Gary A. Lorigan

DEER EPR Measurements for Membrane Protein Structures via Bifunctional Spin Labels and Lipodisq Nanoparticles

Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to those of globular proteins. In this study, we employed a combination of three recently developed methodologies, (1) bifunctional spin labels (BSL), (2) SMA-Lipodisq nanoparticles, and (3) Q band pulsed EPR measurements, to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated an ∼2-fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to those of proteoliposomes and narrower distance distributions for the BSL when compared to those of the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.

Significantly improved sensitivity of Q-band PELDOR/DEER experiments relative to X-band is observed in measuring the intercoil distance of a leucine zipper motif peptide (GCN4-LZ).

Pulsed electron double resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin-labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, we present for the first time a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and higher-quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.

Probing the interaction of polyphenols with lipid bilayers by solid-state NMR spectroscopy

Polyphenols are bioactive natural products that appear to act against a wide range of pathologies. Mechanisms of activity have not been established, but recent studies have suggested that some polyphenols bind to membranes. This study examined the interaction between lipid bilayers and three structurally diverse polyphenols. It was hypothesized that features of the polyphenols such as polarity, molecular size, molecular geometry, and number and arrangement of phenol hydroxyl groups would determine the tendency to interact with the bilayer. The examined compounds included a mixed polyphenol, (-)-epigallocatechin gallate (EGCg); a proanthocyanidin trimer comprising catechin-(4→8)-catechin-(4→8)-catechin (cat₃; and a hydrolyzable tannin, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG). These polyphenols were incorporated at different levels into ²H-labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles (MLVs). ³¹P and ²H solid-state NMR experiments were performed to determine the dynamics of the headgroup region and the hydrophobic acyl chain region of the lipid bilayer upon addition of polyphenols. The chemical shift anisotropy (CSA) width of the ³¹P NMR spectra decreased upon addition of polyphenols. Addition of PGG induces a dramatic reduction on the CSA width compared with the control lipid bilayer sample, whereas addition of cat₃ barely reduces the CSA width. The ²H quadupolar splitting of the lipids also decreased upon addition of polyphenols. At the same concentration, PGG substantially reduced the quadrupolar splitting, whereas cat₃ barely reduced it when compared with the control sample. From a calculation of the order parameters of the acyl chain region of the lipid bilayer, it was concluded that the hydrophobic part of the lipid bilayer was perturbed by PGG, whereas cat₃ did not cause large perturbations. The data suggest that the polarity of the polyphenols affects the interaction between tannins and membranes. The interactions may relate to the biological activities of polyphenols.

Use of electron paramagnetic resonance to solve biochemical problems.

Electron paramagnetic resonance (EPR) spectroscopy is a very powerful biophysical tool that can provide valuable structural and dynamic information about a wide variety of biological systems. The intent of this review is to provide a general overview for biochemists and biological researchers of the most commonly used EPR methods and how these techniques can be used to answer important biological questions. The topics discussed could easily fill one or more textbooks; thus, we present a brief background on several important biological EPR techniques and an overview of several interesting studies that have successfully used EPR to solve pertinent biological problems. The review consists of the following sections: an introduction to EPR techniques, spin-labeling methods, and studies of naturally occurring organic radicals and EPR active transition metal systems that are presented as a series of case studies in which EPR spectroscopy has been used to greatly further our understanding of several important biological systems.

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.

Evidence for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5.

HetR, master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120, stimulates heterocyst differentiation via transcriptional autoregulation and is negatively regulated by PatS and HetN, both of which contain the active pentapeptide RGSGR. However, the direct targets of PatS and HetN remain uncertain. Here, we report experimental evidence for direct binding between HetR and the C-terminal RGSGR pentapeptide, PatS-5. Strains with a hetR allele coding for conservative substitutions at residues 250-256 had altered patterns of heterocysts and, in some cases, reduced sensitivity to PatS-5. Cysteine scanning mutagenesis coupled with electron paramagnetic resonance (EPR) spectroscopy showed quenching of spin label motion at HetR amino acid 252 upon titration with PatS-5, indicating direct binding of PatS-5 to HetR. Gel shift assays indicated that PatS-5 disrupted binding of HetR to a 29 base pair inverted-repeat-containing DNA sequence upstream from hetP. Double electron-electron resonance EPR experiments confirmed that HetR existed as a dimer in solution and indicated that PatS-5 bound to HetR without disrupting the dimer form of HetR. Isothermal titration calorimetry experiments corroborated direct binding of PatS-5 to HetR with a K(d) of 227 nM and a 1:1 stoichiometry. Taken together, these results indicated that PatS-5 disrupted HetR binding to DNA through a direct HetR/PatS interaction. PatS-5 appeared to either bind in the vicinity of HetR amino acid L252 or, alternately, to bind in a remote site that leads to constrained motion of this amino acid via an allosteric effect or change in tertiary structure.

Investigating Structural Changes in the Lipid Bilayer upon Insertion of the Transmembrane Domain of the Membrane-Bound Protein Phospholamban Utilizing 31P and 2H Solid-State NMR Spectroscopy

Phospholamban (PLB) is a 52-amino acid integral membrane protein that regulates the flow of Ca(2+) ions in cardiac muscle cells. In the present study, the transmembrane domain of PLB (24-52) was incorporated into phospholipid bilayers prepared from 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC). Solid-state (31)P and (2)H NMR experiments were carried out to study the behavior of POPC bilayers in the presence of the hydrophobic peptide PLB at temperatures ranging from 30 degrees C to 60 degrees C. The PLB peptide concentration varied from 0 mol % to 6 mol % with respect to POPC. Solid-state (31)P NMR spectroscopy is a valuable technique to study the different phases formed by phospholipid membranes. (31)P NMR results suggest that the transmembrane protein phospholamban is incorporated successfully into the bilayer and the effects are observed in the lipid lamellar phase. Simulations of the (31)P NMR spectra were carried out to reveal the formation of different vesicle sizes upon PLB insertion. The bilayer vesicles fragmented into smaller sizes by increasing the concentration of PLB with respect to POPC. Finally, molecular order parameters (S(CD)) were calculated by performing (2)H solid-state NMR studies on deuterated POPC (sn-1 chain) phospholipid bilayers when the PLB peptide was inserted into the membrane.

Magnetically aligned phospholipid bilayers in weak magnetic fields: optimization, mechanism, and advantages for X-band EPR studies

Our lab is developing a spin-labeled EPR spectroscopic technique complementary to solid-state NMR studies to study the structure, orientation, and dynamics of uniaxially aligned integral membrane proteins inserted into magnetically aligned discotic phospholipid bilayers, or bicelles. The focus of this study is to optimize and understand the mechanisms involved in the magnetic alignment process of bicelle disks in weak magnetic fields. Developing experimental conditions for optimized magnetic alignment of bicelles in low magnetic fields may prove useful to study the dynamics of membrane proteins and its interactions with lipids, drugs, steroids, signaling events, other proteins, etc. In weak magnetic fields, the magnetic alignment of Tm(3+)-doped bicelle disks was thermodynamically and kinetically very sensitive to experimental conditions. Tm(3+)-doped bicelles were magnetically aligned using the following optimized procedure: the temperature was slowly raised at a rate of 1.9K/min from an initial temperature being between 298 and 307K to a final temperature of 318K in the presence of a static magnetic field of 6300G. The spin probe 3beta-doxyl-5alpha-cholestane (cholestane) was inserted into the bicelle disks and utilized to monitor bicelle alignment by analyzing the anisotropic hyperfine splitting for the corresponding EPR spectra. The phases of the bicelles were determined using solid-state 2H NMR spectroscopy and compared with the corresponding EPR spectra. Macroscopic alignment commenced in the liquid crystalline nematic phase (307K), continued to increase upon slowly raising the temperature, and was well-aligned in the liquid crystalline lamellar smectic phase (318K).

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

A differential association of Apolipoprotein E isoforms with the amyloid‐β oligomer in solution

The molecular pathogenesis of disorders arising from protein misfolding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates, whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex misfolding and aggregation that initiates the toxic cascade resulting in Alzheimers disease (AD), we have developed a 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid spin‐labeled amyloid‐β (Aβ) peptide to observe its isoform‐dependent interaction with the apoE protein. Although most individuals carry the E3 isoform of apoE, ∼15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimers. ApoE is consistently associated with the amyloid plaque marker for AD. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We used electron paramagnetic resonance (EPR) spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin‐labeled side chain report on side chain and backbone dynamics as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C‐terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimers risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ. Proteins 2011.