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Amphipathic peptides are accommodated within the diffuse gradient of polarity that characterizes the interfacial regions of phospholipid bilayer membranes. Interfacial membrane interactions are key to the diverse biological functions and activities of these peptides, which encompass a large class of antimicrobial peptides including the helical peptides magainin, melittin, and RTA3 derived from the commensal bacterium Streptococcus mitis. For these peptides in vitro efficacy (high antimicrobial activity with minimal mammalian cell toxicity, equivalent to high potential therapeutic index; PTI), can be broadly understood in relation to the thermodynamics of interfacial binding and membrane disruption in membranes having surface charges that correspond to bacterial and mammalian cell membranes, respectively. Peptides with disrupted amphipathicity resulting from a positively charged amino acid residue on the non-polar helix face, can have greatly enhanced PTI, although a balance of amphipathicity, hydrophobicity and positive charge is required for retention of high antimicrobial activity. These observations are illustrated with recent examples from the literature, and studies on RTA3 and magainin analogues from our laboratories. Despite the identification and optimisation of peptides with very good PTI, a focus on addressing toxicity upon systemic administration and poor in vivo efficacy is likely to be required to translate growing understanding of the relationships between peptide interfacial activity and effects on cells, into novel systemic therapeutics.

Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x-ray crystallographic models. In this article we detail the use of a molecular mechanics approach for suggesting regions of Bacillus stearothermophilus L-lactate dehydrogenase (EC 1.1.1.27) involved in substrate specificity, and hence areas of interest for protein engineers. The approach combines molecular dynamics with energy minimization (MD/EM) to search the conformational space available to a 15-Å sphere of the ternary complex centered on the catalytic histidine. The search is carried out by calculating a 30-ps dynamics trajectory at 300 K and minimizing structures at 1-ps intervals. The protocol has been performed on 14 systems containing different combinations of substrate and mutant /wt LDH. In order to discover which interactions are important in defining substrate specificity, eight conformational parameters representing substrate–active site interactions were measured in each of the 420 minimized structures. These parameters were then compared to the measured catalytic activity of the protein–substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using this methodolgy it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis. Proteins 29:228–239, 1997. © 1997 Wiley-Liss, Inc.

Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x‐ray crystallographic models. In this article we detail the use of a molecular mechanics approach for suggesting regions of Bacillus stearothermophilusL‐lactate dehydrogenase (EC 1.1.1.27) involved in substrate specificity, and hence areas of interest for protein engineers. The approach combines molecular dynamics with energy minimization (MD/EM) to search the conformational space available to a 15‐Å sphere of the ternary complex centered on the catalytic histidine. The search is carried out by calculating a 30‐ps dynamics trajectory at 300 K and minimizing structures at 1‐ps intervals. The protocol has been performed on 14 systems containing different combinations of substrate and mutant /wt LDH. In order to discover which interactions are important in defining substrate specificity, eight conformational parameters representing substrate–active site interactions were measured in each of the 420 minimized structures. These parameters were then compared to the measured catalytic activity of the protein–substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using this methodolgy it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis. Proteins 29:228–239, 1997. © 1997 Wiley‐Liss, Inc.