Session W2: Focus Session: Solvation, Dynamics, and Reactivity in Complex Environments IV

Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.   

Myosin-II sets the optimal response time scale of chemotactic amoeba

The response dynamics of the actin cytoskeleton to external chemical stimuli plays a fundamental role in numerous cellular functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin-II. Here we investigate the role of myosin-II in the response of the actin system to external stimuli. We used a microfluidic device in combination with a photoactivatable chemoattractant to apply stimuli to individual cells with high temporal resolution. We directly compare the actin dynamics in Dictyostelium discodelium wild type (WT) cells to a knockout mutant that is deficient in myosin-II (MNL). Similar to the WT a small population of MNL cells showed self-sustained oscillations even in absence of external stimuli. The actin response of MNL cells to a short pulse of chemoattractant resembles WT during the first 15 sec but is significantly delayed afterward. The amplitude of the dominant peak in the power spectrum from the response time series of MNL cells to periodic stimuli with varying period showed a clear resonance peak at a forcing period of 36 sec, which is significantly delayed as compared to the resonance at 20 sec found for the WT. This shift indicates an important role of myosin-II in setting the response time scale of motile amoeba.   

Fluorescent Dendrimer Nanoconjugates as Advanced Probes for Biological Imaging

Recent advances in fluorescence microscopy have enabled improvements in spatial resolution for biological imaging. However, there is a strong need for development of advanced fluorescent probes to enable a molecular-scale understanding of biological events. In this work, we report the development of a new class of probes for fluorescence imaging based on dye-conjugated dendrimer nanoconjugates. We utilize molecular-scale dendritic scaffolds as fluorescent probes, thereby enabling conjugation of multiple dyes and linkers to the scaffold periphery. In particular, we use polyamidoamine dendrimers as molecular scaffolds, wherein dye conjugation can be varied over a wide range. Single molecule fluorescence imaging shows that dendrimer nanoconjugates are far brighter than single fluorophores, resulting in increased localization precision. In addition, we further developed a new set of remarkably photostable probes by conjugating photoprotective triplet state quenchers directly onto the dendritic scaffold. We observe large increases in the photobleaching times compared to single dyes and reduced transient dark states (blinking). Overall, we believe that these new probes will allow for single molecule imaging over long time scales, enabling new vistas in biological imaging.   

Preferential melting of secondary structures during protein unfolding in different solvents: Competition between hydrophobic solvation and hydrogen bonding

Aqueous binary mixtures such as water-DMSO, water-urea, and water-ethanol are known to serve as denaturants of a host of proteins, although the detailed mechanism is often not known. Here we combine studies on several proteins in multiple binary mixtures to obtain a unified understanding of the phenomenon. We compare with experiments to support the simulation findings. The proteins considered include (i) chicken villin head piece (HP-36), (ii) immunoglobulin binding protein G (GB1), (iii) myoglobin and (iv) lysozyme. We find that for amphiphilic solvents like DMSO, the hydrophobic groups and the strong hydrogen bonding ability of the \textgreater S$=$O oxygen atom act together to facilitate the unfolding. However, the hydrophilic solvents like urea, due to the presence of more hydrophilic ends (C$=$O and two NH$_{\mathrm{2}})$ has a high propensity of forming hydrogen bonds with the side-chain residues and backbone of beta-sheet than the same of alpha helix. Such diversity among the unfolding pathways of a given protein in different chemical environments is especially characterized by the preferential solvation of a particular secondary structure.   

The $gp41_{659-671}$ HIV-1 antibody epitope: a structurally challenging small peptide

We present the results of extensive Molecular Dynamics (MD) simulations of the tridecapeptide corresponding to residues 659-671 of the envelope glycoprotein gp41 of HIV-1, which spans the 2F5 monoclonal antibody epitope ELDKWA. The most recent AMBER force fields ff99SB and ff12SB in both implicit and explicit solvents have been used for a cumulative time longer than 7.2 $\mu s$. We have analyzed the conformational ensembles of the peptide both with and without applied tensile restraints, and found that: (1) The amount of helical populations is important in aqueous solution, but this structure forms part of a flexible conformational ensemble with a rugged free energy landscape with shallow minima, which agrees well with the bulk of the experimental observations; (2) our results are more consistent with the experimental results than those from previous simulations; (3) under uniaxial tension, the disordered peptide first becomes fully helical before melting into turns, loops and $3_{10}$-helices.   

Solvation dynamics and enzyme catalysis in a designed enzyme undergoing directed evolution

We explore whether catalysis of a de novo designed enzyme-substrate complex is correlated to necessary solvent fluctuations to induce a chemical reaction. By studying a designed KEMP Eliminase as it goes through rounds of directed evolution to improve it's catalytic activity, we have found that catalytic activity correlates with an increase in density and structure of water near the active site. This suggests fluctuations in the solvation water near the active site couple to fluctuations in KEMP Eliminase to facilitate the catalytic process. To flesh this idea out, we are studying the progression of vibrational properties and cooperative fluctuations of solvation water by simulating the terahertz observable.   

Environmental Affects on Surfactin Studied Using Multidimensional Infrared Spectroscopy

Surfactin, a cyclic lipopeptide produced by Bacillus subtilis, is a pore forming toxin that has been studied in the literature extensively. It is known to exist in two different conformations, S1 and S2, which are thought to relate to surfactin's pore forming ability. The vibrational characteristics of surfactin have been studied using linear infrared spectroscopy as well as two-dimensional infrared spectroscopy in different environments. The environments probed were specifically chosen to mimic surfactin in an aqueous environment as well as a lipid membrane environment. The vibrational spectra were interpreted using transitional dipole coupling to relate the coupling evident in the data to the structural conformers obtained from NMR data. These measurements have been used to link the structural characteristics of surfactin to different solvent environments to gain insight into surfactin's pore forming ability mechanisms.   

Effect of surfaces in modulating protein folding mechanisms

Protein-surface interactions are ubiquitous in the crowded cytosol, where proteins encounter a variety of surfaces, ranging from membranes surfaces, to the surfaces presented by chaperone molecules. Protein-surface interactions are also at the heart of a number of emerging technologies, including protein micro-arrays, biosensors and biomaterials. The effect of surfaces on protein structure and stability can vary substantially depending on the chemical composition of the surface. In this talk, I will present detailed atomistic simulations of the folding of a small beta-sheet protein in the presence of graphite and titanium oxide surfaces. The role of water-mediated and direct protein-surface interactions in governing protein conformations will be discussed.   

Solvation in Surfactant Films at the Water-Air Interface

Surfactant films at water-air interfaces are used as models for environmental systems and biological membranes. This presentation describes surface sensitive experiments probing solvation of phenylalanine aggregates in surfactant films used as model membranes. Solvation in this complex environment is different from solvation in the bulk aqueous phase, with interesting changes to chemical and morphological structure of these surface films. Consequences of the changes induced in model membranes by the solvation of biological aggregates will be discussed using results of molecular dynamics simulations.   

Temperature Dependent Solvation Dynamics of the Chromophore Environment in the Far-Red Fluorescent Protein mPlum

We used molecular dynamics (MD) simulations to investigate the solvation dynamics of the chromophore environment in the far red fluorescent protein mPlum. Low temperature experiments on mPlum show a reduced Stokes shift compared to the room temperature. This suggests that the flexibility of the chromophore environment is related to the large Stokes shift in the red fluorescent protein mPlum. We performed MD simulations at various temperatures and systematically explored the protein-chromophore hydrogen bond pattern as well as the dynamics of the internal water molecules near the chromophore. We also investigated the dynamics of the hydrogen bond formed between residues E16 and I65, which is considered to play an important role in the observed large red shift in the emission spectrum. We quantify the hydrogen bonding pattern as a function of temperature to show how the Stokes shift in mPlum is correlated to the E16-I65 hydrogen bond dynamics.   

QTES-DFTB dynamics study on the effect of substrate motion on quantum proton transfer in soybean lipoxygenase-1

It has been shown that the proton transfer in the enzymatic active site of soybean lipoxygenase-1 (SLO-1) occurs largely by a quantum tunneling mechanism. This study examined the role of local substrate vibrations on this proton tunneling reaction. We employ an approximate quantum trajectory (QT) dynamics method with linear quantum force. The electronic structure (ES) was calculated on-the-fly with a density functional tight binding (DFTB) method. This QTES-DFTB method scales linearly with number of trajectories, and the calculation of the quantum force is a small addition to the overall cost of trajectory dynamics. The active site was represented as a 44-atom system. Quantum effects were included only for the transferring proton, and substrate nuclei were treated classically. The effect of substrate vibrations was evaluated by freezing or relaxing the substrate nuclei. Trajectory calculations were performed at several temperatures ranging from 250-350 K, and rate constants were calculated through the quantum mechanical flux operator which depends on time-dependent correlation functions. It was found that the substrate motion reliably increases the rate constants, as well as the P/D kinetic isotope effect, by approximately 10\% across all temperatures examined.