Calculating the Free Energy of Association of Transmembrane Helices

AbstractA large number of experimental studies have been devoted to quantifying the interaction between transmembrane (TM) helices in detergent micelles and, more recently, in bilayers. Theoretical calculation of association free energy of TM helices would be useful for predicting the propensity of given sequences to oligomerize and for understanding the difference between association in micelles and in bilayers. In this article, the theoretical foundation for calculating the standard association free energy of TM helices is laid out and is applied to glycophorin A in both micelles and bilayers. The standard association free energy is decomposed into the effective energy, translational, rotational, and conformational entropy terms. The effective energy of association is obtained by molecular dynamics simulations in an implicit membrane model. The translational and rotational entropy of association is calculated from the probability distribution of the translational and rotational degrees of freedom obtained from the molecular dynamics simulations. The side-chain conformational entropy of association is estimated from the probability distribution obtained by rigid rotation of all side-chain dihedral angles. The calculated standard association free energy of glycophorin A in N-dodecylphosphocholine micelles is in good agreement with the experimental value. The translational entropy cost is larger, whereas the rotational entropy cost is smaller in bilayers than in micelles. The standard association free energy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers is calculated to be ∼1.3 kcal/mol more favorable than in N-dodecylphosphocholine micelles, consistent with available experimental data

Membrane Interactions and Pore Formation by the Antimicrobial Peptide Protegrin

AbstractProtegrin is an antimicrobial peptide with a β-hairpin structure stabilized by a pair of disulfide bonds. It has been extensively studied by solid-state NMR and computational methods. Here we use implicit membrane models to examine the binding of monomers on the surface and in the interior of the membrane, the energetics of dimerization, the binding to membrane pores, and the stability of different membrane barrel structures in pores. Our results challenge a number of conclusions based on previous experimental and theoretical work. The burial of monomers into the membrane interior is found to be unfavorable for any membrane thickness. Because of its imperfect amphipathicity, protegrin binds weakly, at most, on the surface of zwitterionic membranes. However, it binds more favorably onto toroidal pores. Anionic charge on the membrane facilitates the binding due to electrostatic interactions. Solid-state NMR results have suggested a parallel NCCN association of monomers in dimers and association of dimers to form octameric or decameric β-barrels. We find that this structure is not energetically plausible for binding to bilayers, because in this configuration the hydrophobic sides of two monomers point in opposite directions. In contrast, the antiparallel NCCN and especially the parallel NCNC octamers are stable and exhibit a favorable binding energy to the pore. The results of 100-ns simulations in explicit bilayers corroborate the higher stability of the parallel NCNC barrel compared with the parallel NCCN barrel. The ability to form pores in zwitterionic membranes provides a rationalization for the peptide’s cytotoxicity. The discrepancies between our results and experiment are discussed, and new experiments are proposed to resolve them and to test the validity of the models

Protein Arcs May Form Stable Pores in Lipid Membranes

AbstractElectron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore

Activity Determinants of Helical Antimicrobial Peptides: A Large-Scale Computational Study

Antimicrobial peptides (AMPs), produced by a wide range of organisms, have attracted attention due to their potential use as novel antibiotics. The majority of these peptides are cationic and are thought to function by permeabilizing the bacterial membrane, either by making pores or by dissolving it (‘carpet’ model). A key hypothesis in the literature is that antimicrobial and hemolytic activity correlate with binding affinity to anionic and zwitterionic membranes, respectively. Here we test this hypothesis by using binding free energy data collected from the literature and theoretical binding energies calculated from implicit membrane models for 53 helical AMPs. We indeed find a correlation between binding energy and biological activity, depending on membrane anionic content: antibacterial activity correlates best with transfer energy to membranes with anionic lipid fraction higher than 30% and hemolytic activity correlates best with transfer energy to a 10% anionic membrane. However, the correlations are weak, with correlation coefficient up to 0.4. Weak correlations of the biological activities have also been found with other physical descriptors of the peptides, such as surface area occupation, which correlates significantly with antibacterial activity; insertion depth, which correlates significantly with hemolytic activity; and structural fluctuation, which correlates significantly with both activities. The membrane surface coverage by many peptides at the MIC is estimated to be much lower than would be required for the ‘carpet’ mechanism. Those peptides that are active at low surface coverage tend to be those identified in the literature as pore-forming. The transfer energy from planar membrane to cylindrical and toroidal pores was also calculated for these peptides. The transfer energy to toroidal pores is negative in almost all cases while that to cylindrical pores is more favorable in neutral than in anionic membranes. The transfer energy to pores correlates with the deviation from predictions of the ‘carpet’ model

Slow Dissociation of a Charged Ligand: Analysis of the Primary Quinone QA Site of Photosynthetic Bacterial Reaction Centers

Reaction centers (RCs) are integral membrane proteins that undergo a series of electron transfer reactions during the process of photosynthesis. In the QA site of RCs from Rhodobacter sphaeroides, ubiquinone-10 is reduced, by a single electron transfer, to its semiquinone. The neutral quinone and anionic semiquinone have similar affinities, which is required for correct in situ reaction thermodynamics. A previous study showed that despite similar affinities, anionic quinones associate and dissociate from the QA site at rates ≈104 times slower than neutral quinones indicating that anionic quinones encounter larger binding barriers (Madeo, J.; Gunner, M. R. Modeling binding kinetics at the QA site in bacterial reaction centers. Biochemistry2005, 44, 10994–11004). The present study investigates these barriers computationally, using steered molecular dynamics (SMD) to model the unbinding of neutral ground state ubiquinone (UQ) and its reduced anionic semiquinone (SQ–) from the QA site. In agreement with experiment, the SMD unbinding barrier for SQ– is larger than for UQ. Multi Conformational Continuum Electrostatics (MCCE), used here to calculate the binding energy, shows that SQ– and UQ have comparable affinities. In the QA site, there are stronger binding interactions for SQ– compared to UQ, especially electrostatic attraction to a bound non-heme Fe2+. These interactions compensate for the higher SQ– desolvation penalty, allowing both redox states to have similar affinities. These additional interactions also increase the dissociation barrier for SQ– relative to UQ. Thus, the slower SQ– dissociation rate is a direct physical consequence of the additional binding interactions required to achieve a QA site affinity similar to that of UQ. By a similar mechanism, the slower association rate is caused by stronger interactions between SQ– and the polar solvent. Thus, stronger interactions for both the unbound and bound states of charged and highly polar ligands can slow their binding kinetics without a conformational gate. Implications of this for other systems are discussed

Structure and Dynamics of a Fusion Peptide Helical Hairpin on the Membrane Surface: Comparison of Molecular Simulations and NMR

The conserved N-terminal residues of the HA2 subunit of influenza hemagglutinin (fusion peptide) are essential for membrane fusion and viral entry. Recent NMR studies showed that the 23-residue fusion peptide forms a helical hairpin that undergoes rocking motion relative to the membrane surface on a nanosecond time scale. To compare with NMR and to obtain a detailed molecular picture of the peptide–membrane interaction, we performed molecular dynamics simulations of the fusion peptide in explicit dimyristoylphosphatidylcholine and with the IMM1 implicit membrane model. To account for low and neutral pH conditions, simulations were performed with acidic groups (E11 and D19) protonated and unprotonated, respectively. The hairpin structure was stable in the simulations, with the N-terminal helix buried more deeply into the hydrophobic membrane interior than the C-terminal helix. Interactions between the tryptophans in the fusion peptide and phospholipid residues contribute to peptide orientation. Higher flexibility of the hairpin was observed in the implicit membrane simulations. Internal correlation functions of backbone N–H vectors were fit to the extended Lipari–Szabo model-free approach to obtain order parameters and correlation times. Good agreement with the NMR results was obtained for orientational fluctuations around the hairpin axis (rotation), but those around the perpendicular axis (tilting) were more limited in the simulations than inferred from the NMR experiments

Solvent Size vs Cohesive Energy as the Origin of Hydrophobicity

Two main physical explanations of hydrophobicity seem to be currently competing. The classical, intuitive view attributes it to the fact that interactions between water molecules are much stronger than those between water and nonpolar groups. The second, "heretic" view attributes it to the small size of the water molecule which increases the entropic cost of opening up a cavity to accommodate the solute. Here we examine the solvation of methane in water and in model liquids that lack one or more of water's properties and report a detailed decomposition of the solvation free energy, enthalpy, entropy, and heat capacity in these solvents. The results fully support the classical view. It is found that fluids with strong intermolecular interactions favor expulsion of methane to its pure phase or to CCl_4, whereas fluids with weak intermolecular interactions do not. However, the specific thermodynamic signature of the hydrophobic effect (entropy driven at room temperature with a large heat capacity change) is a result of the hydrogen-bonding structure of water