Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Agricultural and Biological Engineering

Committee Chair

Ganesan Narsimhan

Committee Member 1

Arun K. Bhunia

Committee Member 2

Osvaldo H. Campanella

Committee Member 3

Nathan S. Mosier

Committee Member 4

Xiao Zhu


As more and more microbes develop resistance to conventional antibiotics and the decline in the approval of new antibiotics in recent years, there is an increasing need for research on identification and design of new therapeutic alternatives. As a result, antimicrobial peptides (AMPs) are becoming one of the most promising alternative options to target pathogens without developing resistance. It is known that AMPs inactivate microorganisms by forming transmembrane pores in cell membrane through adsorption and aggregation. Understanding the detailed mechanism of inactivation by AMPs is necessary for developing new agent for antimicrobial treatment. This study mainly investigates antimicrobial peptides from four aspects: (i) the action of AMP melittin on lipid bilayer through molecular dynamics (MD) simulation, (ii) identification of AMPs from soy protein 7S globulin, (iii) interaction between curcumin and lipid bilayer through both MD simulation and varied experiments, and (vi) comparison of fluorescence dye leakage from liposome by melittin and its mutants. The first part of the study investigated the interaction of multiple melittin peptides with the lipid bilayer. Melittin is a naturally occurring antimicrobial peptide that has the ability to kill bacterial cells through cell membrane penetration leading to pore formation. In this investigation, all atom molecular dynamics (MD) simulation has been carried out to describe the interaction of 2,4 or 6 peptides placed on the surface of 3:1 ratio of 1,2 Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-di-(9Z-octadecenoyl)-sn-glycero3-phospho-(1'-rac-glycerol) (DOPG) lipid bilayer (a mimic of bacterial cell membrane) corresponding to protein-lipid (P/L) ratio of 2/96, 4/162 or 6/166 respectively. MD simulation was also carried out for (i) 1-8 transmembrane peptides (corresponding P/L ratio of 1/128 to 8/128) in a 3:1 ratio of DOPC/DOPG mixed membrane for symmetric as well as asymmetric placement of peptides and (ii) 4-8 transmembrane peptides (corresponding to P/L ratio of 4/128 to 8/128) for pure DOPC lipid bilayer (a mimic of mammalian cell membrane) for asymmetric placement of peptides. This study also quantified the potential of mean force through molecular dynamics (MD) simulation for the addition of melittin into DOPC/DOPG mixed bilayer, a mimic of bacterial membrane, for different extents of insertion into either a bilayer or a pore consisting of three to six transmembrane peptides. Critical P/L ratio of 6/166 was observed for penetration of melittin from the surface with this critical value being 6/128 for water channel formation in case of transmembrane peptides. The energy barrier for insertion of a melittin molecule into the bilayer was highest in the absence of transmembrane peptides and decreased for number of transmembrane peptides from three to six, eventually approaching zero. The decrease in free energy for complete insertion of peptide was found to be higher for larger pore size. Significant water channel formation could be observed only for larger membrane pores. The structure of the pore was found to be toroidal and paraboloid. The prediction of proposed mathematical model agreed fairly well with the computed PMF profiles. The second part of the study proposed a methodology that is based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy β-conglycinin (7S) subunits. Initial screening of peptide segments from soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, one peptide (coded as 7a16) satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to initial structure. The antimicrobial activity of this peptide against L. monocytogenes and E. coli was further confirmed by spot-on-lawn test. The third part of this research studied the interaction between curcumin (CUR) and lipid bilayer. CUR is a widely used natural food ingredient with known ability of targeting microorganism cell membrane, but the detailed mechanism remains unclear. In this study, the interaction of CUR with different types of model lipid bilayer (1-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine (DPPE)), mixture of model lipid bilayer (POPE/POPG with ratio of 3:1), and realistic biological membranes (E. coli and yeast) were investigated by all atom explicit solvent molecular dynamics (MD) simulation over microseconds. CUR readily inserts into different types of model lipid bilayer system in liquid state within the first two hundred nanoseconds of the simulation, staying in the lipid tails region, near the interface of lipid head and lipid tail. Parallel orientation to the membrane surface is found to be more probable than perpendicular orientation for CUR as indicated by the tilt angle distribution. This orientation preference is less significant as the fraction of POPE is increased in the system, likely due to the better water solvation of perpendicular orientation in POPE bilayer. In E. coli and yeast bilayers, tilt angle distributions were similar to that for POPE/POPG mixed bilayer. Water hydration number around CUR in these more realistic bilayers is higher than the corresponding value for POPE/POPG bilayer. In this part, antimicrobial activities of CUR against L. monocytogenes and E. coli were validated using bioassay, viability assay, transmission electron microscopy (TEM), and fluorescence dye leakage experiments. CUR exhibited antimicrobial activity when dissolved in dimethylformamide (DMF). The minimum inhibitory concentration (MIC) of CUR against L. monocytogenes was 78 μg/mL, and the MIC of CUR against E. coli was 156 μg/mL, these values fall in range of reported MIC of CUR. The TEM images indicated CUR may target these two bacteria with different mechanisms: CUR attacked the gram-positive bacteria by disrupting both cell wall and cell membrane, and deactivated the gram-negative bacteria by solubilizing the cell membrane. PMF profiles illustrated an energy barrier for CUR permeated through the bilayer center, which is consistent with CUR density distribution. Existence of multiple CUR molecules inside lipid bilayer could promote permeation of CUR through bilayer center. The last part of the study compared the fluorescence dye leakage of 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by melittin and its mutants. The results indicated: (i) dye leakage is dependent on the concentration of peptide, whereas a critical peptide concentration is needed for pore formation, (ii) a lag time is needed for pore formation above the critical peptide concentration, which implies that pore formation occurred by nucleation, and (iii) the changes of fluorescence intensity are consistent with the antimicrobial activities of these peptides. This technique not only captures the nucleation of pore formation, but also describes the event of growth of pore size. In addition, the balance between net charge and hydrophobicity is essential for design of appropriate pore forming peptides.