Labeling Electron Paramagnetic Resonance Spectroscopy Research Articles

Bovine Serum Albumin Effect on Collapsing PNIPAM Chains in Aqueous Solutions: Spin Label and Spin Probe Study.

The influence of bovine serum albumin (BSA) on collapsing poly(N-isopropylacrylamide) (PNIPAM) chains was studied with turbidimetry and spin probe and spin label electron paramagnetic resonance spectroscopy. An increased ratio of collapsed chains in aqueous solutions in the narrow temperature region near the LCST appeared in the presence of 2.5-10 wt% BSA. The spin probe EPR data indicate that the inner cavities of the BSA dimers are probably responsive to the capture of small hydrophobic or amphiphilic molecules, such as TEMPO nitroxyl radical. The observed features of the structure and dynamics of inhomogeneities of aqueous PNIPAM-BSA solutions, including their mutual influence on the behavior of the polymer and protein below the LCST, should be considered when developing and investigating PNIPAM-based drug delivery systems.

Charge Distribution Patterns of IA3 Impact Conformational Expansion and Hydration Diffusivity of the Disordered Ensemble.

IA3 is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically disordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based on the intrinsically disordered protein (IDP) parameters of fractional net charge (FNC), net charge density per residue (NCPR), and charge patterning (κ), the two domains of IA3 are defined to occupy different domains within conformationally based subclasses of IDPs, thus making IA3 a bimodal domain IDP. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and low-field Overhauser dynamic nuclear polarization (ODNP) spectroscopy results show that these two domains possess different degrees of compaction and hydration diffusivity behavior. This work suggests that SDSL EPR line shapes, analyzed in terms of their local tumbling volume (VL), provide insights into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution patterns within a conformational subclass can impact bound water hydration dynamics, thus possibly offering an alternative thermodynamic property that can encode conformational binding and behavior of IDPs and liquid-liquid phase separations.

Conformational investigation of the asymmetric periplasmic domains of E. coli LptB2FGC using SDSL CW EPR spectroscopy.

The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA-G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptB2FG, is an asymmetric ATP binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptB2FGC binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study will provide insight into the structural asymmetry associated with the newly defined type VI ABC transporter class.

Conformational changes in the activation loop of a bacterial PASTA kinase.

Many bacterial genomes encode a transmembrane protein kinase belonging to the PASTA kinase family, which controls numerous processes in diverse bacterial pathogens, including antibiotic resistance, cell division, stress resistance, toxin production, and virulence. PASTA kinases share a conserved three-part domain architecture, consisting of an extracellular PASTA domain, proposed to sense the peptidoglycan layer status, a single transmembrane helix, and an intracellular Ser/Thr kinase domain. The crystal structures of the kinase domain from two homologous PASTA kinases reveal a characteristic two-lobed structure typical of eukaryotic protein kinases with a centrally located, but unresolved, activation loop that becomes phosphorylated and regulates downstream signaling pathways. We previously identified three sites of phosphorylation on the activation loop (T163, T166, and T168) of IreK, a PASTA kinase from the pathogen Enterococcus faecalis, as well as a distal phosphorylation site (T218) that each influence IreK activity in vivo. Still, the mechanism by which loop phosphorylation regulates PASTA kinase function is yet unknown. Therefore, we utilized site-directed spin labeling (SDSL) and continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to assess the E. faecalis IreK kinase activation loop dynamics, including the effects of phosphorylation on activation loop motion, and the IreK-IreB interaction. Our results reveal that the IreK activation loop occupies a more immobile state when dephosphorylated, and that loop autophosphorylation shifts the loop to a more mobile state that can then enable interaction with IreB, a known substrate.

Characterizing the interactions between the LPS transport protein LptC and the ABC transporter LptB2FG.

Synthesis of the essential glycolipid lipopolysaccharide (LPS) in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following this process, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM). LPS transport to the OM is an essential process and disruption at any stage has detrimental effects on bacterial viability. LptB2FG, the first component of the Lpt system, is a type IV ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane helix, which intercalates between the LptF and LptG transmembrane domains, and has been shown to regulate the ATPase activity of LptB2FG. Despite a high level of structural homology between the periplasmic domains of LptF and LptG, the crystal and cryo-EM structures of LptB2FG in complex with LptC show preferential binding of the periplasmic domain of LptC to the periplasmic domain of LptF. Therefore, the role of LptG in this transport process remains unknown. Additionally, we identified six partial loss of function LptC mutations during an in vivo exploratory mutagenesis study. To further investigate the LptB2FGC complex, site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy, microscale thermophoresis (MST), and an in vitro ATPase assay were employed to characterize differences between the binding partners of periplasmic LptF and LptG along with the mechanistic effects of the LptC loss of function mutants on partner binding and ATPase function or regulation. This work provides evidence suggesting a previously undefined interaction between LptC and LptG while also exploring the role of partial loss of function LptC mutants.

Site-Directed Spin Labeling EPR Spectroscopy for Determination of Albumin Structural Damage and Hypoalbuminemia in Critical COVID-19.

The main factors in the COVID-19 pathology, which can initiate extensive structural changes at the cellular and molecular levels, are the generation of free radicals in abnormal amounts, and oxidative stress. Under "oxidative shock" conditions, the proteins undergo various modifications that affect their function and activity, and as a result distribute malfunctioning protein derivatives in the body. Human serum albumin is a small globular protein characterized by a high overall binding capacity for neutral lipophilic and acidic dosage forms. The albumin concentration is crucial for the maintenance of plasma oncotic pressure, the transport of nutrients, amino acids, and drugs, the effectiveness of drug therapy, and the prevention of drug toxicity. Hypoalbuminemia and structural defects molecule in the protein suggest a risk of changed metabolism and increased plasma concentration of unbound drugs. Therefore, the albumin structural and functional changes accompanied by low protein levels can be a serious prerequisite for ineffective therapy, frequent complications, and high mortality in patients with SARS-CoV-2 infection. The current opinion aims the research community the application of Site-Directed Spin Labeling Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) and 3-Maleimido-PROXYL radical in determining abnormalities of the albumin dynamics and protein concentrations in COVID-19 critical patients.

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.

Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC50 and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (KM/W), and the concentration of the compound in the membrane (cm50) and in the aqueous phase (cw50), which inhibits cell growth by 50%. The KM/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 108 parasites/mL the compounds did not show significantly different results. The cm50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.

Mycobacterium abscessus cell wall and plasma membrane characterization by EPR spectroscopy and effects of amphotericin B, miltefosine and nerolidol

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to characterize the components of the Mycobacterium abscessus massiliense cell envelope and their interactions with amphotericin B (AmB), miltefosine (MIL), and nerolidol (NER). Spin labels analogous to stearic acid and phosphatidylcholine (PC) were distributed on an envelope layer with fluidity comparable to other biological membranes, probably the mycobacterial cell wall, because after treatment with AmB a highly rigid spectral component was evident in the EPR spectra. Methyl stearate analogue spin labels found a much more fluid membrane and did not detect the presence of AmB, except for at very high drug concentrations. Unlike other spin-labeled PCs, the TEMPO-PC spin probe, with the nitroxide moiety attached to the choline of the PC headgroup, also did not detect the presence of AmB. On the other hand, the steroid spin labels were not distributed across the membranes of M. abscessus and, instead, were concentrated in some other location of the cell envelope. Both MIL and NER compounds at 10 μM caused increased fluidity in the cell wall and plasma membrane. Furthermore, NER was shown to have a remarkable ability to extract lipids from the mycobacterial cell wall. The EPR results suggest that the resistance of mycobacteria to the action of AmB must be related to the fact that this drug does not reach the bacterial plasma membrane.

In situ monitoring of protein transfer into nanoscale channels

Protein transfer into nanoscale compartments is critical for many cellular/life processes, yet there are few reports on how compartment properties impact the protein orientation during a transfer. Such a knowledge gap limits a deeper understanding of the protein transfer mechanism, which could be bridged using nanoporous materials. Here, we use a mesoporous silica, a covalent organic framework, and a metal-organic framework with charged, hydrophobic, and neutral surfaces, respectively, to elucidate the impact of channel properties on the transfer of a model protein, lysozyme. Using site-directed spin labeling and time-resolved electron paramagnetic resonance spectroscopy, we reveal that the transfer can be a multi-step process depending on channel properties and depict the relative orientation changes of lysozyme upon transfer into each channel. To the best of our knowledge, this is the first structural insight into protein orientation upon transfer into different compartments, meaningful for the rational design of synthetic materials to host enzymes or mimic the cellular compartments. Protein transfer into nanoscale channels differing in hydrophobicity is demonstrated Multi-step protein transfer is observed for charged and hydrophobic channels Protein changes its orientation depending on the channel surface properties Time-resolved EPR spectroscopy can reveal residue-level details of protein transfer Pan et al. report the orientation changes of a model protein upon transfer into three nanoscale channels that differ in surface hydrophobicity (but a similar diameter). The findings may be useful for guiding the rational design of synthetic materials to host enzymes and/or mimic the cellular compartments.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Localization of metal ions in biomolecules by means of pulsed dipolar EPR spectroscopy.

Metal ions are important for the folding, structure, and function of biomolecules. Thus, knowing where their binding sites are located in proteins or oligonucleotides is a critical objective. X-ray crystallography and nuclear magnetic resonance are powerful methods in this respect, but both have their limitations. Here, a complementary method is highlighted in which paramagnetic metal ions are localized by means of trilateration using a combination of site-directed spin labeling and pulsed dipolar electron paramagnetic resonance spectroscopy. The working principle, the requirements, and the limitations of the method are critically discussed. Several applications of the method are outlined and compared with each other.

Enzyme Immobilization on Graphite Oxide (GO) Surface via One-Pot Synthesis of GO/Metal-Organic Framework Composites for Large-Substrate Biocatalysis.

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often "harsh" reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal-Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.

Identifying β‐arrestin1 conformational states by a non‐GPCR binding partner

The G protein‐coupled receptor (GPCR) C‐X‐C motif chemokine receptor 4 (CXCR4), and its cognate chemokine CXCL12, play essential roles in hematopoiesis, cardiogenesis, neurogenesis and immune responses. In addition, CXCR4 signaling contributes to several aspects of cancer progression and other diseases. Despite its importance CXCR4 signaling remains poorly understood. Because of the critical involvement in human cancers, a comprehensive understanding of CXCR4 signaling is of great therapeutic significance. The present study is focused on a novel CXCR4 signaling pathway thatregulates chemotaxis via the adaptor protein β‐arrestin1 (βarr1). β‐arrestins typically bind to activated GPCRs to negatively regulate signaling by promoting desensitization and internalization, but β‐arrestins also positively promote signaling by interacting with non‐GPCR binding partners. It is relatively well known how β‐arrestins interact with activated GPCRs to promote their internalization and desensitization, however very little is known how β‐arrestins interact with non‐GPCR binding partners to initiate signaling. Previously, our lab showed that βarr1 interacts directly with the adaptor protein STAM1 (signal transducing adaptor molecule) to promote CXCR4‐dependent activation of focal adhesion kinase (FAK), however the mechanism remains unknown. Here, we tested the hypothesis that STAM1 induces a unique conformational signature in βarr1 that facilitates activation of FAK. To test this, we employed Site‐Directed Spin Labeling (SDSL) Electron Paramagnetic Resonance (EPR) spectroscopy. We employed double electron‐electron resonance (DEER) spectroscopy to determine if STAM1 induces a unique conformational signature on βarr1 by incorporating spin labels on selected double cysteine mutants of βarr1 and measuring distances between spin labeled residues upon STAM1 or GPCR binding. STAM1 binding induced similar conformational changes on βarr1 in one double cysteine labeled mutant, but not on two others, as compared to a GPCR. These data suggest that βarr1 undergoes a unique conformational change upon STAM1 binding, in contrast to the well‐established conformational change that occurs upon GPCR binding. To determine if this could be explained by the fact that STAM1 and GPCRs bind to different sites on βarr1 we employed CW(continuous wave)‐EPR spectroscopy to map the STAM1 binding surface on βarr1 by monitoring spectral line‐shape changes of selected reporter sites in βarr1 in the presence or absence of STAM1. Spin labels attached to selected sites on the front surface of βarr1 did not show any side chain immobilization when bound to STAM1, suggesting that STAM1 does not bind to the front surface. In contrast, we identified two residues located on the back surface of βarr1 that showed significant spin label immobilization when bound to STAM1, suggesting STAM1 binds to this surface of βarr1. As GPCRs and other non‐GPCR binding partners typically bind to the front surface of β‐arrestins, our data suggest that STAM1 binds to the back surface on βarr1, which is likely linked to discrete βarr1 conformational dynamics. We propose that STAM1 induces a unique βarr1 conformational signature, which is required for CXCR4‐dependent activation of FAK and chemotaxis.Support or Funding InformationGM106727 06A1

Synthesis, Antileishmanial Activity and Spin Labeling EPR Studies of Novel β-Carboline-Oxazoline and β-Carboline-Dihydrooxazine Derivatives

A series of novel 1-(substituted-phenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8a-8i) and 1-(substituted-phenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9a-9h) derivatives, as well as their respective N-(chloroalkyl)-1-(substituted-phenyl)-9H-β-carboline-3-carboxamide precursors (6a-6i and 7a-7h), were synthesized and evaluated for their in vitro antileishmanial activity against promastigote and intracellular amastigote forms of Leishmania amazonensis. Compounds 8d, 8i, 9e and 9h exhibited significant activity for both promastigote and amastigote forms, with IC50 (50% inhibitory concentration) values ranging from 2.9 to 23.0 µM. In addition, spin label electron paramagnetic resonance (EPR) spectroscopy studies were carried out for the most active compounds against L. amazonensis promastigotes. The studies indicated that the tested compounds cause strong stiffness in the parasite plasma membrane and are capable of inducing internal metalloproteins oxidation of the parasite, resulting in their cross-linking to skeletal proteins. Compounds 8d and 8i produced the largest effect, showing that the presence of oxazoline group at C-3 of β-carboline nucleus is important for antileishmanial activity.

Gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis, EPR Characterization, Rotamer Libraries and Biocompatibility.

The availability of bioresistant spin labels is crucial for the optimization of site‐directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin‐labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide‐ and maleimide‐functionalized spin labels based on the gem‐diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem‐dimethyl analogs by site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem‐diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.

Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy.

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

Site-directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) "locked-off" state of the CO-sensing transcription factor CooA.

The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site-directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs-ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild-type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe132 in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) "locked-off" CooA variants labeled with either MTSL or MAL-6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs-ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) "locked-off" CooA. This observation suggests that, in direct contrast to effector-free CRP, Fe(III) "locked-off" CooA undergoes conformational exchange on the μs-ms timescale. Importantly, we establish MAL-6 as a spin label with a redox-stable linkage that may be utilized to compare conformational dynamics between functional states of CooA.

Probing Conformational Motions Underlying Anesthetic Drug Actions in a Ligand-Gated Ion Channel

General anesthetics and ethanol exert their functional effects on pentameric ligand-gated ion channels (pLGICs) by binding to distinct allosteric sites that are far from the agonist binding sites. While recent high-resolution structures of prokaryotic and eukaryotic pLGICs, solved in the presence of these drugs have illuminated their binding sites, the structural mechanisms underlying how these drugs enhance or inhibit pLGIC function are largely unknown. Recently, a single residue variant in the bacterial pLGIC, GLIC, was identified that is potentiated by ethanol (PNAS 108:12149-12154, 2011). Taking advantage of this GLIC ethanol-sensitive mutant, we are using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy to study motions underlying ethanol potentiation of GLIC currents. Previous studies have shown that agonist-mediated channel opening is associated with an inward tilting of the extracellular binding domain (ECD). Here, we examined if, and how, the ECD moves in response to ethanol binding in the transmembrane channel domain. Individual cysteine residues were introduced in the ECD of an ethanol-sensitive GLIC construct (F14’A) at the top of β-strands (β1, 2, 5, 6, 8), labeled with the spin probe MTSL and ethanol-induced motions were measured using double electron-electron resonance (DEER) spectroscopy and compared to agonist (proton)-induced gating motions. DEER distance distributions for spin labels attached to N19C (β1), S93C (β5) and Q101C (β6) revealed that the attached probes moved closer together at pH 3.0, indicating that the inner β-sheet of GLIC ECD tilts inwards upon agonist activation. Interestingly, DEER distance distributions for N19 (β1) and S93 (β5) showed no significant changes in the presence of ethanol, indicating that structural mechanisms underlying ethanol allosteric drug modulation of pLGIC function are different than agonist-mediated motions that lead to channel activation.

Characterization of and lipopolysaccharide binding to the E. coli LptC protein dimer.

Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O-antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB2 complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site-directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration-independent N-terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C-terminal regions showing a greater affinity for LPS than the N-terminal domain, which has similar dissociation constants to LptA.