Probing Membrane Insertion Activity of Antimicrobial Polymers via Coarse-grain Molecular Dynamics

Knowledge of the mechanism of action of antimicrobial agents is crucial for the development of new compounds to combat microbial pathogens. To this end, computational studies on the interaction of known membrane-active antimicrobial polymers with phospholipid bilayers reveal spontaneous membrane insertion and cooperative action at low and high concentrations, respectively. In late-stage attack, antimicrobials cross the membrane core and occasionally align to provide a stepping-stone pathway for water permeation; this suggests a possible new mode of action that does not depend on pore formation for transport to and across the inner leaflet. The computations rationalize the observed activity of a new class of antimicrobial compounds.     

De novo design of a redox-active minimal rubredoxin mimic

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.     

Computational design of a self-assembling β-peptide oligomer

The first computationally designed self-assembling oligomer consisting of exclusively β-amino acids (βAAs) is presented. The packing of a β-3(14) helix into coiled-coils of varying stoichiometries as a function of amino acid sequence is examined. β-Peptides with hVal repeating every third residue in the sequence appeared to have a strong propensity to pack into hexameric bundles. The designed sequence was synthesized and characterized with CD spectroscopy, NMR, and analytical ultracentrifugation, suggesting that the peptide adopts a well-folded hexameric structure.     

Polar networks control oligomeric assembly in membranes

Polar interactions have a profound influence on membrane stability and structure. A membrane-solubilized GCN4 peptide, MS-1, is used to study the impact of polar networks. Amide functionalities from amino acid side chains have been shown to promote peptide oligomerization, but lacked specificity. Herein, the hydrogen bonding interactions of an Asn side chain are coupled with the hydroxyl of Ser or Thr to generate a polar network. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicate that a trimer assembly is established where each membrane-embedded hydrogen bond contributes 1 kcal mol-1.     

De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.     

Controlling the conformation of arylamides: computational studies of intramolecular hydrogen bonds between amides and ethers or thioethers

The role of an ortho-alkylthioether group in controlling the conformation around the ring-N bonds of meta-connected arylamide oligomers is studied. Density functional theory (DFT) geometries of model compounds, including acetanilide, an ether acetanilide, and a thioether acetanilide, and their corresponding diamides, show that for either monoamide or diamide the alkyl side chain of the thioether should be perpendicular to the aryl plane, whereas for the ether monoamide, the alkyl side chain is in the aryl plane. DFT ring-N torsional potentials and constrained geometries of the model compounds demonstrate that carbonyl-S repulsion leads to a high torsional barrier and that intramolecular N-H...S and C-H...O hydrogen bonds and ring-amide conjugation lead to N-H having a preferred orientation in the benzene plane pointing towards S. The N-H bond lengthens and the ortho-ring C-H bond shortens in a regular pattern in the approach to the preferred orientation. Calculated IR frequencies for the N-H stretch show a clear red shift between model compounds without and with the thioether side chain.     

Simulated evolution of emergent chiral structures in polyalanine

The relationship between monomer chirality and polymer structure has been studied using both theoretical and experimental methods. Atomistic models, such as the ones employed in computational protein folding and design, can be used to study the relationship between monomer chirality and the properties of polypeptides. Using a simulated evolution approach that combines side-chain epimerization with backbone flexibility, we recapitulate the relationship between basic forces that drive secondary structure formation and sequence homochirality. Additionally, we find heterochiral motifs including a C-terminal helix capping interaction and stable helix-reversals that result in bent helix structures. Our studies show that simulated evolution of chirality with backbone flexibility can be a powerful tool in the design of novel heteropolymers with tuned stereochemical properties.