Temperature Dynamics of Single Molecular Antifreeze Protein

Tuesday, February 14, 2017 E-cadherin lattices predict a two-phase elastic response in which E-cadherin complexes first straighten and then unbind without unfolding. Cisinteractions are predicted to play a minor role in the mechanical response of these complexes. Simulations of the desmoglein/desmocollin systems show that forces required to unbind both the homodimer of desmoglein-2 and a canonical heterodimer of desmoglein-2 and desmocollin-1 are similar, even in the presence of a salt-bridge shown to be important for the heterodimer interaction. As with the E-cadherin system, both the desmoglein dimer and desmoglein/desmocollin systems exhibited unbinding after an initial straightening of both molecules, with no unfolding. Overall, our simulations provide insights into the molecular mechanics of adherens junctions and desmosomes. 1586-Plat Towards a Rational Design of Macrolide Antibiotics in Order to Combat Bacterial Resistance Anna Pavlova1, Jerry M. Parks2, Adegboyega K. Oyelere3, James C. Gumbart1. 1 Physics, Georgia Institue of Technology, Atlanta, GA, USA, 2Center of Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 3Chemistry & Biochemistry, Georgia Institue of Technology, Atlanta, GA, USA. Macrolides, which bind in the protein exit tunnel of the bacterial ribosome, are one of the most prescribed classes of antibiotics.However, macrolide resistance caused by ribosomal modifications at base A2058 in segment 23S is a growing problem. It has been foundthat attachment of aromatic moieties can increasemacrolide activity against both wild-type and macrolide-resistant ribosomes due to additional aromatic interactions with rRNA, resulting in new antibiotics, e.g., telithromycin. Our previous simulations of the stalling peptideSecM in the ribosome revealed stable aromatic interactions between residue W155 in SecM and base A751 in segment23S, inspiring the development of azithromycin derivatives containing indole-analog moieties that could mimic these interactions. Several of these derivatives showed improved activity against wild-typeEscherichia coli compared to azithromycin, although, a better understanding of the structure-activity relationship for the different moietiesis needed for further improvement. Here, we used molecular dynamics simulations to study erythromycin and azithromycin in wild-type and A2058-modified, E. coli ribosomes. The ribosomal modifications resulted in less favorable interactions between base 2058 and the desosamine sugar of the macrolides as well as a greater displacement of the macrolides, explaining the causes for resistance. Additionally, two of the azithromycin derivatives noted above were simulated in the wild-type ribosome. We found that the added indole-analog moieties adopted different geometries when interacting with base A751, which could explain the differences in their activity. Our results illustrate the utility of MD simulations in the design of a new generation of macrolides that can overcome bacterial resistance. 1587-Plat Fast Forward Protein Folding Maxwell I. Zimmerman, Gregory R. Bowman. Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO, USA. Molecular dynamics simulations are increasingly used to understand complex phenomenon such as protein folding; simulations can provide full atomistic details on a level that is currently unavailable to many experimental approaches. Despite this utility, computational resources often limit the timescales accessible to simulations and prevent the observation of protein folding events (i.e. even the fastest folding events occur in the ms-ms regime, yet typical desktop computers only simulate a few ns per day). In lieu of increasing computer power by orders of magnitude, better algorithms for reaching these timescales must be developed. Adaptive sampling algorithms reduce required simulation time by taking advantage of many simulations run in parallel, for which simulations can be pieced together with the construction of a Markov State Model. Adaptive sampling schemes can be described as an undirected approach that follow four main steps: 1) run simulations, 2) build a Markov State Model from current data, 3) analyze the discovered states, and 4) start new simulations from structures that have been analyzed. Structures that are used to restart simulations are traditionally chosen to optimize some statistical ranking. Here, we present a new sampling scheme that additionally ranks states based on physical traits and can follow gradients in conformational space, which we demonstrate reduces the simulation time required to observe protein folding events. In contrast to simulation techniques that perturb energy landscapes, such as steered molecular dynamics, this new sampling scheme provides thermodynamically accurate

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conformational transitions and pathways to folded states, since simulations are always at equilibrium. 1588-Plat Temperature Dynamics of Single Molecular Antifreeze Protein Rio Okada1,2, Tatsuya Arai3,4, Daichi Fukami3,4, Yuhuku Matsushita1, Jae-won Chang1, Hiroshi Sekiguchi5, Noboru Ohta5, Tadashi Mori6, Masaki Nishijima7, Keisuke Miyazawa8, Takeshi Fukuma8, Keigo Ikezaki1, Sakae Tsuda3,4, Yuji C. Sasaki1,5. 1 Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan, 2National Institute of Advanced Industrial Science and Technology (AIST), OPERANDO-OIL, Tokyo, Japan, 3Graduate School of Life Science, The University of Hokkaido, Hokkaido, Japan, 4Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hokkaido, Japan, 5Research & Utilization Division, Japan Synchrotron Radiation Research Institute, SPring-8, Hyogo, Japan, 6Graduate School of Engineering, The University of Osaka, Osaka, Japan, 7Office for University-Industry Collaboration, The University of Osaka, Osaka, Japan, 8 School of Electrical and Computer Engineering, The University of Kanazawa, Kanazawa, Japan. AntiFreeze Proteins (=AFPs) bind to a surface of ice crystals and inhibit their growth. Some living organisms; fishes, insects and fungus, at low-temperature environment have several different types of AFPs. AFPs protect their body from freezing damages. To clarify the antifreeze effect of lpAFP (isolated from longsnout poacher, a fish living in the sea of Okhotsk) at single molecular level, we performed the single molecular observation using diffracted X-ray tracking (=DXT). DXT is the method to observe single molecular motions using X-rays and gold nanocrystals as probe. Gold nanocrystals are labeled on AFPs, and when irradiated with X-rays, diffracted spots from gold nanocrystals can be observed. We can observe the motion of the AFPs by tracking these spots. We performed the DXT experiments at SPring-8 BL40XU, and the spatial and time resolutions are the order of milli-radian and micro-second. In this study, we used lpAFP isolated from longsnout poacher, and observed the temperature dependency of the lpAFP’s single molecular brownian motion. As a result, lpAFP’s motion increased at 5  C. Next, to ensure the relationship between the adsorption property and the maximum brownian motion, we observed the temperature dependency of the adsorption affinity to AgI thin film as ice surface. As a result, the adsorption amount to AgI of lpAFP was the largest at 2 C. These two experiments suggest that there is a strong correlation between lpAFP’s molecular motion and adsorption property. We acquired the temperature dependency of lpAFP using other experiments; circular dichroism (CD), atomic force microscopy (AFM), dynamic light scattering (DLS), and small angle X-ray scattering (SAXS, performed at SPring-8 BL40B2). In poster session, we discuss the detail experimental procedure and results.

Platform: Mitochondria in Cell Life and Death 1589-Plat Charcot-Marie-Tooth Type2A Mfn2 Domain-Specific Mutants Deferentially Alter Mitochondrial Fusion Dynamics and Motility Veronica Eisner1, Diego Troncoso1, Pamela Rojas1, Josefa Vial1, Mauricio Castro1, Sergio Henrı´quez1, Rita Horvath2. 1 Universidad Cato´lica de Chile, Santiago, Chile, 2Newcastle University, Newcastle, United Kingdom. Mitochondrial function relays on the balance between fusion and fission events that determine mitochondrial communication. Mutations of outer mitochondrial membrane (OMM) fusion protein Mfn2, cause motor neuron degeneration disease Charcot-Marie-Tooth Type 2 (CMT2). Also, Mfn2 plays a controversial role in the ER-mitochondrial communication. Thus, it is unknown if Mfn2 CMT2-associated domain-specific mutations alter mitochondrial fusion dynamics, ER/mitochondria communication, or both. Here we study mitochondrial fusion dynamics in human fibroblasts isolated from skin samples of control and CMT2-confirmed patients from the Centre for Neuromuscular Diseases Biobank, Newcastle. The cells were transfected mitochondrial matrix-targeted mtDsRed and photo-switchable protein mtPAGFP. By means of 408 nm mediated photo-conversion and time series confocal imaging, we studied mitochondrial continuity and fusion events (f.e.) frequency. We evaluated two domain-specific Mfn2 mutants: L248H (GTPase domain) and M376V (GTPase to HR1 connector). Mfn2-L248H carrying fibroblasts display hyperfused mitochondria and f.e. frequency inhibition 0.42 f.e./min as opposed to 1.2 f.e./min in control fibroblasts. Moreover, Mfn2-M378V fibroblasts showed inhibition of both mitochondrial continuity and f.e. frequency (0.45 f.e./min). In addition, the ultrastructure of control and Mfn2-M378V fibroblasts, evaluated by Transmission Electron