Assessing topology and surface orientation of an antimicrobial peptide magainin 2 using mechanically aligned bilayers and electron paramagnetic resonance spectroscopy.

Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label.

Probing topology and dynamics of the second transmembrane domain (M2δ) of the acetyl choline receptor using magnetically aligned lipid bilayers (bicelles) and EPR spectroscopy.

Characterizing membrane protein structure and dynamics in the lipid bilayer membrane is very important but experimentally challenging. EPR spectroscopy offers a unique set of techniques to investigate a membrane protein structure, dynamics, topology, and distance constraints in lipid bilayers. Previously our lab demonstrated the use of magnetically aligned phospholipid bilayers (bicelles) for probing topology and dynamics of the membrane peptide M2δ of the acetyl choline receptor (AchR) as a proof of concept.

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous efforts have been devoted toward the development and application of EPR spectroscopic techniques to study the structure of biological systems such as membrane proteins and peptides.

Architecture of the fungal nuclear pore inner ring complex.

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96.

Enhancement of electron spin echo envelope modulation spectroscopic methods to investigate the secondary structure of membrane proteins.

This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an α-helical secondary structure with ESEEM spectroscopy using a (2)H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins.

Distance measurements on a dual-labeled TOAC AChR M2δ peptide in mechanically aligned DMPC bilayers via dipolar broadening CW-EPR spectroscopy.

A membrane alignment technique has been used to measure the distance between two TOAC nitroxide spin labels on the membrane-spanning M2δ, peptide of the nicotinic acetylcholine receptor (AChR), via CW-EPR spectroscopy. The TOAC-labeled M2δ peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides.

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy.

Wild-type phospholamban (WT-PLB), a Ca(2+)-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca(2+)-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles.

Time-resolved EPR immersion depth studies of a transmembrane peptide incorporated into bicelles.

The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2δ AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n=5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n=5, 7, 12, and 16), and M2δ AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits.

Determining the helical tilt of membrane peptides using electron paramagnetic resonance spectroscopy.

Theoretical calculations of hyperfine splitting values derived from the EPR spectra of TOAC spin-labeled rigid aligned alpha-helical membrane peptides reveal a unique periodic variation. In the absence of helical motion, a plot of the corresponding hyperfine splitting values as a function of residue number results in a sinusoidal curve that depends on the helical tilt angle that the peptide makes with respect to the magnetic field. Motion about the long helical axis reduces the amplitude of the curve and averages out the corresponding hyperfine splitting values.

Comparing the structural topology of integral and peripheral membrane proteins utilizing electron paramagnetic resonance spectroscopy.

The alignment of membrane proteins provides pertinent structural and dynamic information. Structural topology data gleaned from such studies can be used to determine the functional mechanisms associated with a wide variety of integral membrane proteins. In this communication, we successfully demonstrate, for the first time, the determination of the structural topology and helical tilt of an antimicrobial peptide magainin 2 using aligned X-band spin-label EPR spectroscopic techniques.

DNA cleavage by photolysis of aryl sulfoxides.

Aryl sulfoxides have been identified as a class of organic compounds capable of inducing DNA cleavage in the presence of UV light. Phenyl sulfoxide and methyl phenyl sulfoxide were both shown to cleave pBR322 DNA at concentrations of 180 and 360 microM, respectively. Radical trapping studies indicate carbon-centered radicals are the active cleavage species.