In 1981, I came to MCW as a Muscular Dystrophy Association postdoctoral fellow, developing advanced electron paramagnetic resonance (EPR) spin labeling techniques to study membrane dynamics. I joined the MCW faculty in 1986.
PhD, Chemistry, University of Kentucky, Lexington, KY, 1981
BS, Chemistry and Biology, Western Kentucky University, Bowling Green, KY, 1976
We utilize site-directed spin labeling (SDSL) EPR spectroscopy and other biophysical and biochemical techniques to work on problems related to the membrane interactions of peptides and proteins and the structure and dynamics of membrane proteins.
Mechanism of Activation and Membrane Interactions of Pseudomonas toxin ExoU
ExoU is a 74 kDa protein produced by the Gram-negative bacterial pathogen, Pseudomonas aeruginosa. ExoU is injected directly into eukaryotic cells by a needle-like Type III secretion system (T3SS). Once inside the eukaryotic cell, ExoU functions as a phospholipase to disrupt membranes and facilitate dissemination of the bacterial infection. Activation of ExoU requires interaction with ubiquitin. Our site-directed spin label EPR studies in conjunction with NMR, mutagenesis, and biochemical analyses have provided an initial indication of the ubiquitin binding domain. Current aims of this research are to fully characterize the ExoU-ubiquitin binding interface and to understand how ExoU interacts with its target membrane substrate. These studies will facilitate the development of novel inhibitors of ExoU as a means to attenuate the virulence of P. aeruginosa infections.
Mechanisms of Antimicrobial Peptides: Membrane Interactions
Antibiotic resistance is an increasingly serious problem in the treatment of infectious disease. During the past two decades, a large number of peptides with potent antibacterial, antiviral, and antifungal properties have been identified from a wide range of both vertebrate and invertebrate species. These antimicrobial peptides (AMPs) are an important component of the innate arm of host resistance, serving as a first line of defense against infection. Despite being evolutionarily ancient, resistance to AMPs has only rarely been observed. Consequently, there is great interest in the development of these peptides for the treatment of drug-resistant infections.
Models of Transmembrane Channel Formation
(A) Peptide a-helices (cylinders) initially associate parallel to the membrane surface, either superficially (left) or embedded just below the aqueous interface. (B) Peptides continue to accumulate at or near the bilayer surface, disrupting lipid packing and causing membrane thinning. This step may or may not involve peptide-peptide aggregation. Once a critical peptide/lipid ratio is reached, peptides either (C) insert into the membrane as a barrel-stave type pore or (D) induce the localized formation of toroidal pores.
Our laboratory is involved in elucidating the mechanisms by which AMPs disrupt bacterial membrane structure, determining the basis of AMP selectivity for microbial cells, and developing more effective antimicrobial peptides and peptidomimetics. Whereas classical antibiotics generally target cell wall synthesis, protein translation, or some other highly specific target, AMPs are believed to function by directly disrupting the microbial cell membrane. Peptides are prepared using either recombinant DNA methods or solid-phase chemical synthesis, and their interactions with model membranes (liposomes) and cells are characterized by a variety of physical techniques including circular dichroism, fluorescence, and EPR site-directed spin labeling (SDSL). Our fundamental hypothesis is that a more complete understanding of peptide structure and dynamic interactions with the membrane will allow the design and development of improved AMPs and related antibiotics for the treatment of infections by existing drug-resistant strains such as Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA).
Samantha Kohn, Graduate Student
(Feix JB, Kohn S, Tessmer MH, Anderson DM, Frank DW.) Cell Biochem Biophys. 2019 Mar;77(1):79-87.
(Tessmer MH, Anderson DM, Pickrum AM, Riegert MO, Moretti R, Meiler J, Feix JB, Frank DW.) Proc Natl Acad Sci U S A. 2018 01 16;115(3):525-530.
(Herneisen AL, Sahu ID, McCarrick RM, Feix JB, Lorigan GA, Howard KP.) Biochemistry. 2017 11 07;56(44):5955-5963.
(Fischer AW, Anderson DM, Tessmer MH, Frank DW, Feix JB, Meiler J.) ACS Omega. 2017 Jun 30;2(6):2977-2984.
(Tessmer MH, Anderson DM, Buchaklian A, Frank DW, Feix JB.) J Biol Chem. 2017 02 24;292(8):3411-3419.
(Kim SS, Upshur MA, Saotome K, Sahu ID, McCarrick RM, Feix JB, Lorigan GA, Howard KP.) Biochemistry. 2015 Dec 15;54(49):7157-67.
(Anderson DM, Feix JB, Frank DW.) PLoS Pathog. 2015 Jul;11(7):e1004944.
(Anderson DM, Sato H, Dirck AT, Feix JB, Frank DW.) J Bacteriol. 2015 Feb;197(3):529-41.
(Schultz KM, Feix JB, Klug CS.) Protein Sci. 2013 Nov;22(11):1639-45.
(Anderson DM, Feix JB, Monroe AL, Peterson FC, Volkman BF, Haas AL, Frank DW.) J Biol Chem. 2013 Sep 13;288(37):26741-52.
(Anderson DM, Schmalzer KM, Sato H, Casey M, Terhune SS, Haas AL, Feix JB, Frank DW.) Mol Microbiol. 2011 Dec;82(6):1454-67.
(Mainali L, Feix JB, Hyde JS, Subczynski WK.) J Magn Reson. 2011 Oct;212(2):418-25.