National Biomedical Electron Paramagnetic Resonance Center

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Research Highlight #127
Antimicrobial Peptides

Jimmy B. Feix
Department of Biophysics and National Biomedical EPR Center, Medical College of Wisconsin, Milwaukee, WI
[Additional Grant Support: GM068829]

Infectious disease remains the leading cause of mortality worldwide.  A significant aspect of this problem is the continuing rise of infections that are resistant to most, if not all, conventional antibiotics.   The increasing prevalence of drug-resistant infections impacts our ability to effectively treat cancer, kidney disease, AIDS, and a variety of other disorders.  To meet this challenge it is essential that new drug targets be identified for the treatment of infectious disease, and that new classes of antibiotics be developed.

Our studies are focused on the development of antibiotics based on small, naturally occurring antimicrobial peptides that have been found in a wide variety of both vertebrate and invertebrate species.  These peptides are capable of providing a rapid and broad-spectrum response against a wide variety of pathogens.  Because the specificity of these peptides is based on recognition of general properties of the cell membrane, the emergence of resistance is exceedingly rare and full activity against infectious agents that are resistant to conventional antibiotics is typically retained.

A limiting factor in our ability to further enhance the efficacy of these peptides is the lack of detailed knowledge about their mechanism of action, and in particular the manner in which they interact with and disrupt the cell membrane.  Using site-directed spin labeling EPR technology we have completed an initial study characterizing the interaction of a lead peptide, designated CM15, with model lipid bilayers that mimic the bacterial membrane (1).  This study lays the foundation for further analysis of structure-function-activity relationships and optimization of peptide antibiotics.

The abstract of the paper detailing the initial study follows:

The interaction of antimicrobial peptides with membranes is a key factor in determining their biological activity.  In this study we have synthesized a series of minimized cecropin-mellitin hybrid peptides each containing a single cysteine residue, modified the cysteine with the sulfhydryl-specific methanethiosulfonate spin-label, and used electron paramagnetic resonance spectroscopy to measure membrane-binding affinities and determine the orientation and localization of peptides bound to membranes that mimic the bacterial cytoplasmic membrane.  All of the peptides were unstructured in aqueous solution but underwent a significant conformational change upon membrane binding that diminished the rotational mobility of the attached spin-label.  Apparent partition coefficients were similar for five of the six constructs examined, indicating that location of the spin-label had little effect on peptide binding as long as the attachment site was in the relatively hydrophobic C-terminal domain.  Depth measurements based on accessibility of the spin-labeled sites to oxygen and nickel ethylenediaminediacetate indicated that at high lipid/peptide ratios these peptides form a single  -helix, with the helical axis aligned parallel to the bilayer surface and immersed  5 Å below the membrane-aqueous interface.  Such a localization would provide exposure of charged/polar residues on the hydrophilic face of the amphipathic helix to the aqueous phase, and allow the nonpolar residues along the opposite face of the helix to remain immersed in the hydrophobic phase of the bilayer.  These results are discussed with respect to the mechanism of membrane disruption by antimicrobial peptides.

1. Bhargava, K. and Feix, J. B. "Membrane binding, structure, and localization of cecropin-mellitin hybrid peptides: A site-directed spin labeling study." Biophys. J. 86: 329-336, 2004.



Research Highlight #126
Membrane Microdomains as Detected by "Discrimination by Oxygen Transport" Based on Pulse EPR Spin Labeling

Witold K. Subczynski1, Anna Wisniewska1, James S. Hyde1, and Akihiro Kusumi2
1Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI; 2Department of Biological Science, Institute for Advanced Research, Nagoya University, Nagoya, Japan
[Additional Grant Support: EY015526]

During the last year membranes made from binary mixtures of phosphatidylcholine and cholesterol or sphingomyelin and cholesterol as well as from ternary raft-forming mixtures were investigated using a pulse electron paramagnetic resonance (EPR) spin-labeling method named "discrimination by oxygen transport (DOT)".  Data obtained for membranes made from binary mixtures of phosphatidylcholine and cholesterol or sphingomyelin and cholesterol indicate that we are able to discriminate and characterize solid-ordered, liquid-disordered and liquid-ordered domains in these membranes.  Also preliminary results obtained for membranes made from ternary raft-forming mixtures clearly show the presence of two distinct membrane domains in these membranes: slow oxygen transport domain which was ascribed as a raft domain, and high oxygen transport domain which was ascribed as bulk lipids.

The research was supported in part by the National Eye Institute, and is directed to the fiber cell plasma membrane of the eye lens.  It has been shown that the level of sphingomyelin is elevated in a cataractous fiber cell membrane, which may lead to the formation of raft domains.  Thus, in both aged and cataractous fiber cells, conditions favor the formation of membrane raft domains.  We intend to study the changes in the fiber cell plasma membrane of the eye lens that occurs during maturation and aging.  Studies of the effects of lipid composition on significant raft characteristics are important in order to understand the physiological functions of rafts in normal cells and in aged and cataractous fiber cells of the mammalian eye lens.  It is noted that age-related nuclear cataracts are a primary cause of blindness in the elderly in third world countries.

We would like to put special emphasis on the DOT method itself (which was developed in the National Biomedical EPR Center for observation of raft dynamics in influenza viral membrane; Kawasaki et al., Biophys. J. 80, 738-748 (2001)).  This method employs pulse EPR with dual probes, a nitroxide spin probe (attached to the specific positions in a lipid) and molecular oxygen, in which bimolecular collision of the molecular oxygen (fast relaxing species) with the nitroxide moiety of the spin label (slow relaxing species) accelerates the relaxation of the nitroxide spin.  The bimolecular collision rate is proportional to 1/ T1 (Air) – 1/ T1 (N2), where T1 represents the spin-lattice relaxation time of the spin label in the presence of atmospheric air or nitrogen gas.  The typical spin-lattice relaxation time of lipid spin labels falls in the range of 1 to 10 µs, and can fall to 0.1 µs in the presence of oxygen.  The signal-to-noise ratio is good enough that changes as small as 10% can be measured.  Thus the time scale of the DOT method is approximately 0.1 to 100 µs.  This indirect method permits discrimination of membrane domains when the collision rates (oxygen diffusion-concentration products) differ.  Additionally, membrane domains can be characterized by profiles of the oxygen diffusion-concentration product in situ, which provides useful information about dynamics of each domain. These results were presented during the meeting "Biophysical Society Discussions: Probing Membrane Microdomains" in Asilomar, California, 2004.



Research Highlight #125
Mitochondrially-Targeted Nitroxides—A New Class of SOD Mimetics

Joy Joseph, Srigiridhar Kotamraju, Shasi V. Kalivendi, Simmy Thomas, Anuradha Dhanasekaran, and B. Kalyanaraman
[Additional Grant Support: HL073056]

The mitochondrial respiratory chain is a potential source of reactive oxygen species (ROS) (e.g., superoxide and hydrogen peroxide).  Thus, it is conceivable that mitochondria are more vulnerable to oxidative damage than other cellular organelles.  Oxidant-mediated mitochondrial dysfunction was linked to apoptotic cell death in several neurodegenerative and cardiovascular diseases.  Because most conventional antioxidants ( a-tocopherol and N-acetyl cysteine) do not significantly accumulate within mitochondria, effectiveness is limited.  Emerging literature data show that antioxidants covalently coupled to a triphenylphosphonium cation are preferentially taken up by the mitochondria.  The large membrane potential of 150-180 mV (negative inside) across the mitochondrial inner membrane can be used to deliver molecules into mitochondria.  Mito-carboxy proxyl was synthesized by covalently coupling a triphenylphosphonium cation to carboxy proxyl.  Alkylphosphonium nitroxides have previously been used to measure transmembrane potentials and membrane dynamics.  Localization of nitroxide in the mitochondria was monitored by EPR. We found that Mito-carboxy proxyl (Mito-CP) is considerably more effective in inhibiting mitochondrial oxidative damage at much lower concentrations than the "untargeted" carboxy proxyl (CP).  We propose that mitochondria-targeted nitroxides are a new class of targeted antioxidants that can mimic manganese SOD, a superoxide dismutating enzyme present in the mitochondrial matrix.

Mitochondrial role in oxidative cell signaling is a rapidly growing area of research. Mitochondria generated ROS has been implicated in various neurodegenerative diseases such as Parkinson's, ALS, Alzheimer's, and Friedreich ataxia. Nitroxides (e.g., Tempol) have been used to mitigate oxidative damage in animal models. Currently, we are testing the therapeutic efficacy of mito-nitroxides in ALS animal models.



Research Highlight #124
RF Phase Shifts and Elliptical Polarization in Tissue with Surface Coils

James S. Hyde1, Jason W. Sidabras1, Richard R. Mett1, Ben Williams2, Artur Sucheta2, Harold M. Swartz2
1Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI; 2Department of Diagnostic Radiology, Dartmouth Medical School, Hanover, NH
[Additional Grant Support: R01 EB002032, R01 EB001417]

In vivo EPR is generally carried out in the RF frequency range of 250 MHz to 1 GHz, while MRI scanners with frequencies as high as 400 MHz for humans and 500 MHz for small animals exist.  Thus, the frequencies for in vivo EPR and MRI overlap. Surface RF coils are widely used in MRI, but were only recently introduced for EPR (1).  We report the use of finite-element modeling of RF fields in tissues at a frequency of 1.2 GHz in a context of in vivo EPR.  In spite of the vast literature of surface coils in MRI, we found no use of finite-element solutions of Maxwell's equations in order to obtain the RF field and phase distributions in tissue.  Because of the overlap of EPR and MRI frequencies, our work is relevant to both modalities.

The geometry is illustrated in Fig. 1.  The circular surface coil of diameter 2r = 1 cm is described in (1).  It is placed on the end of a cylinder of tissue-equivalent polyacrylamide gel as described in (2) using a complex dielectric constant of ?Ã = 49.5 +j25.8.  The static magnetic field is along the z-axis, and the y-axis passes through the center of the coil and coincides with the axis of the cylindrical phantom.  We consider an imaging plane that is perpendicular to y and displaced from the coil by a distance r.  In this plane, only components of H1, the RF magnetic field, that are perpendicular to the static magnetic field H0, i.e., H1x and H1y are relevant for magnetic resonance.  Simulations were carried out using Ansoft HFSS 9.2 (Pittsburgh, PA).
 We have observed in free space that the RF field is linearly polarized throughout.  The complex dielectric constant of the phantom causes the RF electric field in the imaging plane to be elliptically polarized except along the y-axis, where it remains linearly polarized.  An elliptically polarized RF field can be represented by a superposition of two circularly polarized fields rotating in opposite directions.  Only that component rotating in the direction of precession of the spins is EPR or MRI active.  This basic physics does not seem to have been previously enunciated and is the central finding reported here.

Figure 2a shows an image of the circularly polarized field in one rotational sense and Fig. 2b in the opposite sense.  Only one of these can be active in magnetic resonance, depending on the north and south pole orientation of the static magnetic field.  The regions of sensitivity are off-set from the coil axis.  The orientation of the static field can effectively be changed in this problem by moving the coil from one end of the phantom to the other.  Images of a uniform phantom acquired in these two coil positions do not superimpose.  There is widespread interest in parallel acquisition of MRI images using multiple coils at increasingly higher static magnetic field strength.  It is apparent to us, based on this study, that finite-element modeling of the solutions to Maxwell's equations including the complex permittivity of tissue is a requisite for effective MRI coil design.  EPR resonator design strategies can serve as a model for high frequency MRI coil design since historically the radio frequencies have been higher in EPR.

1. Salikhov, I., Hirata, H., Walczak, T., Swartz, H.  J. Magn. Reson. 164, 54-59 (2003).
2. Andreuccetti, D., Bini, M., Ignesti, A., Olmi, R., Rubino, N., Vanni, R. IEEE Trans. Biomed. Engin. 35, 275-277 (1988).

Research Highlight #123
Secondary Mode of Copper Binding in the Prion Protein: Contributions from S-Band EPR

M. Chattopadhyay1, D. Newell1, W.E. Antholine2, E. Aronoff-Spencer3, G. Legname4, G.J. Gerfen3, J. Peisach3, and G.L. Millhauser1
1University of California, Santa Cruz; 2Medical College of Wisconsin; 3Albert Einstein College of Medicine; 4University of California, San Francisco
[Additional Grant Support: GM065790]

Low frequency EPR, particularly S-band (3.4 GHz), is a well-known method to resolve nitrogen and proton superhyperfine from signals from type 2 copper complexes.  This method has been used by us to study the binding of cupric ion to fragments that mimic amino acid sequences in the prion protein (1, 2).  In new studies with Prof. Glenn Millhauser at the University of California, Santa Cruz, it was found that the binding of cupric ion to the major site in the octarepeat is blocked and binding to a second cupric binding site is enhanced (3) by methylating an amide nitrogen.  S-band spectra of Cu2+ added to KKRPHGmeGGW containing 15N-labeled glycines were obtained.  The MI=-1/2 line is shown in Fig. 1.  The five superhyperfine lines are consistent with two nitrogen donor atoms. None of the glycines participate in the Cu2+ binding.  On the basis of these results, a structure where histidine utilizes both its imidazole ring and amide nitrogen is proposed (Fig. 2).  The broadening of S-band spectra in samples prepared in 17O-enriched water is consistent with binding of at least one oxygen ligand from water.  The proposed model, if correct, could lead to pharmaceutical intervention in human prion disease. 



1. Burns, C. S., Aronoff-Spencer, E., Dunham, C. M., Lario, P., Avdievich, N. I., Antholine, W. E., Olmstead, M. M., Vrielink, A., Gerfen, G., Peisach, J., Scott, W. G., and Millhauser, G. L., 2002, Biochemistry, 41, 3991-4001.
2. Burns, C. S., Aronoff-Spencer, E., Legname, G., Prusiner, S. B.,Antholine, W. E., Gerfen, G. J., Peisach, J., and Millhauser, G. I., Biochemistry, 2003, 42, 6794-6803.
3. Chattopadhyay, M., Newill, D., Antholine, W. E., Aronoff-Spencer, E., Legname, G., Gerfen, G. J., Peisach, J., and Millhauser, G. L., Modes of copper binding in the prion protein, American Chemical Society Meeting, Anaheim, CA, March 28-April 1, 2004.
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