Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

Abstract

Since their initial discovery, 30 years ago, antimicrobial peptides (AMPs) have been intensely investigated as a possible solution to the increasing problem of drug-resistant bacteria. The interaction of antimicrobial peptides with the cellular membrane of bacteria is the key step of their mechanism of action. Fluorescence spectroscopy can provide several structural details on peptide-membrane systems, such as partition free energy, aggregation state, peptide position and orientation in the bilayer, and the effects of the peptides on the membrane order. However, these "low-resolution" structural data are hardly sufficient to define the structural requirements for the pore formation process. Molecular dynamics simulations, on the other hand, provide atomic-level information on the structure and dynamics of the peptide-membrane system, but they need to be validated experimentally. In this review we summarize the information that can be obtained by both approaches, highlighting their versatility and complementarity, suggesting that their synergistic application could lead to a new level of insight into the mechanism of membrane destabilization by AMPs.

How Many Antimicrobial Peptide Molecules Kill a Bacterium? The Case of PMAP-23

“…In these cases, titration with bacterial cells did not cause significant variations in the fluorescent signal (Supplementary Figure 3), demonstrating the absence of any relevant scattering-related artifacts. 17,21 This conclusion was confirmed also by following peptide/bacteria association through variations in the dansyl fluorescence time-decay, which is not affected by scattering (Supplementary Figure 4). We calculated the fraction of peptide bound to bacterial membranes from the a Determined as the fraction of colony forming units (CFU) in a sample incubated for 2 h with different peptide concentrations, as compared with the CFU of the control at time zero, i.e., (4.5 ± 0.5) × 10 8 CFU/mL.…”

“…To determine peptide binding to bacterial cells, we used fluorescence spectroscopy, due to the high sensitivity of this technique. 17 We ensured selective detection of the peptide signal by labeling PMAP-23 at the N-terminus with a dansyl (5-(dimethylamino)naphthalene-1-sulfonyl chloride) moiety, a probe extremely sensitive to the environment polarity that can be excited at wavelengths where intrinsic fluorophores do not absorb. 18 Labeling slightly affected peptide activity, causing an increase in the MIC by a factor of 2 (data not shown).…”

Integrated Approach to Structure-Based Enzymatic Drug Design: Molecular Modeling, Spectroscopy, and Experimental Bioactivity

“…37 Computer-aided drug design forms part of the research strategy called structure-based design, which is an iterative combination of experimental and computer-based approaches. The results of both of these approaches can be combined to generate hypotheses and ideas (and test them) about the Table 2 Infrared spectroscopy a Signals from amide groups (CONH) in proteins are quite prominent and extensively used to determine structural information about proteins; phosphodiester groups are sensitive to hydrogen bonding; lipids can be studied through the ester group 1, 5, and 6 NMR spectroscopy Measures 1 H, 13 C, 15 N, and even some magnetic resonance signals of metal ions in biomolecules; it can provide structural information (through-space proton−proton interactions (NOE), backbone torsional angles, etc.) to study inhibitor/enzymes complexes; it provides solution-phase information and is a relatively fast technique 1, 3.2.2, 3.4, 5, and 6…”

Designing Antimicrobial Peptide: Current Status

“…electrostatics) 37 . MD simulations are making a noticeable progress clarifying peptide-bilayer interactions mechanisms 52 . Nevertheless, the results should be validated using experimental data due to the empirical nature of force fields and their lack of "utter" prediction accuracy 37 .…”