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Exploration of Weak Interactions between Folate and Glycine-Betaine
50a
Sunday, February 28, 2016
with its ligand 1-adamantanecarboxylic acid. Our volumetric measurements showed that the binding constant is significantly greater in a solution of 5% DMSO than in pure water. Our molecular dynamics simulations provided the vision at the molecular scale of the effect of DMSO in the binding event as well as associated free energy values in the presence and absence of 5% DMSO. This work should help in providing a better rationalization in the design of potent hydrophobic ligands knowing the DMSO contribution to their binding properties. 261-Pos Board B41 Atomic Mechanisms of Stabilizing and Destabilizing Co-Solvents on Protein Stability Cristiano L. Dias, Zhaoqian Su. Physics, New Jersey Institute of Technology, Newark, NJ, USA. Urea and trimethylamine n-oxide (TMAO) are small molecules known to destabilize and stabilize, respectively, the structure of proteins when added to aqueous solution. To unravel the molecular mechanisms of these cosolvents on protein structure we perform explicit all-atom molecular dynamics simulations of extended poly-alanine and poly-leucine dimers. We use an umbrella sampling protocol to compute the potential of mean force (PMF) of dimers at different concentrations of urea and TMAO. We find that the large nonpolar side chain of leucine is affected by both urea and TMAO whereas backbone atoms and alanine’s side chain are not. Urea is found to occupy positions between leucine’s side chains that are not accessible to water. This accounts for extra Lennard-Jones bonds between urea and side chains that provide the enthalpic driving force for unfolding. 262-Pos Board B42 FCS on Proteins in Crowded Environments Alyazan Albarghash1,2, Daryan Kempe1, Niklas Ole Junker1, Birgit Simone Hillebrecht1, Friedemann Melchior Landmesser1, Jo¨rg Fitter1,2. 1 I. Physikalisches Institut I.A., RWTH Aachen University, Aachen, Germany, 2Institute for Complex Systems - Molecular Biophysics (ICS-5), Forschungszentrum Ju¨lich, Ju¨lich, Germany. The most of our knowledge about proteins is mainly gained from in-vitro measurements. However, the results obtained from in-vitro measurements are likely to be deviated from the explicit natural characteristics of the proteins in the living cell. Yet, the technical difficulty of labeling proteins inside the living cell hinders carrying out in-vivo fluorescence spectroscopic studies. An approach to get closer to the complex nature of the cellular cytoplasm is to roughly mimic it by an artificial crowded-environment. Fluorescence Correlation Spectroscopy (FCS) is utilized to study diffusion effects of fluorescently labeled molecules. The implication of FCS in studying proteins mobility in a crowded-medium is limited by several technical obstacles such as distortion of the molecule detection volume[1] and switching from Brownian diffusion regime to hindered diffusion[2] regime. A pre-characterization of the viscosity and refractive index of investigated solutions and their effects on the validity of the measurement has been carried out [3]. Further, FCS was utilized to investigate the diffusional phenomena of various proteins dissolved in solutions containing classes of crowders [4]. References [1] C. B. Mu¨ller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding, Soft Matter, vol. 5, pp. 13581366, 2009. [2] Phillips, R. J. (2000). A hydrodynamic model for hindered diffusion of proteins and micelles in hydrogels. Biophysical Journal, 79(6), 3350-3353. [3] Junker N. (2014). Bachelor project. RWTH Aachen. [4] Hillebrecht B. S. (2015). Bachelor project. RWTH Aachen. 263-Pos Board B43 Modeling Macromolecular Crowding through Translational and Rotational Diffusion of Small Molecular Probes Megan Currie1, Brenden Berry1, Taylor Ward1, Erin D. Sheets1,2, Ahmed A. Heikal1. 1 Department of Chemistry & Biochemistry, University of Minnesota, Duluth, Duluth, MN, USA, 2Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota, Duluth, Duluth, MN, USA. Living cells are crowded with macromolecules such as proteins, DNA, RNA, biomembranes, actin filaments, and a wide range of organelles. Macromolecular crowding in living cells is believed to affect processes such as protein folding, biochemical reactions and diffusion, which are essential for cell survival, function, and ultimately biomedical implications. Although a large body of work using myriad model systems and experimental methods has been done, the role of crowding on molecular processes is not fully understood. In this contribution, we used polymers (Ficoll-70) and proteins (BSA, ovalbumin) as biomimetic crowding agents as a means to quantify their effect on
the diffusion mechanism of a small molecular probe (namely, rhodamine green, RhG). To distinguish between crowding and viscosity effects on diffusion, control studies were carried out on glycerol-enriched buffer under the same experimental conditions. The viscosity of these crowding and control environments was quantified independently using viscometer and rheology methods. In contrast with glycerol-rich buffers, the rotational diffusion measurements using time-resolved anisotropy indicate binding reaction (specific or transient due to weak interactions) that is dependent on the concentration and type of the crowding agents. These results on the fast time scale guide our modeling of the translational diffusion of RhG using fluorescence correlation spectroscopy (FCS) as a function of crowding agents. Our results on both translational and rotational diffusion studies of RhG indicate deviation from the StokesEinstein model as a function of the concentration and type of the crowding agents. Our studies represent a step forward in the collective effort aimed at elucidating the role of macromolecular crowding towards quantitative cell biology. 264-Pos Board B44 Effect of Co-Solutes on Model Reaction Equilibria: Might Changes in the Free Energy of Bulk Water be the Underlying Cause? Daryl K. Eggers. Chemistry, San Jose State University, San Jose, CA, USA. Studies that characterize binding interactions in the presence of other solutes are necessary for understanding molecular interactions in vivo, including drug interactions. Unfortunately the binding equations of classical thermodynamics were derived for ideal dilute solutions, whereas biological solutions deviate greatly from the ideal assumption. This work examines model binding reactions in the presence of various co-solutes at molar concentrations using isothermal titration calorimetry and solubility measurements. The results are analyzed with original equations that acknowledge the participation of water molecules in the balanced reaction. The thermodynamic framework treats the free energy of bulk water as a variable that depends on the chemistry and concentration of the co-solute. A major concept behind this approach is the idea that changes in reaction equilibria (K values or transfer free energies) may be attributed to changes in the desolvation energy of the reactants; an equilibrium can shift in either direction, depending on whether the co-solute increases or decreases the average free energy of the bulk water because this term is a defining component of the desolvation energy. The outcomes of this work are consistent with the hypothesis that co-solutes do not need to interact directly with one of the reactants in order to alter the equilibrium, though preferential interactions between solute and reactant may be apparent in many systems. 265-Pos Board B45 Exploration of Weak Interactions between Folate and Glycine-Betaine Purva P. Bhojane, Michael R. Duff, Elizabeth E. Howell. Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA. In vitro studies with two different dihydrofolate reductases (EcDHFR, E.coli chromosomal and R67 DHFR, plasmid encoded) have shown that weak interactions between osmolytes and the substrate, dihydrofolate, decreases its affinity towards these enzymes. The unique changes in binding affinity with water activity for each osmolyte indicate preferential interactions between osmolyte and folate and its derivatives. Characterization of these interactions is essential for better understanding of in vivo effects of folate and its various redox states with available functional groups inside the cell. Quantitation of weak interactions between folate and glycine-betaine using a vapor pressure osmometry method yields a preferential interaction coefficient, or m23/RT value. This provides a scale for measuring the preference of folate for glycine-betaine relative to water. The predicted m23/RT value for folate using an accessible surface area calculation indicates equal preference for water and glycine-betaine. Experimental measurements found a folate concentration dependence of the m23/RT values, consistent with dimerization of folate. Studies with other model compounds suggest aromatic rings prefer to interact with glycine-betaine as compared to water. Our results also indicate neutral folate preferentially interacts with glycinebetaine whereas the anionic form excludes glycine-betaine. Can m23/RT values be used to predict osmotic stress effects on ligand binding? In some cases, yes. However, the caveat is whether all the ligand atoms are used in binding. As glutamate excludes glycine-betaine, calculation of the m23/RT value for polyglutamylated folates (pteroyltetra-g-glutamate (PG4)) predicts an overall exclusion of glycine-betaine. This should translate into tighter binding of PG4 to DHFR. Our studies found glycine-betaine addition weakens binding of both folate and PG4 to R67 DHFR to similar extents. Similar effects were observed for folate binding to EcDHFR. These results indicate that the additional glutamates do not contribute to binding to DHFR.
Sunday, February 28, 2016
with its ligand 1-adamantanecarboxylic acid. Our volumetric measurements showed that the binding constant is significantly greater in a solution of 5% DMSO than in pure water. Our molecular dynamics simulations provided the vision at the molecular scale of the effect of DMSO in the binding event as well as associated free energy values in the presence and absence of 5% DMSO. This work should help in providing a better rationalization in the design of potent hydrophobic ligands knowing the DMSO contribution to their binding properties. 261-Pos Board B41 Atomic Mechanisms of Stabilizing and Destabilizing Co-Solvents on Protein Stability Cristiano L. Dias, Zhaoqian Su. Physics, New Jersey Institute of Technology, Newark, NJ, USA. Urea and trimethylamine n-oxide (TMAO) are small molecules known to destabilize and stabilize, respectively, the structure of proteins when added to aqueous solution. To unravel the molecular mechanisms of these cosolvents on protein structure we perform explicit all-atom molecular dynamics simulations of extended poly-alanine and poly-leucine dimers. We use an umbrella sampling protocol to compute the potential of mean force (PMF) of dimers at different concentrations of urea and TMAO. We find that the large nonpolar side chain of leucine is affected by both urea and TMAO whereas backbone atoms and alanine’s side chain are not. Urea is found to occupy positions between leucine’s side chains that are not accessible to water. This accounts for extra Lennard-Jones bonds between urea and side chains that provide the enthalpic driving force for unfolding. 262-Pos Board B42 FCS on Proteins in Crowded Environments Alyazan Albarghash1,2, Daryan Kempe1, Niklas Ole Junker1, Birgit Simone Hillebrecht1, Friedemann Melchior Landmesser1, Jo¨rg Fitter1,2. 1 I. Physikalisches Institut I.A., RWTH Aachen University, Aachen, Germany, 2Institute for Complex Systems - Molecular Biophysics (ICS-5), Forschungszentrum Ju¨lich, Ju¨lich, Germany. The most of our knowledge about proteins is mainly gained from in-vitro measurements. However, the results obtained from in-vitro measurements are likely to be deviated from the explicit natural characteristics of the proteins in the living cell. Yet, the technical difficulty of labeling proteins inside the living cell hinders carrying out in-vivo fluorescence spectroscopic studies. An approach to get closer to the complex nature of the cellular cytoplasm is to roughly mimic it by an artificial crowded-environment. Fluorescence Correlation Spectroscopy (FCS) is utilized to study diffusion effects of fluorescently labeled molecules. The implication of FCS in studying proteins mobility in a crowded-medium is limited by several technical obstacles such as distortion of the molecule detection volume[1] and switching from Brownian diffusion regime to hindered diffusion[2] regime. A pre-characterization of the viscosity and refractive index of investigated solutions and their effects on the validity of the measurement has been carried out [3]. Further, FCS was utilized to investigate the diffusional phenomena of various proteins dissolved in solutions containing classes of crowders [4]. References [1] C. B. Mu¨ller, T. Eckert, A. Loman, J. Enderlein, and W. Richtering, Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding, Soft Matter, vol. 5, pp. 13581366, 2009. [2] Phillips, R. J. (2000). A hydrodynamic model for hindered diffusion of proteins and micelles in hydrogels. Biophysical Journal, 79(6), 3350-3353. [3] Junker N. (2014). Bachelor project. RWTH Aachen. [4] Hillebrecht B. S. (2015). Bachelor project. RWTH Aachen. 263-Pos Board B43 Modeling Macromolecular Crowding through Translational and Rotational Diffusion of Small Molecular Probes Megan Currie1, Brenden Berry1, Taylor Ward1, Erin D. Sheets1,2, Ahmed A. Heikal1. 1 Department of Chemistry & Biochemistry, University of Minnesota, Duluth, Duluth, MN, USA, 2Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota, Duluth, Duluth, MN, USA. Living cells are crowded with macromolecules such as proteins, DNA, RNA, biomembranes, actin filaments, and a wide range of organelles. Macromolecular crowding in living cells is believed to affect processes such as protein folding, biochemical reactions and diffusion, which are essential for cell survival, function, and ultimately biomedical implications. Although a large body of work using myriad model systems and experimental methods has been done, the role of crowding on molecular processes is not fully understood. In this contribution, we used polymers (Ficoll-70) and proteins (BSA, ovalbumin) as biomimetic crowding agents as a means to quantify their effect on
the diffusion mechanism of a small molecular probe (namely, rhodamine green, RhG). To distinguish between crowding and viscosity effects on diffusion, control studies were carried out on glycerol-enriched buffer under the same experimental conditions. The viscosity of these crowding and control environments was quantified independently using viscometer and rheology methods. In contrast with glycerol-rich buffers, the rotational diffusion measurements using time-resolved anisotropy indicate binding reaction (specific or transient due to weak interactions) that is dependent on the concentration and type of the crowding agents. These results on the fast time scale guide our modeling of the translational diffusion of RhG using fluorescence correlation spectroscopy (FCS) as a function of crowding agents. Our results on both translational and rotational diffusion studies of RhG indicate deviation from the StokesEinstein model as a function of the concentration and type of the crowding agents. Our studies represent a step forward in the collective effort aimed at elucidating the role of macromolecular crowding towards quantitative cell biology. 264-Pos Board B44 Effect of Co-Solutes on Model Reaction Equilibria: Might Changes in the Free Energy of Bulk Water be the Underlying Cause? Daryl K. Eggers. Chemistry, San Jose State University, San Jose, CA, USA. Studies that characterize binding interactions in the presence of other solutes are necessary for understanding molecular interactions in vivo, including drug interactions. Unfortunately the binding equations of classical thermodynamics were derived for ideal dilute solutions, whereas biological solutions deviate greatly from the ideal assumption. This work examines model binding reactions in the presence of various co-solutes at molar concentrations using isothermal titration calorimetry and solubility measurements. The results are analyzed with original equations that acknowledge the participation of water molecules in the balanced reaction. The thermodynamic framework treats the free energy of bulk water as a variable that depends on the chemistry and concentration of the co-solute. A major concept behind this approach is the idea that changes in reaction equilibria (K values or transfer free energies) may be attributed to changes in the desolvation energy of the reactants; an equilibrium can shift in either direction, depending on whether the co-solute increases or decreases the average free energy of the bulk water because this term is a defining component of the desolvation energy. The outcomes of this work are consistent with the hypothesis that co-solutes do not need to interact directly with one of the reactants in order to alter the equilibrium, though preferential interactions between solute and reactant may be apparent in many systems. 265-Pos Board B45 Exploration of Weak Interactions between Folate and Glycine-Betaine Purva P. Bhojane, Michael R. Duff, Elizabeth E. Howell. Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA. In vitro studies with two different dihydrofolate reductases (EcDHFR, E.coli chromosomal and R67 DHFR, plasmid encoded) have shown that weak interactions between osmolytes and the substrate, dihydrofolate, decreases its affinity towards these enzymes. The unique changes in binding affinity with water activity for each osmolyte indicate preferential interactions between osmolyte and folate and its derivatives. Characterization of these interactions is essential for better understanding of in vivo effects of folate and its various redox states with available functional groups inside the cell. Quantitation of weak interactions between folate and glycine-betaine using a vapor pressure osmometry method yields a preferential interaction coefficient, or m23/RT value. This provides a scale for measuring the preference of folate for glycine-betaine relative to water. The predicted m23/RT value for folate using an accessible surface area calculation indicates equal preference for water and glycine-betaine. Experimental measurements found a folate concentration dependence of the m23/RT values, consistent with dimerization of folate. Studies with other model compounds suggest aromatic rings prefer to interact with glycine-betaine as compared to water. Our results also indicate neutral folate preferentially interacts with glycinebetaine whereas the anionic form excludes glycine-betaine. Can m23/RT values be used to predict osmotic stress effects on ligand binding? In some cases, yes. However, the caveat is whether all the ligand atoms are used in binding. As glutamate excludes glycine-betaine, calculation of the m23/RT value for polyglutamylated folates (pteroyltetra-g-glutamate (PG4)) predicts an overall exclusion of glycine-betaine. This should translate into tighter binding of PG4 to DHFR. Our studies found glycine-betaine addition weakens binding of both folate and PG4 to R67 DHFR to similar extents. Similar effects were observed for folate binding to EcDHFR. These results indicate that the additional glutamates do not contribute to binding to DHFR.