Covalently Circularized Nanodiscs : EM and NMR Applications

Monday, February 13, 2017 and pre-cardiac cells located within bilateral regions of the mesoderm called heart fields (HFs) fold and fuse along the embryonic midline. Then both layers lengthen axially as the anterior intestinal portal (AIP) moves downward. Finally, the fused HFs thicken and expand to create the HT. Here, we combine experiments on chick embryos with computational modeling to explore physical mechanisms of heart tube formation. According to our hypothesis, differential anisotropic growth between mesoderm and endoderm drives ventral folding, and contraction along the AIP generates tension to elongate the HT. We test this hypothesis using biochemical perturbations of cell proliferation and contractility, as well as computational modeling. In embryos exposed to the mitotic inhibitor aphidicolin, little or no folding occurred, confirming that cell proliferation is required to initiate folding of HFs. To determine the effects of actomyosin contraction, we cultured Hamburger-Hamilton stage 5 embryos in media containing the myosin inhibitor blebbistatin. During incubation, folding occurred in treated embryos, but AIP descension proceeded at a progressively slower rate, showing that AIP downward motion is generated mainly by AIP contraction. Simulating these mechanisms in our computational model produces morphology in reasonable agreement with experiments. For the last phase, adding circumferential growth causes the HF to expand, creating the primitive HT, while radial growth increases the thickness of the heart wall. In conclusion, results of our study support our hypothesis for the creation of the heart tube. 1497-Pos Board B565 Covalently Circularized Nanodiscs : EM and NMR Applications Mahmoud L. Nasr1, Julia Simon1, Zhao Zhao2, Mike Strauss1, William Shih3, James Hogle1, Gerhard Wagner1. 1 BCMP, Harvard Medical School, Boston, MA, USA, 2Wyss Institute, Harvard Medical School, Boston, MA, USA, 3BCMP and Wyss Institute, Harvard Medical School, Boston, MA, USA. A traditional nanodisc is composed of a nanometer-sized phospholipid bilayer patch encircled by two copies of membrane scaffold protein (MSP). To date, the utility of this system for structural studies has been limited by the heterogeneity in size and only small nanodiscs could be assembled with the currently available MSP. Here, we present novel nanodiscs encircled by DNA scaffold and covalently circularized MSP variants (cMSPs). The use of cMSPs enhanced the homogeneity, thermal stability and proteolytic resistance of the nanodiscs. Moreover, the use of cMSPs and DNA scaffold enabled us to extend the size of nanodiscs up to 100 nm in diameter. Interestingly, we were able to manipulate the shape of the new nanodiscs and produce polygonal as well as elliptical shaped nanodiscs. In this study, we show that reconstitution into cNDs enhanced the quality of NMR spectra for both VDAC-1, a beta-barrel membrane protein, and the G protein-coupled receptor NTR1, an alpha-helical membrane protein. Using our method, we have created 50 nm nanodiscs and used them to study poliovirus entry and RNA translocation. A 50 nm nanodisc is sufficiently large to accommodate multiple copies of the poliovirus receptor, and has enough surface area to act as a surrogate membrane for the RNA translocation complex during viral uncoating. Virus binding to nanodisc-CD155 complex and subsequent insertion of viral components into and across the membrane were confirmed by EM. To obtain a high-resolution structure for the RNA translocation complex we conducted single-particle cryo-EM studies. The method for making these novel nanodiscs as well as the NMR and CryoEM data will be will be presented and discussed. 1498-Pos Board B566 Molecular Dynamics of Adenosine Triphosphate Interacting with Lipid Membranes Abhinav Ramkumar, Xiaoling Leng, Ryan Z. Lybarger, Horia I. Petrache. Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN, USA. Adenosine triphosphate (ATP) provides the chemical energy needed in most biological processes from metabolic reactions to cellular mechanics. Hydrolysis of ATP that cleaves phosphate bonds is the mechanism by which energy is released to the environment, resulting in lower energy derivative forms of ATP as adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Within biological cells, this chemical reaction often takes place in the vicinity of lipid membranes. Biophysical experiments by x-ray scattering and NMR spectroscopy have indicated that ATP binds to lipid membranes primarily through the adenine ring, leaving the phosphate chains available for hydrolysis. However, the exact dynamics of ATP, and in particular the possible cooperativity between bound ATP molecules is still unknown. Furthermore, biochemical studies suggest the possibility of ATP functioning as a neurotransmitter which further motives a molecular study of its interaction with lipid membranes. All-atom molecular dynamics simulations at microsecond timescales

305a

reveal that ATP, ADP, and AMP bind to lipid headgroups cooperatively and this behavior generates significant electrostatic charging of membranes even at low concentrations that are typical in biological cells and can affect the transmission of the action potential in neurons. 1499-Pos Board B567 Novel Photochromic Compound Composed of Azobenzene and Spiropyran to Control the Function of Kinesin Eg5 Kentaro Saito. SOKA University, Tokyo, Japan. Kinesin Eg5 is a plus-end-directed microtubule-based motor that is essential for bipolar spindle formation during eukaryotic cell division. As Eg5 overexpressed in various malignant tumors, it has been considered as a potential target for cancer treatment. Loop L5 of Eg5 is a key region determining ATPase activity and motor function. Photochromic molecules undergo reversible isomerization in response to ultraviolet and visible light irradiation. Azobenzene exhibits cis-trans isomerization upon UV and visible light irradiations. Spiropyran also shows spiro-merocyanine isomerization upon UV and visible light irradiations. We have previouly introduced photochromic molecules azobenzene derivatives or spiropyran derivatives into L5 and succeeded to control the Eg5 ATPase activity using light irradiation. In this study, we designed and synthesized a novel photochromic thiol reactive compounds composed of azobenzene and spiropyran derivatives, IASA in order to control the function of Eg5 photo reversibly. IASP undergoes reversible isomerization among different three states. UV and visible light irradiations induced Cis-Mero state and Trans-Spiro state, respectively. In the dark, IASP exhibited Trans-Mero state. Therefore, it is expected that the different isomer states of IASP at the incorporated functional site of Eg5 may induce different conformational change of Eg5 resulting alteration of Eg5 ATPase and motor activities. The isomerizations of IASA were monitored by measuring absorption spectra. IASA was incorporated into the reactive single cysteine residue of Eg5 mutant. We tried to examine the photo reversible alteration of ATPase activity of Eg5 modified with IASA. 1500-Pos Board B568 Microwave Dielectric Properties of Conductive Liquids Christopher E. Bassey1, Madeson Claiborne1, Kaylee Garcia2. 1 Mathematics and Physics, Azusa Pacific University, Azusa, CA, USA, 2 Engineering and Computer Science, Azusa Pacific University, Azusa, CA, USA. The propagation of electromagnetic waves in material-loaded transmission lines is a standard technique for determining the dielectric properties of materials. Knowledge of the dielectric properties of materials in turn provides information on their quality, purity, and water content. However, in conductive media, measurements are difficult due to short-circuit effects. Also, the effect of conductivity on the dielectric properties of materials is still being studied. The goal of this work is to investigate the influence of conductivity on electromagnetic wave propagation, and to provide experimental data on the dielectric properties of various concentrations of saline solutions. We used AGILENT E5071C Automatic Network Analyzer (ANA), which is an instrument for measuring the transmission and reflection coefficients of electromagnetic signals as a function of frequency. The ANA, in conjunction with a dielectric probe, was used to measure the electrical properties of pure and conductive liquids. We made measurements between 500 MHz and 8.5 GHz frequency range, at an average room temperature of 21.0 o C. We gradually changed the conductivity of distilled water by adding calculated masses of sodium chloride (NaCl) to obtain saline solutions between 0.001 and 0.100 M. We then measured the corresponding values of dielectric constant, loss factor and loss tangent. Results showed a decrease in dielectric constant with conductivity and an increase in in loss factor with conductivity. The saline solutions also showed a greater dispersion than the distilled water. This work has useful applications in biophysics, soil science, bioengineering and environmental engineering. 1501-Pos Board B569 On the Viscoelastic Properties of the Brain Tissue with Indentation Technique Aref Samadi-Dooki, George Z. Voyiadjis, Rhett W. Stout. Louisiana State University, Baton Rouge, LA, USA. Variety of methods have been used for characterization of the mechanical properties of the brain at the tissue level. Nevertheless, the obtained results from different researchers are extremely scattered and sometimes in contrast to one another. While brain tissue is extremely soft, its mechanical properties are quite a challenge to obtain them. In this study, the accurate analysis of the mechanical heterogeneity of the brain tissue is performed through dynamic and pseudo-static indentation techniques. This study is aimed at evaluating the viscoelastic response of the brain and presenting its anisotropy, inhomogeneity,