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Proteins of halophilic organisms that accumulate molar concentrations of KCl in their cytoplasm have much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity: either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations, which would stabilize the folded protein. In the course of this Ph.D. study, we investigated these possibilities using atomistic molecular dynamics simulations and classical force fields. High quality parameters describing the interaction between K+ and carboxylate groups present in acidic amino acids are indispensable for this study. We first evaluated the quality of the default parameters for these ions within the widely used AMBER ff14SB force field for proteins and found that they perform poorly. We propose new parameters, which reproduce solution activity derivatives of potassium acetate solutions up to 2 mol/kg and the distances between potassium ions and carboxylate groups observed in x-ray structures of proteins. To understand the role of acidic amino acids in protein hydration, we investigated this aspect for 5 halophilic proteins in comparison with 5 mesophilic ones. Our results do not support the necessity of acidic amino acids to keep folded proteins hydrated. Proteins with a larger fraction of acidic amino acids indeed have higher hydration levels. However, the hydration level of each protein is identical at low (b_KCl = 0.15 mol/kg) and high (b_KCl = 2 mol/kg) KCl concentration. It has also been proposed that cooperative interactions between acidic amino acids with nearby hydrated cations stabilize the folded protein and slow down its solvation shell; according to this theory, the cations would be preferentially excluded from the unfolded structure. We investigate this possibility through extensive free energy calculation simulations. We find that cooperative interactions between neighboring acidic amino acids exist and are mediated by the ions in solution but are present in both folded and unfolded structures of halophilic proteins. The translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; therefore, such a cooperative effect does not result in unusually slow solvent dynamics as has been suggested.
Membrane adhesion is a fundamental biological process in which membranes are attached to neighboring membranes or surfaces. Membrane adhesion emerges from a complex interplay between the binding of membrane-anchored receptors/ligands and the membrane properties. In this work, we study membrane adhesion mediated by lipid-anchored saccharides using microsecond-long full-atomistic molecular dynamics simulations. Motivated by neutron scattering experiments on membrane adhesion via lipid-anchored saccharides, we investigate the role of LeX, Lac1, and Lac2 saccharides and membrane fluctuations in membrane adhesion.
We study the binding of saccharides in three different systems: for saccharides in water, for saccharides anchored to essentially planar membranes at fixed separations, and for saccharides anchored to apposing fluctuating membranes. Our simulations of two saccharides in water indicate that the saccharides engage in weak interactions to form dimers. We find that the binding occurs in a continuum of bound states instead of a certain number of well-defined bound structures, which we term as "diffuse binding".
The binding of saccharides anchored to essentially planar membranes strongly depends on separation of the membranes, which is fixed in our simulation system. We show that the binding constants for trans-interactions of two lipid-anchored saccharides monotonically decrease with increasing separation. Saccharides anchored to the same membrane leaflet engage in cis-interactions with binding constants comparable to the trans-binding constants at the smallest membrane separations. The interplay of cis- and trans-binding can be investigated in simulation systems with many lipid-anchored saccharides. For Lac2, our simulation results indicate a positive cooperativity of trans- and cis-binding. In this cooperative binding the trans-binding constant is enhanced by the cis-interactions. For LeX, in contrast, we observe no cooperativity between trans- and cis-binding. In addition, we determine the forces generated by trans-binding of lipid-anchored saccharides in planar membranes from the binding-induced deviations of the lipid-anchors. We find that the forces acting on trans-bound saccharides increase with increasing membrane separation to values of the order of 10 pN.
The binding of saccharides anchored to the fluctuating membranes results from an interplay between the binding properties of the lipid-anchored saccharides and membrane fluctuations. Our simulations, which have the same average separation of the membranes as obtained from the neutron scattering experiments, yield a binding constant larger than in planar membranes with the same separation. This result demonstrates that membrane fluctuations play an important role at average membrane separations which are seemingly too large for effective binding. We further show that the probability distribution of the local separation can be well approximated by a Gaussian distribution. We calculate the relative membrane roughness and show that our results are in good agreement with the roughness values reported from the neutron scattering experiments.