While many antimicrobial peptides (AMPs) disrupt bacterial membranes, some translocate into bacteria and interfere with intracellular processes. Buforin II and DesHDAP1 are thought to kill bacteria by interacting with nucleic acids. Here, molecular modeling and experimental measurements are used to show that neither nucleic acid binding peptide selectively binds DNA sequences. Simulations and experiments also show that changing lysines to arginines enhances DNA binding, suggesting that including additional guanidinium groups is a potential strategy to engineer more potent AMPs. Moreover, the lack of binding specificity may make it more difficult for bacteria to evolve resistance to these and other similar AMPs.

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides' mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties. (C) 2011 Elsevier B.V. All rights reserved.

Buforin II (BF2) is an antimicrobial peptide that is hypothesized to kill bacteria by entering cells and binding nucleic acids. To further investigate this proposed mechanism, we used computer modeling and experimental measurements to consider the interactions between BF2 and DNA. Computational and experimental results imply that the peptide forms specific interactions with DNA. Moreover, we observe a general correlation between DNA affinity and antimicrobial activity for a series of BF2 variants. Thus, our results support the proposed mechanism for BF2 and provide a useful approach for evaluating the nucleic acid interactions of other antimicrobial peptides. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mutations that alter the phenotypic behavior of the Escherichia coli mechanosensitive channel of small conductance (MscS) have been identified; however, most of these residues play critical roles in the transition between the closed and open states of the channel and are not directly involved in lipid interactions that transduce the tension response. In this study, we use molecular dynamic simulations to predict critical lipid interacting residues in the closed state of MscS. The physiological role of these residues was then investigated by performing osmotic downshock assays on MscS mutants where the lipid interacting residues were mutated to alanine. These experiments identified seven residues in the first and second transmembrane helices as lipid-sensing residues. The majority of these residues are hydrophobic amino acids located near the extracellular interface of the membrane. All of these residues interact strongly with the lipid bilayer in the closed state of MscS, but do not face the bilayer directly in structures associated with the open and desensitized states of the channel. Thus, the position of these residues relative to the lipid membrane appears related to the ability of the channel to sense tension in its different physiological states.

Molecular dynamics (MD) simulations play an increasingly important role in many aspects of biochemical research but are often not part of the biochemistry curricula at the undergraduate level. This article discusses the pedagogical value of exposing students to MD simulations and provides information to help instructors consider what software and hardware resources are necessary to successfully introduce these simulations into their courses. In addition, a brief review of the MD-based activities in this issue and other sources are provided. (c) 2016 by The International Union of Biochemistry and Molecular Biology, 44:118-123, 2016.

As computational modeling plays an increasingly central role in biochemical research, it is important to provide students with exposure to common modeling methods in their undergraduate curriculum. This article describes a series of computer labs designed to introduce undergraduate students to energy minimization, molecular dynamics simulations, and homology modeling. These labs were created as part of a one-semester course on the molecular modeling of biochemical systems. Students who completed these activities felt that they were an effective component of the course, reporting improved comfort with the conceptual background and practical implementation of the computational methods. Although created as a component of a larger course, these activities could be readily adapted for a variety of other educational contexts. As well, all of these labs utilize software that is freely available in an academic environment and can be run on fairly common computer hardware, making them accessible to teaching environments without extensive computational resources.

Thimet oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of thimet oligopeptidase in which Gly or Tyr residues in the 599-611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in k(cat) observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.