Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.

An ideal bioanalytical method would allow carrying out sensitive, selective, reproducible, fast, and in situ chemical measurements of the composition, structure and function of complex biological systems. Physical chemistry possesses advanced structure analyzing techniques which are suitable to solve, at a sub-molecular level, the structure of biomolecules. However, the prerequisite of the presence of the electrolyte solution limits the number of applicable structure analyzing techniques. Infrared spectroscopy is one of the most powerful analytical techniques used to identify the structure of biomolecules, monitor structural changes under various experimental environments and physical states. In addition, infrared reflection absorption spectroscopy (IRRAS) has been successfully applied to study supramolecular films at the solid|liquid interface. Assemblies of biomolecules belong to a particularly demanding because they bring specific for the maintenance of their functions experimental requirements which have to be taken into account planning in situ spectroelectrochemical experiments. In this paper potnential of in situ IRRAS under electrochemical control for studies of structural changes in assemblies of biomolecules such as lipid bilayers and protein films is described. Studies of different classes of biomolecules bring certain experimetnal conditions concerning the electrolyte composition, pH value, temperature or chemical nature of the solid support, which have to be fulfilled. Obviously, these conditons may significantly restrict the applicability of the in situ PM IRRAS. The selection of the solid substrate with required optical and electrical properties, the optical window and the electrolyte composition have to be adapted to the needs of biomolecules. Adjustement of experimetnal conditions as well as possibilites of the enlargement of the in situ PM IRRAS for studies of biomimetic assemblies is reviewed in this paper. Furthermore, experimetnal resutls reporting potenial driven changes in models of cell membranes and protein films are reviewed. Pioneering studies aiminig at the determination of structural changes in lipid membranes exposed to physiological electric fields and interacting simultaneously with protiens are described.