The objectives of this project are to understand the fundamental mechanism of the interaction between antimicrobial peptides (AMP) and lipid membranes and to understand the basic mechanism of pore formation in lipid membranes by AMPs. To accomplish the first goal, the Principal Investigator (PI) will perform all atom computer simulations in order to calculate the free energy of peptide adsorption and its permeation across the lipid membrane. The microscopically detailed information that will be obtained from simulations will allow the PI to determine the energetic gain upon peptide adsorption and the pathway of peptide permeation across the membrane. The second specific goal of the project will be accomplished by performing simulations on systems containing multiple peptides and a bilayer. The bilayer will contain either one kind of a lipid or will be composed of a mixture of lipids having biological significance. The role of some specific lipids present in the membrane will be carefully considered. For example, the recent experimental study of cholesterol influence on action of AMPs produced controversial results and interpretations. The molecular detailed picture provided by simulations will be extremely useful to resolve these controversies. To understand the molecular architecture of pores in cell membranes, the PI will also perform comparative simulations on systems containing different AMPs. In general, simulations performed by the PI will provide a link between experiment, phenomenological theory and the microscopic details responsible for AMP action in membranes.
Broader Impacts Work on the project will provide essential training in the biophysics of membranes for undergraduate and graduate students and postdoctoral fellows. The results from the research will be discussed in the lectures the PI gives in a course on membranes which is taught as a module in the Molecular and Cellular Biophysics program at the University of North Carolina. A less quantitative version of these lectures is given in the Biophysical Society summer course in biophysics for minority students. The objective of this course is to introduce minority students from colleges and universities lacking formal programs in biophysics to the exciting world of mechanistic and structural biology, and the possibility of a career in biophysics. Some of the students from the course also get hands on experience in membrane biophysics by performing research in the PI's laboratory. The PI is also participating in the work of the International Graduate Research Training Group, which includes faculty and students, mainly from Germany and North Carolina. Under the framework of this Group's activities, students from the laboratories in Germany will be able to visit the PI's laboratory and work on the projects related to the study of peptide/membrane interactions, while students from the PI's group will be able to visit Germany.
Computer simulation techniques such as Monte Carlo and/or Molecular Dynamics that are used to study biological processes have substantially improved over the past few years. Simulations play a particular important role in cases where experimental techniques are not able to provide atomistic information. One example is biological membranes and proteins that interact with them. A large number of proteins and membranes can fall under this category, however the focus of our research is antimicrobial peptides. Antimicrobial peptides (AMPs) are a group of proteins that belong to the innate immune system in organisms. They are the front line of general defense against exogenous organisms before a more specific response is established. Their target is the biological membrane, where they interact and interfere with the natural processes occurring in the membrane. Our research is concentrated on the study of the interaction between model biological membranes consisting of phospholipid bilayers and AMPs such as melittin. The tool we use is the Molecular Dynamics computer simulation technique. Melittin is one of the most widely studied members of the AMP family; it is a protein with 26 amino acids and it is the most predominant component of the bee venom. Its main function is to increase membrane permeability and when the target of melittin is a unicellular organism this function can be lethal, since membrane serves as the main protection for the organism. The mechanism by which antimicrobial peptides, including melittin, work is not clearly understood and the molecular details of this process need to be discovered. Experiments indicated that melittin disrupts membranes by creating pores at concentrations above a critical peptide to lipid ratio. Our simulations were performed at peptide/lipid ratios below and above critical and we observed pores only when the ratio was above critical. Pores were created when peptides were assembled on the membrane surface and they formed a shape similar to a "wedge" which facilitated membrane disruption. We also observed a reorientation of peptides from the surface to a trans-membrane state assembling into a disordered pore. A flux of water molecules flowing through the pore could also be observed. After a period of ~100-200 nanoseconds the pore disassembled, thus confirming the transient character of the pore (see Figure 1). Experiments indicate that in the case of melittin the period when pores have a transient character is followed by an equilibrium state with permanent pores made out of four to seven melittin molecules. The detailed molecular architecture of pores is not known; are the pores regular or chaotic in their geometry? Can simulations help in answering this question? Unfortunately we cannot follow by simulations the whole process of transient pore creation with the subsequent transition to an equilibrium situation containing permanent melittin pores, because this process is too long in terms of molecular time computers are able to simulate. But we can prepare pores with different architecture and study what kind of a pore produces results that are most consistent with experiments. We did exactly this and observed that pores where peptides have a symmetric arrangement with their N-termini located on both leaflets of the bilayer produced pore architecture consistent with experimental results (Figure 2). To understand the thermodynamic and kinetic factors responsible for pore creation in bilayers we performed free energy calculations for melittin reorientation from the surface to the trans-membrane state. We observed that when melittin/lipid ratio was below critical a large free energy barrier exist for the reorientation. This barrier is substantially reduced when melittin/lipid ratio is above critical. We also observed that free energy barrier for the reorientation of the second melittin, given that the first melittin is already in a trans-membrane state, is small (Figure 3). This tells us that pore creation by melittin peptides has a collective or cooperative character. As of today we know that around a thousand of different AMPs exist. Do they act in the same way? Experiments indicate that although all of them destroy membranes, the detailed mechanism of action is different. For example, some experiments indicate that an AMP magainin-2 creates large pores, but peptides do not line up the walls of the pores. Our simulations indeed showed that magainin-2 creates large pores but the walls of these pores are made of peptides. Work on the projects supported by the NSF provided essential training in interdisciplinary field of biophysics for undergraduate students, graduate students and postdoctoral fellows. During the last four years covered by the grant, four undergraduate, three graduate and three post-doctoral fellows worked in the laboratory on projects related to peptide/membrane interactions. It should be also mentioned that the work we do on understanding the fundamental issues related to the peptide-membrane interactions provides knowledge needed to construct new synthetic antibiotics; a very important task now, when the present generation of antibiotics is losing its potency.
