Bulletin of the American Physical Society
APS March Meeting 2015
Volume 60, Number 1
Monday–Friday, March 2–6, 2015; San Antonio, Texas
Session B48: Focus Session: Physics of Protein Interactions |
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Sponsoring Units: DBIO Chair: Megan Valentine, University of California, Santa Barbara Room: 217C |
Monday, March 2, 2015 11:15AM - 11:27AM |
B48.00001: ABSTRACT WITHDRAWN |
Monday, March 2, 2015 11:27AM - 11:39AM |
B48.00002: Coarsening of protein clusters on subcellular drops exhibits strong and sudden size selectivity Aidan Brown, Andrew Rutenberg Autophagy is an important process for the degradation of cellular components, with receptor proteins targeting substrates to downstream autophagy machinery. An important question is how receptor protein interactions lead to their selective accumulation on autophagy substrates. Receptor proteins have recently been observed in clusters, raising the possibility that clustering could affect autophagy selectivity. We investigate the clustering dynamics of the autophagy receptor protein NBR1. In addition to standard receptor protein domains, NBR1 has a ``J'' domain that anchors it to membranes, and a coiled-coil domain that enhances self-interaction. We model coarsening clusters of NBR1 on the surfaces of a polydisperse collection of drops, representing organelles. Despite the disconnected nature of the drop surfaces, we recover dynamical scaling of cluster sizes. Significantly, we find that at a well-defined time after coarsening begins, clusters evaporate from smaller drops and grow on larger drops. Thus, coarsening-driven size selection will localize protein clusters to larger substrates, leaving smaller substrates without clusters. This provides a possible physical mechanism for autophagy selectivity, and can explain reports of size selection during peroxisome degradation. [Preview Abstract] |
Monday, March 2, 2015 11:39AM - 11:51AM |
B48.00003: Mechanism of bacterial membrane poration by Antimicrobial Peptides Ankita Arora, Abhijit Mishra Bacterial resistance to conventional antibiotics is a major health concern. Antimicrobial peptides (AMPs), an important component of mammalian immune system, are thought to utilize non-specific interactions to target common features on the outer membranes of pathogens; hence development of resistance to such AMPs may be less pronounced. Most AMPs are amphiphilic and cationic in nature. Most AMPs form pores in the bacterial membranes causing them to lyse, however, the exact mechanism is unknown. Here, we study the AMP CHRG01 (KSSTRGRKSSRRKK), derived from human $\beta $ defensin 3 (hBD3) with all Cysteine residues substituted with Serine. Circular Dichorism studies indicate that CHRG01 shows helicity and there is change in helicity as it interacts with the lipid membrane. The AMP was effective against different species of bacteria. Leakage of cellular components from bacterial cells observed by SEM and AFM indicates AMP action by pore formation. Confocal microscopy studies on giant vesicles incubated with AMP confirm poration. The effect of this AMP on model bacterial membranes is characterized using Small Angle X-ray scattering and Fluorescence spectroscopy to elucidate the mechanism behind antimicrobial activity. [Preview Abstract] |
Monday, March 2, 2015 11:51AM - 12:27PM |
B48.00004: Dissecting EB1-microtubule interactions from every direction: using single-molecule visualization and static and dynamic binding measurements Invited Speaker: Benjamin Lopez EB1 is an important microtubule associating protein (MAP) that acts as a master coordinator of protein activity at the growing plus-end of the microtubule. We can recapitulate the plus-end binding behavior of EB1 along the entire length of a static microtubule using microtubules polymerized in the presence of the nonhydrolyzable GTP analogs GMPCPP and GTP$\gamma $S instead of GTP. Through the use of single-molecule TIRF imaging we find that EB1 is highly dynamic (with a sub-second characteristic binding lifetime) and continuously diffusive while bound to the microtubule. We measure the diffusion coefficient, $D$, through linear fitting to mean-squared displacement of individually labeled proteins, and the binding lifetime, $\tau $, by fitting a single exponential decay to the probability distribution of trajectory lifetimes. In agreement with measurements of other diffusive MAPs, we find that $D$ increases and $\tau $ decreases with increasing ionic strength. We also find that $D$ is sensitive to the choice of GTP analog: EB1 proteins bound to GTP$\gamma $S polymerized microtubules have a $D$ half of that found with GMPCPP polymerized microtubules. To compare these single-molecule measurements to the bulk binding behavior of EB1, we use TIRF imaging to measure the intensity of microtubules coated with EB1-GFP as a function of EB1 concentration. We find that EB1 binding is cooperative and both the quantity of EB1 bound and the dissociation constant are sensitive to GTP analog and ionic concentration. The correlation between binding affinity and $D$ and the cooperative nature of EB1-microtubule binding leads to a decrease in $D$ with increasing EB1 concentration. Interestingly, we also find an increase in $\tau $ at high EB1 concentrations, consistent with attractive EB1-microtubule interactions driving the cooperativity. To further understand the nature of the cooperativity we estimate the interaction energy by measuring the association and dissociation rates ($k_{\mathrm{on}}$ and $k_{\mathrm{off}}$ respectively) at different concentrations of EB1. [Preview Abstract] |
Monday, March 2, 2015 12:27PM - 12:39PM |
B48.00005: A Compete-and-Survive Mechanism Explains the Single FtsZ-Ring Formation Ganhui Lan, Li-Ping Xiong Cytokinesis is a critical step in cell reproduction. In bacterial cells, this process is mediated by the cytoskeletal Z ring which is assembled from FtsZ filaments that are ``anchored'' to the cell membrane through ZipA/FtsA molecules. Fluorescence Recovery after Photobleaching experiments have shown that the Z ring is highly dynamic, with recovery half time of 8 $\sim$ 30 seconds, yet has a rather persistent overall structure. But it is unclear how a single narrow dynamic Z ring emerges from a big pool of cytoplasmic FtsZ molecules. Here, we developed a rule-based molecular model with FtsZ and ZipA/FtsA molecules, by explicitly considering the elementary assembling events of molecules and their diffusion. Our model can not only efficiently reproduce the Z ring with experimentally observed statistical properties, but provide a convenient way to combine biochemical dynamic and physical assembling processes within the same spatiotemporal modeling framework. In agreement with experiments, we showed that the spontaneous self-assembling process relies on the molecular ``stoichiometry'': either high or low FtsZ to ZipA/FtsA ratios would result in multiple Z rings or aggregated bundles. Our \textit{in silico} FRAP experiment further yields a recovery half time comparable to experimental results. These results indicate that the rapid turnover dynamics prevents the FtsZ molecules from being sequestered by small FtsZ bundles dispersed over the membrane, allowing single Z ring to emerge and mature. This dynamic colocalization mechanism provides cells a simple way for spatial regulation. [Preview Abstract] |
Monday, March 2, 2015 12:39PM - 12:51PM |
B48.00006: Characterizing the statistical properties of protein surfaces Ji Hyun Bak, Anne-Florence Bitbol, William Bialek Proteins and their interactions form the body of the signaling transduction pathway in many living systems. In order to ensure the accuracy as well as the specificity of signaling, it is crucial that proteins recognize their correct interaction partners. How difficult, then, is it for a protein to discriminate its correct interaction partner(s) from the possibly large set of other proteins it may encounter in the cell? An important ingredient of recognition is shape complementarity. The ensemble of protein shapes should be constrained by the need for maintaining functional interactions while avoiding spurious ones. To address this aspect of protein recognition, we consider the ensemble of proteins in terms of their three-dimensional shapes, more precisely in terms of their solvent-excluded surfaces. We take into account all high-resolution structures from E.coli non-DNA-binding cytoplasmic proteins that can be retrieved from the Protein Data Bank. We aim to characterize the statistical properties of the ensemble of protein surfaces, including the dimensionality of the space of surfaces. [Preview Abstract] |
Monday, March 2, 2015 12:51PM - 1:03PM |
B48.00007: Microfluidic free-flow electrophoresis for the discovery and characterisation of calmodulin binding partners Therese Herling, Sara Linse, Tuomas Knowles Non-covalent and transient protein-ligand interactions are integral to cellular function and malfunction. Key steps in signalling and regulatory pathways rely on reversible non-covalent protein-protein binding or ion chelation. Here we present a microfluidic free-flow electrophoresis method for detecting and characterising protein-ligand interactions in solution. We apply this method to probe the binding equilibria of calmodulin, a central protein to calcium signalling pathways. In this study we characterise the specific binding of calmodulin to phosphorylase kinase, a known target, and creatine kinase, which we identify as a putative binding partner through a protein array screen and surface plasmon resonance experiments. We verify the interaction between calmodulin and creatine kinase in solution using free-flow electrophoresis and investigate the effect of calcium and sodium chloride on the calmodulin-ligand binding affinity in free solution without the presence of a potentially interfering surface. Our results demonstrate the general applicability of quantitative microfluidic electrophoresis to characterise binding equilibria between biomolecules in solution. [Preview Abstract] |
Monday, March 2, 2015 1:03PM - 1:15PM |
B48.00008: Atomic force microscopy based nanoassay: a new method to study $\alpha $-Synuclein-dopamine bioaffinity interactions Stefania Corvaglia, Barbara Sanavio, Barbara Sorce, Alessandro Bosco, Stefania Sabella, Pierpaolo Pompa, Giacinto Scoles, Loredana Casalis Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular $\alpha $-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of $\alpha $-Synuclein on gold to study the early stages of $\alpha $-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-$\alpha $-Synuclein adducts. [Preview Abstract] |
Monday, March 2, 2015 1:15PM - 1:27PM |
B48.00009: Non-covalent interactions between ATP and RecA DNA-repairing proteins: DFT and semiempirical calculations Jorge Rodriguez The role of Bacterial RecA in the structural maintenance of genomes and the genetic information they carry has been established. In particular, the RecA \quad DNA-repairing protein from \textit{D. Radiodurans}, a radiation-resistant bacteria, is crucial for \quad the repair of double strand breaks (DSBs). We have performed semi-empirical free-energy calculations and QM/MM calculations to study their non-covalent interactions with ATP and ADP. Such studies provide insight into the mechanisms of ATP/ADP $\to $ RecA energy transfer and, therefore, about specific functional uses of incoming energy for DNA repairing mechanisms. We present a detailed analysis of the non-covalent interactions which minimize the interaction Gibbs free energies leading to the most stable non-covalent binding sites. Van der Waal, hydrogen bonding and electrostatic interactions has been quantified which provides a detailed insight into the mechanisms of ATP-RecA interaction. Further, possible chemical interactions and functional roles of RecA proteins are explored based on the previously mentioned studies. \textit{Acknowledgements}: Funded, in part, by DTRA award 106339 (JHR). Dr. Mark C. Palenik and Mrs. Lora Beard are gratefully acknowledged [Preview Abstract] |
Monday, March 2, 2015 1:27PM - 1:39PM |
B48.00010: Photo-Activated Localization Microscopy of Single Carbohydrate Binding Modules on Cellulose Nanofibers Amy Hor, Daryl Dagel, QuocAnh Luu, Madhusudan Savaikar, Shi-You Ding, Steve Smith Photo Activated Localization Microscopy (PALM) is used to conduct an in vivo study of the binding affinity of polysaccharide-specific Carbohydrate Binding Modules (CBMs) to insoluble cellulose substrates. Two families of CBMs, namely {\it Tr}CBM1 and {\it Ct}CBM3, were modified to incorporate photo-activatable mCherry fluorescent protein (PAmCherry), and exposed to highly crystalline {\it Valonia} cellulose nano-fibrils. The resulting PALM images show CBMs binding along the nano-fibril long axis in a punctuated linear array, localized with, on average, 10 nm precision. Statistical analysis of the binding events results in nearest neighbor distributions between CBMs. A comparison between {\it Tr}CBM1 and {\it Ct}CBM3 reveals a similarity in the nearest neighbor distribution peaks but differences in the overall binding density. The former is attributed to steric hindrance among the CBMs on the nano-fibril whereas the latter is attributed to differences in the CBMs' binding strength. These results are compared to similar distributions derived from TEM measurements of dried samples of {\it Ct}CBM3-CdSs quantum dot bioconjugates and AFM images of {\it Ct}CBM3-GFP bound to similar {\it Valonia} nano-fibrils. [Preview Abstract] |
Monday, March 2, 2015 1:39PM - 2:15PM |
B48.00011: Force spectroscopy of biomolecular folding and binding: theory meets experiment Invited Speaker: Olga Dudko Conformational transitions in biological macromolecules usually serve as the mechanism that brings biomolecules into their working shape and enables their biological function. Single-molecule force spectroscopy probes conformational transitions by applying force to individual macromolecules and recording their response, or ``mechanical fingerprints,'' in the form of force-extension curves. However, how can we decode these fingerprints so that they reveal the kinetic barriers and the associated timescales of a biological process? I will present an analytical theory of the mechanical fingerprints of macromolecules. The theory is suitable for decoding such fingerprints to extract the barriers and timescales. The application of the theory will be illustrated through recent studies on protein-DNA interactions and the receptor-ligand complexes involved in blood clot formation. [Preview Abstract] |
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