Publications by authors named "Likai Song"

40 Publications

The helix 0 of endophilin modifies membrane material properties and induces local curvature.

Biochim Biophys Acta Biomembr 2020 10 11;1862(10):183397. Epub 2020 Jun 11.

Department of Physics, University of South Florida, Tampa, FL 33620, United States of America. Electronic address:

The amphipathic helix 0 of endophilin (i.e., H0-Endo) is important to membrane binding, but its function of curvature generation remains controversial. We used electron paramagnetic resonance (EPR) spectroscopy to study effects of H0-Endo on membrane material properties. We found that H0-Endo reduced lipid chain mobility and increased bilayer polarity, i.e., making the bilayer interior more polar. Lipid-dependent examination revealed that anionic lipids augmented the effect of H0-Endo, while cholesterol had a minimal impact. Our EPR spectroscopy of magnetically aligned bicelles showed that as the peptide-to-lipid ratio increased, the lipid chain orientational order decreased gradually, followed by a sudden loss. We discuss an interfacial-bound model of the amphipathic H0-Endo to account for all EPR data. We used atomic force microscopy and fluorescence microscopy to explore membrane morphological changes. We found that H0-Endo caused the formation of micron-sized holes in mica-supported planar bilayers. Hole formation is likely caused by two competing forces - the adhesion force exerted by the substrate represses bilayer budging, whereas the line tension originating from peptide clustering has a tendency of destabilizing bilayer organization. In the absence of substrate influences, membrane curvature induction was manifested by generating small vesicles surrounding giant unilamellar vesicles. Our results of membrane perforation and vesiculation suggest that the functionality of H0-Endo is more than just coordinating membrane binding of endophilin.
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http://dx.doi.org/10.1016/j.bbamem.2020.183397DOI Listing
October 2020

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

Proc Natl Acad Sci U S A 2019 11 17;116(45):22556-22566. Epub 2019 Oct 17.

Laboratory of Immunobiology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115;

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.
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http://dx.doi.org/10.1073/pnas.1912427116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842640PMC
November 2019

Effects of glassing matrix deuteration on the relaxation properties of hyperpolarized C spins and free radical electrons at cryogenic temperatures.

J Chem Phys 2019 Jun;150(23):234307

Department of Physics, University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, USA.

Glassing matrix deuteration could be a beneficial sample preparation method for C dynamic nuclear polarization (DNP) when large electron paramagnetic resonance (EPR) width free radicals are used. However, it could yield the opposite DNP effect when samples are doped with small EPR width free radicals. Herein, we have investigated the influence of solvent deuteration on the C nuclear and electron relaxation that go along with the effects on C DNP intensities at 3.35 T and 1.2 K. For C DNP samples doped with trityl OX063, the C DNP signals decreased significantly when the protons are replaced by deuterons in glycerol:water or DMSO:water solvents. Meanwhile, the corresponding solid-state C T relaxation times of trityl OX063-doped samples generally increased upon solvent deuteration. On the other hand, C DNP signals improved by a factor of ∼1.5 to 2 upon solvent deuteration of samples doped with 4-oxo-TEMPO. Despite this C DNP increase, there were no significant differences recorded in C T values of TEMPO-doped samples with nondeuterated or fully deuterated glassing matrices. While solvent deuteration appears to have a negligible effect on the electron T relaxation of both free radicals, the electron T relaxation times of these two free radicals generally increased upon solvent deuteration. These overall results suggest that while the solid-phase C DNP signals are dependent upon the changes in total nuclear Zeeman heat capacity, the C relaxation effects are related to H/H nuclear spin diffusion-assisted C polarization leakage in addition to the dominant paramagnetic relaxation contribution of free radical centers.
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http://dx.doi.org/10.1063/1.5096036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588520PMC
June 2019

CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores.

Methods Mol Biol 2019 ;1877:257-303

Department of Biochemistry & Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.

The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.
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http://dx.doi.org/10.1007/978-1-4939-8861-7_18DOI Listing
May 2019

The T Cell Antigen Receptor α Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations.

Immunity 2018 11 30;49(5):829-841.e6. Epub 2018 Oct 30.

Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Initial molecular details of cellular activation following αβT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αβTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαβ and CD3 associations.
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http://dx.doi.org/10.1016/j.immuni.2018.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249037PMC
November 2018

Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein Nanoparticles.

ACS Omega 2018 Aug 21;3(8):9586-9597. Epub 2018 Aug 21.

National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310, United States.

Modulations of synaptic membranes play an essential role in the physiological and pathological functions of the presynaptic protein α-synuclein (αSyn). Here we used solution atomic force microscopy (AFM) and electron paramagnetic resonance (EPR) spectroscopy to investigate membrane modulations caused by αSyn. We used several lipid bilayers to explore how different lipid species may regulate αSyn-membrane interactions. We found that at a protein-to-lipid ratio of ∼1/9, αSyn perturbed lipid bilayers by generating semi-transmembrane defects that only span one leaflet. In addition, αSyn coaggregates with lipid molecules to produce ∼10 nm-sized lipoprotein nanoparticles. The obtained AFM data are consistent with the apolipoprotein characteristic of αSyn. The role of anionic lipids was elucidated by comparing results from zwitterionic and anionic lipid bilayers. Specifically, our AFM measurements showed that anionic bilayers had a larger tendency of forming bilayer defects; similarly, our EPR measurements revealed that anionic bilayers exhibited more substantial changes in lipid chain mobility and bilayer polarity. We also studied the effect of cholesterol. We found that cholesterol increased the capability of αSyn in inducing bilayer defects and altering lipid chain mobility and bilayer polarity. These data can be explained by an increase in the lipid headgroup-headgroup spacing and/or specific cholesterol-αSyn interactions. Interestingly, we found an inhibitory effect of the cone-shaped phosphatidylethanolamine lipids on αSyn-induced bilayer remodeling. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization and suppress lipid extraction. Our results of lipid-dependent membrane modulations are likely relevant to αSyn functioning.
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http://dx.doi.org/10.1021/acsomega.8b01462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120733PMC
August 2018

Cholesterol and phosphatidylethanolamine lipids exert opposite effects on membrane modulations caused by the M2 amphipathic helix.

Biochim Biophys Acta Biomembr 2019 01 30;1861(1):201-209. Epub 2018 Jul 30.

National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, United States. Electronic address:

Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.
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http://dx.doi.org/10.1016/j.bbamem.2018.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260955PMC
January 2019

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from .

ACS Catal 2016 Dec 12;6(12):8036-8047. Epub 2016 Oct 12.

Department of Chemistry, Kansas State University, Manhattan, KS, 66506, USA.

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases, in which a catalytic distal aspartate is involved in HO activation to catalyze oxidations in acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial DyP from is among the most active DyPs and shows activity toward phenolic lignin model compounds (, , 23447). Based on the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II-like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the DyP catalyzes oxidation of large-size substrates multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in DyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the crosslinking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class DyP is minimal possibly due to its extremely small solvent accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.
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http://dx.doi.org/10.1021/acscatal.6b01952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751956PMC
December 2016

Transition Metal Doping Reveals Link between Electron T Reduction and C Dynamic Nuclear Polarization Efficiency.

J Phys Chem A 2017 Dec 21;121(48):9221-9228. Epub 2017 Nov 21.

Department of Physics, The University of Texas at Dallas , Richardson, Texas 75080, United States.

Optimal efficiency of dissolution dynamic nuclear polarization (DNP) is essential to provide the required high sensitivity enhancements for in vitro and in vivo hyperpolarized C nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI). At the nexus of the DNP process are the free electrons, which provide the high spin alignment that is transferred to the nuclear spins. Without changing DNP instrumental conditions, one way to improve C DNP efficiency is by adding trace amounts of paramagnetic additives such as lanthanide (e.g., Gd, Ho, Dy, Tb) complexes to the DNP sample, which has been observed to increase solid-state C DNP signals by 100-250%. Herein, we have investigated the effects of paramagnetic transition metal complex R-NOTA (R = Mn, Cu, Co) doping on the efficiency of C DNP using trityl OX063 as the polarizing agent. Our DNP results at 3.35 T and 1.2 K show that doping the C sample with 3 mM Mn-NOTA led to a substantial improvement of the solid-state C DNP signal by a factor of nearly 3. However, the other transition metal complexes Cu-NOTA and Co-NOTA complexes, despite their paramagnetic nature, had essentially no impact on solid-state C DNP enhancement. W-band electron paramagnetic resonance (EPR) measurements reveal that the trityl OX063 electron T was significantly reduced in Mn-doped samples but not in Cu- and Co-doped DNP samples. This work demonstrates, for the first time, that not all paramagnetic additives are beneficial to DNP. In particular, our work provides a direct evidence that electron T reduction of the polarizing agent by a paramagnetic additive is an essential requirement for the improvement seen in solid-state C DNP signal.
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http://dx.doi.org/10.1021/acs.jpca.7b09448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793213PMC
December 2017

Enhanced Efficiency of C Dynamic Nuclear Polarization by Superparamagnetic Iron Oxide Nanoparticle Doping.

J Phys Chem C Nanomater Interfaces 2017 Sep 17;121(35):19505-19511. Epub 2017 Aug 17.

Department of Physics, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX 75080 USA.

Attainment of high NMR signal enhancements is crucial to the success of or hyperpolarized NMR or imaging (MRI) experiments. In this work, we report on the use of a superparamagnetic iron oxide nanoparticle (SPION) MRI contrast agent Feraheme (ferumoxytol) as a beneficial additive in C samples for dissolution dynamic nuclear polarization (DNP). Our DNP data at 3.35 T and 1.2 K reveal that addition of 11 mM elemental iron concentration of Feraheme in trityl OX063-doped 3 M [1-C] acetate samples resulted in a substantial improvement of C DNP signal by a factor of almost 3-fold. Concomitant with the large DNP signal increase is the narrowing of the C microwave DNP spectra for samples doped with SPION. W-band electron paramagnetic resonance (EPR) spectroscopy data suggest that these two prominent effects of SPION doping on C DNP can be ascribed to the shortening of trityl OX063 electron as explained within the thermal mixing DNP model. Liquid-state C NMR signal enhancements as high as 20,000-fold for SPION-doped samples were recorded after dissolution at 9.4 T and 297 K, which is about 3 times the liquid-state NMR signal enhancement of the control sample. While the presence of SPION in hyperpolarized solution drastically reduces C , this can be mitigated by polarizing smaller aliquots of DNP samples. Moreover, we have shown that Feraheme nanoparticles (~30 nm in size) can be easily and effectively removed from the hyperpolarized liquid by simple mechanical filtration, thus one can potentially incorporate an in-line filtration for these SPIONS along the dissolution pathway of the hyperpolarizer-a significant advantage over other DNP enhancers such as the lanthanide complexes. The overall results suggest that the commercially-available and FDA-approved Feraheme is a highly efficient DNP enhancer that could be readily translated for use in clinical applications of dissolution DNP.
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http://dx.doi.org/10.1021/acs.jpcc.7b06408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876865PMC
September 2017

Operando EPR for Simultaneous Monitoring of Anionic and Cationic Redox Processes in Li-Rich Metal Oxide Cathodes.

J Phys Chem Lett 2017 Sep 14;8(17):4009-4016. Epub 2017 Aug 14.

Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.

Anionic redox chemistry offers a transformative approach for significantly increasing specific energy capacities of cathodes for rechargeable Li-ion batteries. This study employs operando electron paramagnetic resonance (EPR) to simultaneously monitor the evolution of both transition metal and oxygen redox reactions, as well as their intertwined couplings in LiMnO, LiNiMnO, and LiNiMnCoO cathodes. Reversible O/O redox takes place above 3.0 V, which is clearly distinguished from transition metal redox in the operando EPR on LiMnO cathodes. O/O redox is also observed in LiNiMnO, and LiNiMnCoO cathodes, albeit its overlapping potential ranges with Ni redox. This study further reveals the stabilization of the reversible O redox by Mn and e hole delocalization within the Mn-O complex. The interactions within the cation-anion pairs are essential for preventing O from recombination into gaseous O and prove to activate Mn for its increasing participation in redox reactions. Operando EPR helps to establish a fundamental understanding of reversible anionic redox chemistry. The gained insights will support the search for structural factors that promote desirable O redox reactions.
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http://dx.doi.org/10.1021/acs.jpclett.7b01425DOI Listing
September 2017

C Dynamic Nuclear Polarization Using a Trimeric Gd Complex as an Additive.

J Phys Chem A 2017 Jul 28;121(27):5127-5135. Epub 2017 Jun 28.

Department of Physics, University of Texas at Dallas , 800 West Campbell Road, Richardson, Texas 75080 United States.

Dissolution dynamic nuclear polarization (DNP) is one of the most successful techniques that resolves the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) by amplifying the signal by several thousand-fold. One way to further improve the DNP signal is the inclusion of trace amounts of lanthanides in DNP samples doped with trityl OX063 free radical as the polarizing agent. In practice, stable monomeric gadolinium complexes such as Gd-DOTA or Gd-HP-DO3A are used as beneficial additives in DNP samples, further boosting the DNP-enhanced solid-state C polarization by a factor of 2 or 3. Herein, we report on the use of a trimeric gadolinium complex as a dopant in C DNP samples to improve the C DNP signals in the solid-state at 3.35 T and 1.2 K and consequently, in the liquid-state at 9.4 T and 298 K after dissolution. Our results have shown that doping the C DNP sample with a complex which holds three Gd ions led to an improvement of DNP-enhanced C polarization by a factor of 3.4 in the solid-state, on par with those achieved using monomeric Gd complexes but only requires about one-fifth of the concentration. Upon dissolution, liquid-state C NMR signal enhancements close to 20 000-fold, approximately 3-fold the enhancement of the control samples, were recorded in the nearby 9.4 T high resolution NMR magnet at room temperature. Comparable reduction of C spin-lattice T relaxation time was observed in the liquid-state after dissolution for both the monomeric and trimeric Gd complexes. Moreover, W-band electron paramagnetic resonance (EPR) data have revealed that 3-Gd doping significantly reduces the electron T of the trityl OX063 free radical, but produces negligible changes in the EPR spectrum, reminiscent of the results with monomeric Gd-complex doping. Our data suggest that the trimeric Gd complex is a highly beneficial additive in C DNP samples and that its effect on DNP efficiency can be described in the context of the thermal mixing mechanism.
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http://dx.doi.org/10.1021/acs.jpca.7b03869DOI Listing
July 2017

Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy.

J Phys Chem B 2017 05 10;121(19):5058-5071. Epub 2017 May 10.

National High Magnetic Field Laboratory, Florida State University , Tallahassee, Florida 32310, United States.

A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with the membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. A force spectroscopy experiment shows that PrP106-126 reduces Young's modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid atomic vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intrachain conformation, while the interchain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the interchain interaction, while the intrachain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs.
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http://dx.doi.org/10.1021/acs.jpcb.7b02772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770145PMC
May 2017

Influence of Dy and Tb doping on C dynamic nuclear polarization.

J Chem Phys 2017 Jan;146(1):014303

Department of Physics, University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080, USA.

Dynamic nuclear polarization (DNP) is a technique that uses a microwave-driven transfer of high spin alignment from electrons to nuclear spins. This is most effective at low temperature and high magnetic field, and with the invention of the dissolution method, the amplified nuclear magnetic resonance (NMR) signals in the frozen state in DNP can be harnessed in the liquid-state at physiologically acceptable temperature for in vitro and in vivo metabolic studies. A current optimization practice in dissolution DNP is to dope the sample with trace amounts of lanthanides such as Gd or Ho, which further improves the polarization. While Gd and Ho have been optimized for use in dissolution DNP, other lanthanides have not been exhaustively studied for use in C13 DNP applications. In this work, two additional lanthanides with relatively high magnetic moments, Dy and Tb, were extensively optimized and tested as doping additives for C13 DNP at 3.35 T and 1.2 K. We have found that both of these lanthanides are also beneficial additives, to a varying degree, for C13 DNP. The optimal concentrations of Dy (1.5 mM) and Tb (0.25 mM) for C13 DNP were found to be less than that of Gd (2 mM). W-band electron paramagnetic resonance shows that these enhancements due to Dy and Tb doping are accompanied by shortening of electron T of trityl OX063 free radical. Furthermore, when dissolution was employed, Tb-doped samples were found to have similar liquid-state C13 NMR signal enhancements compared to samples doped with Gd, and both Tb and Dy had a negligible liquid-state nuclear T shortening effect which contrasts with the significant reduction in T when using Gd. Our results show that Dy doping and Tb doping have a beneficial impact on C13 DNP both in the solid and liquid states, and that Tb in particular could be used as a potential alternative to Gd in C13 dissolution DNP experiments.
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http://dx.doi.org/10.1063/1.4973317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218971PMC
January 2017

Impact of Ho(3+)-doping on (13)C dynamic nuclear polarization using trityl OX063 free radical.

Phys Chem Chem Phys 2016 Aug 18;18(31):21351-9. Epub 2016 Jul 18.

Department of Physics, University of Texas at Dallas, Richardson, TX 75080, USA.

We have investigated the effects of Ho-DOTA doping on the dynamic nuclear polarization (DNP) of [1-(13)C] sodium acetate using trityl OX063 free radical at 3.35 T and 1.2 K. Our results indicate that addition of 2 mM Ho-DOTA on 3 M [1-(13)C] sodium acetate sample in 1 : 1 v/v glycerol : water with 15 mM trityl OX063 improves the DNP-enhanced (13)C solid-state nuclear polarization by a factor of around 2.7-fold. Similar to the Gd(3+) doping effect on (13)C DNP, the locations of the positive and negative (13)C maximum polarization peaks in the (13)C microwave DNP sweep are shifted towards each other with the addition of Ho-DOTA on the DNP sample. W-band electron spin resonance (ESR) studies have revealed that while the shape and linewidth of the trityl OX063 ESR spectrum was not affected by Ho(3+)-doping, the electron spin-lattice relaxation time T1 of trityl OX063 was prominently reduced at cryogenic temperatures. The reduction of trityl OX063 electron T1 by Ho-doping is linked to the (13)C DNP improvement in light of the thermodynamic picture of DNP. Moreover, the presence of Ho-DOTA in the dissolution liquid at room temperature has negligible reduction effect on liquid-state (13)C T1, in contrast to Gd(3+)-doping which drastically reduces the (13)C T1. The results here suggest that Ho(3+)-doping is advantageous over Gd(3+) in terms of preservation of hyperpolarized state-an important aspect to consider for in vitro and in vivo NMR or imaging (MRI) experiments where a considerable preparation time is needed to administer the hyperpolarized (13)C liquid.
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http://dx.doi.org/10.1039/c6cp03954eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199769PMC
August 2016

Selective Membrane Disruption Mechanism of an Antibacterial γ-AApeptide Defined by EPR Spectroscopy.

Biophys J 2016 04;110(8):1789-1799

National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida. Electronic address:

γ-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. It is not clear how γ-AApeptides interact with bacterial membranes and alter lipid assembly, but such information is essential to understanding their antimicrobial activities and guiding future design of more potent and specific antimicrobial agents. Using electron paramagnetic resonance techniques, we characterized the membrane interaction and destabilizing mechanism of a lipo-cyclic-γ-AApeptide (AA1), which has broad-spectrum antibacterial activities. The analyses revealed that AA1 binding increases the membrane permeability of POPC/POPG liposomes, which mimic negatively charged bacterial membranes. AA1 binding also inhibits membrane fluidity and reduces solvent accessibility around the lipid headgroup region. Moreover, AA1 interacts strongly with POPC/POPG liposomes, inducing significant lipid lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon AA1 binding for liposomes mimicking mammalian cell membranes, which consist of neutral lipids and cholesterol. Our findings suggest that AA1 interacts and disrupts bacterial membranes through a carpet-like mechanism. The results showed that the intrinsic features of γ-AApeptides are important for their ability to disrupt bacterial membranes selectively, the implications of which extend to developing new antibacterial biomaterials.
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http://dx.doi.org/10.1016/j.bpj.2016.02.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850324PMC
April 2016

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields.

J Magn Reson 2016 Apr 15;265:188-96. Epub 2016 Feb 15.

Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA. Electronic address:

High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(∼94 GHz) and D-band (∼140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to α-helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ∼50 μL, concentration sensitivities of 2-20 μM were achieved, representing a ∼10-fold enhancement compared to a cylindrical TE011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.
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http://dx.doi.org/10.1016/j.jmr.2016.02.007DOI Listing
April 2016

Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN(-) binding defined by EPR-based hybrid method.

Sci Rep 2016 Jan 28;6:20025. Epub 2016 Jan 28.

National Laboratory for Physical Science at Microscale, School of Life Science, University of Science and Technology of China, and High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, 230027, P. R. China.

Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN(-), which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.
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http://dx.doi.org/10.1038/srep20025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730233PMC
January 2016

Binding of MgtR, a Salmonella transmembrane regulatory peptide, to MgtC, a Mycobacterium tuberculosis virulence factor: a structural study.

J Mol Biol 2014 Jan 17;426(2):436-46. Epub 2013 Oct 17.

National High Magnetic Field Laboratory, Tallahassee, FL 32306, USA; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA; Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA. Electronic address:

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.
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http://dx.doi.org/10.1016/j.jmb.2013.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947350PMC
January 2014

Disruption of helix-capping residues 671 and 674 reveals a role in HIV-1 entry for a specialized hinge segment of the membrane proximal external region of gp41.

J Mol Biol 2014 Mar 26;426(5):1095-108. Epub 2013 Sep 26.

Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.
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http://dx.doi.org/10.1016/j.jmb.2013.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934758PMC
March 2014

Immunogenicity of membrane-bound HIV-1 gp41 membrane-proximal external region (MPER) segments is dominated by residue accessibility and modulated by stereochemistry.

J Biol Chem 2013 Nov 18;288(44):31888-901. Epub 2013 Sep 18.

From the Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.

Structural characterization of epitope-paratope pairs has contributed to the understanding of antigenicity. By contrast, few structural studies relate to immunogenicity, the process of antigen-induced immune responses in vivo. Using a lipid-arrayed membrane-proximal external region (MPER) of HIV-1 glycoprotein 41 as a model antigen, we investigated the influence of physicochemical properties on immunogenicity in relation to structural modifications of MPER/liposome vaccines. Anchoring the MPER to the membrane via an alkyl tail or transmembrane domain retained the MPER on liposomes in vivo, while preserving MPER secondary structure. However, structural modifications that affected MPER membrane orientation and antigenic residue accessibility strongly impacted induced antibody responses. The solvent-exposed MPER tryptophan residue (Trp-680) was immunodominant, focusing immune responses, despite sequence variability elsewhere. Nonetheless, immunogenicity could be readily manipulated using site-directed mutagenesis or structural constraints to modulate amino acid surface display. These studies provide fundamental insights for immunogen design aimed at targeting B cell antibody responses.
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http://dx.doi.org/10.1074/jbc.M113.494609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814781PMC
November 2013

Electron spin resonance studies of trityl OX063 at a concentration optimal for DNP.

Phys Chem Chem Phys 2013 Jun;15(24):9800-7

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

We have performed temperature-dependent electron spin resonance (ESR) measurements of the stable free radical trityl OX063, an efficient polarizing agent for dissolution dynamic nuclear polarization (DNP), at the optimum DNP concentration (15 mM). We have found that (i) when compared to the W-band electron spin-lattice relaxation rate T1e(-1) of other free radicals used in DNP at the same concentration, trityl OX063 has slower T1e(-1) than BDPA and 4-oxo-TEMPO. At T > 20 K, the T1e(-1)vs. T data of trityl OX063 appears to follow a power law dependence close to the Raman process prediction whereas at T < 10 K, electronic relaxation slows and approaches the direct process behaviour. (ii) Gd(3+) doping, a factor known to enhance DNP, of trityl OX063 samples measured at W-band resulted in monotonic increases of T1e(-1) especially at temperatures below 20-40 K while the ESR lineshapes remained essentially unchanged. (iii) The high frequency ESR spectrum can be fitted with an axial g-tensor with a slight g-anisotropy: g(x) = g(y) = 2.00319(3) and g(z) = 2.00258(3). Although the ESR linewidth D monotonically increases with field, the temperature-dependent T1e(-1) is almost unchanged as the ESR frequency is increased from 9.5 GHz to 95 GHz, but becomes faster at 240 GHz and 336 GHz. The ESR properties of trityl OX063 reported here may provide insights into the efficiency of DNP of low-γ nuclei performed at various magnetic fields, from 0.35 T to 12 T.
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http://dx.doi.org/10.1039/c3cp50186hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698225PMC
June 2013

Dissolution DNP-NMR spectroscopy using galvinoxyl as a polarizing agent.

J Magn Reson 2013 Feb 19;227:14-9. Epub 2012 Nov 19.

Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, 75390, United States.

The goal of this work was to test feasibility of using galvinoxyl (2,6-di-tert-butyl-α-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy) as a polarizing agent for dissolution dynamic nuclear polarization (DNP) NMR spectroscopy. We have found that galvinoxyl is reasonably soluble in ethyl acetate, chloroform, or acetone and the solutions formed good glasses when mixed together or with other solvents such as dimethyl sulfoxide. W-band electron spin resonance (ESR) measurements revealed that galvinoxyl has an ESR linewidth D intermediate between that of carbon-centered free radical trityl OX063 and the nitroxide-based 4-oxo-TEMPO, thus the DNP with galvinoxyl for nuclei with low gyromagnetic ratio γ such as (13)C and (15)N is expected to proceed predominantly via the thermal mixing process. The optimum radical concentration that would afford the highest (13)C nuclear polarization (approximately 6% for [1-(13)C]ethyl acetate) at 3.35 T and 1.4 K was found to be around 40 mM. After dissolution, large liquid-state NMR enhancements were achieved for a number of (13)C and (15)N compounds with long spin-lattice relaxation time T(1). In addition, the hydrophobic galvinoxyl free radical can be easily filtered out from the dissolution liquid when water is used as the solvent. These results indicate that galvinoxyl can be considered as an easily available free radical polarizing agent for routine dissolution DNP-NMR spectroscopy.
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http://dx.doi.org/10.1016/j.jmr.2012.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552151PMC
February 2013

The efficiency of DPPH as a polarising agent for DNP-NMR spectroscopy.

RSC Adv 2012 Jan 25;2(33):12812-12817. Epub 2012 Oct 25.

Advanced Imaging Research Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390 USA. ; Tel: +1-214-645-2750.

The free radical 2,2-diphenyl-1-pycrylhydrazyl (DPPH) was tested as a polarising agent for fast dissolution dynamic nuclear polarisation (DNP) NMR spectroscopy. DPPH was found to be reasonably soluble in sulfolane and the optimum concentration for DNP is 20-40 mM depending upon whether short polarisation times or the maximum signal intensity is needed. W-band ESR measurements revealed that the ESR linewidth D of DPPH is intermediate between that of BDPA and 4-oxo-TEMPO. Several thousand-fold NMR signal enhancements in the liquid-state were achieved for (13)C, (15)N, (89)Y, and (109)Ag compounds, demonstrating that DPPH can be added to the list of polarising agents for DNP-NMR spectroscopy. Furthermore, the hydrophobic DPPH free radical can be easily filtered out from the dissolution liquid when water is used as the dissolution solvent.
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http://dx.doi.org/10.1039/C2RA21853DDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507463PMC
January 2012

Antibody mechanics on a membrane-bound HIV segment essential for GP41-targeted viral neutralization.

Nat Struct Mol Biol 2011 Oct 16;18(11):1235-43. Epub 2011 Oct 16.

Laboratory of Immunobiology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain complementarity-determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.
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http://dx.doi.org/10.1038/nsmb.2154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253551PMC
October 2011

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation.

Proc Natl Acad Sci U S A 2011 May 2;108(20):8218-23. Epub 2011 May 2.

National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310, USA.

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.
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http://dx.doi.org/10.1073/pnas.1014137108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100986PMC
May 2011

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling.

Proc Natl Acad Sci U S A 2011 Feb 24;108(5):1868-73. Epub 2011 Jan 24.

Department of Microbiology and Molecular Genetics and Medicine, Harvard Medical School, Boston, MA 02115, USA.

The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin--lethal factor (LF) and edema factor (EF)--across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1' of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.
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http://dx.doi.org/10.1073/pnas.1018965108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033316PMC
February 2011

Distance and dynamics determination by W-band DEER and W-band ST-EPR.

Eur Biophys J 2010 Mar 9;39(4):711-9. Epub 2009 Dec 9.

National High Magnetic Field Laboratory, Institute of Molecular Biophysics, Department of Biological Science, Department of Biochemistry and Chemistry, Florida State University, Tallahassee, FL 32310, USA.

To explore high-field EPR in biological applications we have compared measurements of dynamics with X-band (9 GHz) and W-band (94 GHz) saturation transfer EPR (ST-EPR) and distance determination by X and W-band DEER. A fourfold increase of sensitivity was observed for W-band ST-EPR compared with X-band. The distance measurements at both fields showed very good agreement in both the average distances and in the distance distributions. Multifrequency EPR thus provides an additional experimental dimension to facilitate extraction of distance populations. However, the expected orientational selectivity of W-band DEER to determine the relative orientation of spins has not been realized, most likely because of the large orientational disorder of spin labels on the protein surface.
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http://dx.doi.org/10.1007/s00249-009-0565-3DOI Listing
March 2010

Interactions between lipids and human anti-HIV antibody 4E10 can be reduced without ablating neutralizing activity.

J Virol 2010 Jan 11;84(2):1076-88. Epub 2009 Nov 11.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA.

Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.
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http://dx.doi.org/10.1128/JVI.02113-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798360PMC
January 2010

Solubilization and characterization of the anthrax toxin pore in detergent micelles.

Protein Sci 2009 Sep;18(9):1882-95

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115, USA.

Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self-associates to form a heptameric ring-shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore-like state. N-tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion-competent 440-kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore-like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14-solubilized pore showed an extended, mushroom-shaped structure. Circular dichroïsm measurements revealed an increase in alpha helix and a decrease in beta structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2beta2-2beta3 loop transforms into the transmembrane segment of the beta-barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to alpha structure is dependent on residues of the luminal 2beta11-2beta12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.
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http://dx.doi.org/10.1002/pro.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777363PMC
September 2009
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