatures of their native environments. Of the extant sequences, the sequence derived from E. coli had the smallest change in stability across environments, likely because it evolved to function in the highly charged bacteria cytosol. At the melting temperature, the folding kinetics of the five sequences are identical and the folding kinetics of the extant sequences are unchanged in cells compared to in vitro. These results demonstrate the significance of quinary interactions in consensus sequences, highlighting the importance of incorporating quinary structure into models for consensus sequence design. |
Monday, March 14, 2022 12:06PM - 12:18PM |
B06.00002: Optimizing kinetics of hetero-subunit assembly using differentiable models Adip Jhaveri In macromolecular assemblies ranging from ribosomes to viral capsids, kinetic trapping produces long-lived intermediates that reduce yield of the target, functional products. Cells can exploit energy-consuming pathways to prevent trapping, but this comes at a cost. Here, we quantify how the threat of kinetic trapping expands with complexity due to topology, size, or heterogeneity of assemblies, from trimers and hetero-tetramers to larger assemblies. We exploit efficient automatic differentiation of kinetic models to 'discover' assembly pathways that can maximize yield while avoiding errors and traps. We show how assemblies can use distinct strategies for avoiding traps, where hierarchies of rates (and binding energies) for dimerization steps offers in some ways a simpler and more flexible evolutionary strategy than pathways that require cooperativity. Our results further show which highly connected subunits must be most severely constrained. We use these results to assess how active control by enzymes could provide more robust strategies to optimize yield than perfect optimization of stochastic interactions. Our results provide strategies for reducing trapping and maximizing assembly yield at minimal cost, borrowing techniques used in machine learning to find optimal assembly pathways despite fixed topologies and energetics. |
Monday, March 14, 2022 12:18PM - 12:30PM |
B06.00003: Nature of size fluctuations in filamentous cytoskeletal structures Aldric Rosario, Shane G McInally, Bruce L Goode, Jane Kondev Cells control the size of their cytoskeletal structures, such as microtubule-based cilia and actin cables, by mechanisms that are still not fully understood. Balance-point models of length control, for which there is ample experimental support in a variety of systems [1], assume length-dependent rates of filament assembly and/or disassembly. When the two competing rates are equal, then a single, stable fixed-point length is reached. Using a master equation approach, we find that length fluctuations, small deviations in length produced by stochastic addition, and removal of building blocks/fragments during filament assembly and disassembly, are Gaussian independent of the nature of feedback. The variance of the distribution is given by the product of the steady-state filament length and the size of the fragments added or removed. Comparing our calculations to the experimentally measured fluctuations of flagella length in unicellular organisms (Giardia and Chlamydomonas) provides new information about the molecular mechanisms involved in cytoskeletal filament length control. In both, we find that the measured length fluctuations support the idea that assembly and disassembly events occur in bursts, where a large number of proteins (tubulin dimers or short polymers) are added and/or removed at a time. |
Monday, March 14, 2022 12:30PM - 12:42PM |
B06.00004: Predicting Timescales of DNA Mediated Protein Assembly Mankun Sang, Margaret E Johnson Transcription initiation pioneers are proteins that are necessary to help remodel DNA into an exposed structure that supports gene transcription. For the GAGA pioneer factor, experiments have recently shown that its ability to form larger oligomers is impacted by DNA binding, with a direct impact on its residence times and recruitment of additional proteins needed for transcription. To study the collective dynamics and microscopic mechanisms that control this process, we need models that can capture the spatial and temporal dynamics of localization and assembly on and around DNA. Here we implemented a reaction-diffusion model to simulate proteins binding and forming higher-order complexes on chromatin. It allows dynamic tracking over a long timescale (milliseconds to seconds) with high spatial resolution and predicts how residence times on DNA are controlled by higher-order clustering of the pioneer proteins. We found that the binding rates between pioneers themselves as well as between protein and DNA are key parameters controlling their arrival times and lifetimes on DNA. Our results capture the distribution and diffusion of pioneer factors across the genome, with direct implications for how their dynamics impact productive transcription. |
Monday, March 14, 2022 12:42PM - 12:54PM |
B06.00005: Active nuclear transport in living cells: dynamics and steady-state profiles of cargo concentrations Alex S Rautu, Michael J Shelley The transport of cargo into and out of the nucleus is crucial for the functioning of all eukaryotes cells. Although the migration of ions and small molecules can occur diffusively, the nuclear transport of larger macromolecules generally requires an active process. Large protein channels known as nuclear pores embedded into nuclear envelope regulate the passage of many proteins and RNA molecules. However, their movement through nuclear pores does not require energy input, the nuclear transport being powered in large part by the free energy transduction from the Ran cycle. At the energetic cost of guanosine triphosphate hydrolysis, the system of nuclear pores and corresponding transport receptors is able to generate steep concentration gradients across the nuclear envelope, thereby controlling the flux and directionality of nucleocytoplasmic transport. Here, a physical model of this mechanism is described that allows us to determine the temporal and spatial distributions of transport receptors and cargo proteins within the nucleus, and their role in the regulation of other cellular processes such as chromatin remodeling and transcription is discussed. |
Monday, March 14, 2022 12:54PM - 1:06PM |
B06.00006: Acentrosomal spindles assemble from branching nucleation near chromosomes Bernardo Gouveia, Sagar U Setru, Joshua W Shaevitz, Howard A Stone, Sabine Petry Microtubules are generated at centrosomes, chromosomes, and within mitotic and meiotic spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains to be learned about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy (TIRFM) to find that chromosomes alone can indeed form spindles, and that this occurs predominantly through branching microtubule nucleation. By knocking out motor activity, we show that the organization of the resulting branched networks are consistent with a theoretical model whereby the effectors for branching nucleation are released in a gradient around chromosomes. Moreover, the microtubule nucleation rate in spindles scales with the amount of chromatin. Motors serve to self-organize these chromosomal branched networks into proper spindles, which we rationalize using a model of opposing motors. |
Monday, March 14, 2022 1:06PM - 1:18PM |
B06.00007: The roles of stoichiometric crowding and protein-protein interactions in accelerating translation elongation rates in E. coli Jennifer L Hofmann, Roseanna N Zia Translation elongation in Escherichia coli is a complex physico-chemical process, involving diffusive transport, entropic exclusion, and combinatoric matching between ribosomes and tRNAs. Previous work from our group has demonstrated that ‘stoichiometric crowding’ – the relative abundances and volume fractions of translation molecules – improves diffusive search times and drives an increase in ribosomal productivity as a function of cellular growth rate [Maheshwari et al., in review]. This physical phenomenon is insensitive to changes in chemical kinetics, though the overall elongation rate is underpredicted. Here, we extend our representative E. coli cytoplasm by incorporating attractions between EF-Tu–GFP–tRNA and ribosomal L7/L12 subunits, which are hypothesized to ‘pre-load’ ternary complexes onto ribosomes. We demonstrate the structural and dynamic implications of pre-loading, which ultimately combines with stoichiometric crowding to facilitate faster translation elongation rates. |
Monday, March 14, 2022 1:18PM - 1:30PM |
B06.00008: LIM domain proteins localize to strained actin, focal adhesions and adherens junctions in a tension-dependent manner Shailaja Seetharaman, Jonathan Winkelman, Yvonne Beckham, Margaret Gardel The actomyosin is coupled to mechanosensitive adhesion components such as focal adhesions (FAs) and adherens junctions (AJs), and can generate forces that maintain overall tissue homeostasis. A ubiquitous LIM (Lin11, Isl- 1, and Mec-3) domain family of proteins is associated with actomyosin and adhesions; for e.g., LIM protein Zyxin localizes to FAs, and is also seen at stress fiber strain sites (SFSS). The mechanisms by which LIM proteins sense forces and bind to strained actin or adhesive structures remain unknown. Here, we show that several members of the LIM family including Zyxin, Paxillin, FHL-2 and LIMD1 bind to SFs, FAs or AJs in a contractility-dependent manner. Specifically, the LIM regions of 18 different LIM proteins localize to SFSS. Similarly, paxillin-like 1 (Pxl1) protein from fission yeast also binds to strained actin in mammalian cells. This suggests an evolutionarily conserved mechanism by which the LIM domains recognize and bind to SFs or adhesive structures upon mechanical stress. We hypothesize that the force-sensitivity of LIM proteins could arise from the organization of the LIM region, amino acid substitutions, or post-translational modifications within the LIM region. We are currently exploring the molecular and mechanotransductive mechanisms by which the different LIM proteins sense mechanical stress and are recruited to distinct cellular structures. We are also investigating the effects of this force-sensitive recruitment of LIM proteins on downstream cellular processes. |
Monday, March 14, 2022 1:30PM - 1:42PM |
B06.00009: Measuring Weak Protein-Protein Interactions Using a Mechanically Transduced Immunosorbent Assay Joshua P Steimel, Christopher J Petell, Joe Harrison, Brian Strahl, Michael Pappas Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry, yet many critical interactions are unmeasurable due to a scarcity of reagents and limitations in the affinity ranges that can be measured. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein coated particles on a protein functionalized substrate. The translational displacement scales with the effective friction induced by the PPI interaction, producing a mechanical signal to indicate binding event. METRIS can measure interactions across a wide range of affinities, $10^{-3}-10^{-15}$M, has high resolution, and measures $\Delta \Delta$G differences of approximately 0.4$\frac{kcal}{mol}$, and utilizes a small amount of reagents, 20 pmol. METTRIS can provides quantitative insights into the interplay between epigentic modifications and can be utilized a new breakthrough in diagnostics. |
Monday, March 14, 2022 1:42PM - 1:54PM |
B06.00010: Evolutionary advantage of a dissociative search mechanism in DNA mismatch repair Kyle Crocker, James London, Andres A Medina, Richard Fishel, Ralf Bundschuh Protein complexes involved in DNA mismatch repair diffuse along dsDNA as sliding clamps in order to locate a hemimethylated incision site. They have been observed to use a dissociative mechanism, in which two proteins, while continuously remaining attached to the DNA, sometimes associate into a single complex sliding on the DNA and sometimes dissociate into two independently sliding proteins. Here, we study the probability that these complexes locate a given target site via a semi-analytic, Monte Carlo calculation that tracks the association and dissociation of the sliding complexes. We compare such probabilities to those obtained using a nondissociative diffusive scan in the space of physically realistic diffusion constants, hemimethylated site distances, and total search times to determine the regions in which dissociative searching is more or less efficient than nondissociative searching. We conclude that the dissociative search mechanism is advantageous in the majority of the physically realistic parameter space, suggesting that the dissociative search mechanism confers an evolutionary advantage. |
Monday, March 14, 2022 1:54PM - 2:06PM |
B06.00011: Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics Dimitrios Vavylonis, Shuting Zhang The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), binding of the complex to the side of actin filaments, and recruitment of actin monomers. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied the C-alpha based model Kim and Hummer that retains amino-acid level information and allows coarse-grained molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent cryo-EM structures of the Arp2/3 complex branch bound to a mother actin filament to represent the activated form of Arp2/3 complex. We studied its interactions with the VCA domain of the NPF Wiskott-Aldrich syndrome protein (WASp) and actin monomers. We found stable configurations with one or two actin monomers bound along the branch filament direction and with VCA domain associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. The results further suggest that the interaction between the two actin subunits, and the flexibility of actin D-loop contribute to the stability of the complex. We also performed simulations of active Arp2/3 complex bound to a mother actin filament. The bound conformation remained stable during the simulations, which reflect the contribution of each subunit to the binding. D-loop flexibility of actin subunits on the mother filament further stabilized the binding. |
Monday, March 14, 2022 2:06PM - 2:18PM |
B06.00012: Overlooked transcription factor crosslinking and steric forces can coordinate chromatin and TAD organization Adam R Lamson, Wen Yan, Alex S Rautu, Michael J Shelley Polymer simulations are increasingly effective at interpreting the often beautiful hierarchical patterns that arise in chromatin conformation capture (3C,Hi-C,Micro-C) assays. While a number of models and theories can reconstruct these patterns in silco, many use fitted coarse-grained potentials and ignore microscopic mechanisms involved in the interactions between chromatin, transcription factors (TFs), and the surrounding nucleoplasm. Here, I investigate how steric forces between TFs expand and isolate genomic regions, while individually modeled crosslinking proteins condense other regions. These simulations provide a microscopic interpretation of the potentials used in other methods, allowing us to explore the importance of physical constants, such as TF binding rates and binding strength, in creating specific yet stable chromatin domains. |
Monday, March 14, 2022 2:18PM - 2:30PM |
B06.00013: Understanding the Unfolding Mechanisms of ??D Crystallin using Molecular Dynamics Simulations DEEPSHIKHA GHOSH, Kandarp Sojitra, Manish Agarwal, Mithun Radhakrishna γD-Crystallins being the longest-lived proteins are responsible for maintaining the transparency and refractivity of the lens. The unfolding of γD-Crys results in large insoluble aggregates which leads to cataracts. However, the unfolding pathway of γD-Crys is still unclear due to experimental constraints. One of the approaches to address this is through understanding the pathways of aggregation as well as determining the major forces driving the formation of these aggregates using Molecular Dynamic Simulations. Our initial results show that isolated greek key motifs of γD-Crys tend to unfold while the domains are found to be stable. This indicates that the domains are stabilized by strong hydrophobic intermotif interactions, whereas the motifs are destabilized when exposed to solvent by disrupting the hydrophobic core. Further, our mutation studies show that by replacing the hydrophobic residues Ile3 and Trp42 with charged residues the domain unfolds, which infers that Ile3 and Trp42 play a major role in stabilizing the protein. Our findings can help to predict potential drug targets, which will help γD-crystallin to retain its native conformation and provide an economically viable solution to treat cataracts. |
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