Publications by authors named "Linda Columbus"

41 Publications

TM1385 from Thermotoga maritima functions as a phosphoglucose isomerase via cis-enediol-based mechanism with active site redundancy.

Biochim Biophys Acta Proteins Proteom 2021 04 7;1869(4):140602. Epub 2021 Jan 7.

Department of Chemistry, University of Virginia, Charlottesville, VA, USA. Electronic address:

Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.
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http://dx.doi.org/10.1016/j.bbapap.2021.140602DOI Listing
April 2021

Imaging Flow Cytometry Analysis of CEACAM Binding to Opa-Expressing Neisseria gonorrhoeae.

Cytometry A 2020 10 2;97(10):1081-1089. Epub 2020 Jun 2.

Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia, 22903, USA.

Human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are a family of receptors that mediate intercellular interactions. Pathogenic bacteria have ligands that bind CEACAMs on human cells. Neisseria gonorrhoeae (Gc) encodes numerous unique outer membrane opacity-associated (Opa) proteins that are ligands for one or more CEACAMs. CEACAMs that are expressed on epithelial cells facilitate Gc colonization, while those expressed on neutrophils affect phagocytosis and consequent intracellular survival of Gc. Since Opa protein expression is phase-variable, variations in receptor tropism affect how individual bacteria within a population interact with host cells. Here we report the development of a rapid, quantitative method for collecting and analyzing fluorescence intensity data from thousands of cells in a population using imaging flow cytometry to detect N-CEACAM bound to the surface of Opa-expressing Gc. We use this method to confirm previous findings regarding Opa-CEACAM interactions and to examine the receptor-ligand interactions of Gc expressing other Opa proteins, as well as for other N-CEACAM proteins. © 2020 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.24037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062897PMC
October 2020

The Fluidity of Phosphocholine and Maltoside Micelles and the Effect of CHAPS.

Biophys J 2019 05 30;116(9):1682-1691. Epub 2019 Mar 30.

Department of Chemistry, University of Virginia, Charlottesville, Virginia. Electronic address:

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.e., fluidity). The nitroxide probe rotational dynamics in longer chain detergents is more restricted than in shorter chain detergents, and maltoside micelles are more restricted than phosphocholine micelles. Furthermore, the micelle microviscosity can be modulated with mixtures, as demonstrated with mixtures of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate with n-dodecylphosphocholine, n-tetradecylphosphocholine, n-decyl-β-D-maltoside, or n-dodecyl-β-D-maltoside. These results indicate that observed differences in membrane protein stability in these detergents could be due to fluidity in addition to the already determined structural differences.
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http://dx.doi.org/10.1016/j.bpj.2019.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506624PMC
May 2019

Quantifying Carcinoembryonic Antigen-like Cell Adhesion Molecule-Targeted Liposome Delivery Using Imaging Flow Cytometry.

Mol Pharm 2019 06 13;16(6):2354-2363. Epub 2019 May 13.

Carcinoembryonic antigen-like cell adhesion molecules (CEACAMs) are human cell-surface proteins that can exhibit increased expression on tumor cells and are thus a potential target for novel tumor-seeking therapeutic delivery methods. We hypothesize that engineered nanoparticles containing a known interaction partner of CEACAM, Neisseria gonorrhoeae outer membrane protein Opa, can be used to deliver cargo to specific cellular targets. In this study, the cell association and uptake of protein-free liposomes and Opa proteoliposomes into CEACAM-expressing cells were measured using imaging flow cytometry. A size-dependent internalization of liposomes into HeLa cells was observed through endocytic pathways. Opa-dependent, CEACAM1-mediated uptake of liposomes into HeLa cells was observed, with limited colocalization with endosomal and lysosomal trafficking compartments. Given the overexpression of CEACAM1 on several distinct cancers and interest in using CEACAM1 as a component in treatment strategies, these results support further pursuit of investigating Opa-dependent specificity and the internalization mechanism for therapeutic delivery.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b01274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6740330PMC
June 2019

Heterocellular Contact Can Dictate Arterial Function.

Circ Res 2019 05;124(10):1473-1481

From the Robert M. Berne Cardiovascular Research Center (X.S., C.A.R., T.C.S.K., A.S.K., Y.Y., M.E.G., A.K.B., B.E.I.), University of Virginia School of Medicine, Charlottesville.

Rationale: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs.

Objective: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling.

Methods And Results: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation.

Conclusions: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540980PMC
May 2019

Refinement of Highly Flexible Protein Structures using Simulation-Guided Spectroscopy.

Angew Chem Int Ed Engl 2018 12 27;57(52):17110-17114. Epub 2018 Nov 27.

Departments of Biomedical Engineering and Molecular Physiology, University of Virginia, Box 800886, Charlottesvile, VA, 22908, USA.

Highly flexible proteins present a special challenge for structure determination because they are multi-structured yet not disordered, so their conformational ensembles are essential for understanding function. Because spectroscopic measurements of multiple conformational populations often provide sparse data, experiment selection is a limiting factor in conformational refinement. A molecular simulations- and information-theory based approach to select which experiments best refine conformational ensembles has been developed. This approach was tested on three flexible proteins. For proteins where a clear mechanistic hypothesis exists, experiments that test this hypothesis were systematically identified. When available data did not yield such mechanistic hypotheses, experiments that significantly outperform structure-guided approaches in conformational refinement were identified. This approach offers a particular advantage when refining challenging, underdetermined protein conformational ensembles.
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http://dx.doi.org/10.1002/anie.201810462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424112PMC
December 2018

Low- q Bicelles Are Mixed Micelles.

J Phys Chem Lett 2018 Aug 25;9(15):4469-4473. Epub 2018 Jul 25.

Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904 , United States.

Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.
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http://dx.doi.org/10.1021/acs.jpclett.8b02079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353637PMC
August 2018

Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.

Nat Commun 2017 10 31;8(1):1201. Epub 2017 Oct 31.

Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, 23298, USA.

Cellular protein homeostasis depends on heat shock proteins 70 kDa (Hsp70s), a class of ubiquitous and highly conserved molecular chaperone. Key to the chaperone activity is an ATP-induced allosteric regulation of polypeptide substrate binding and release. To illuminate the molecular mechanism of this allosteric coupling, here we present a novel crystal structure of an intact human BiP, an essential Hsp70 in ER, in an ATP-bound state. Strikingly, the polypeptide-binding pocket is completely closed, seemingly excluding any substrate binding. Our FRET, biochemical and EPR analysis suggests that this fully closed conformation is the major conformation for the ATP-bound state in solution, providing evidence for an active release of bound polypeptide substrates following ATP binding. The Hsp40 co-chaperone converts this fully closed conformation to an open conformation to initiate productive substrate binding. Taken together, this study provided a mechanistic understanding of the dynamic nature of the polypeptide-binding pocket in the Hsp70 chaperone cycle.
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http://dx.doi.org/10.1038/s41467-017-01310-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662698PMC
October 2017

Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX).

Hypertension 2016 12 31;68(6):1494-1503. Epub 2016 Oct 31.

From the Department of Molecular Physiology and Biological Physics (T.C.S.K., C.M., M.C., M.P., M.Y., B.E.I.), Robert M. Berne Cardiovascular Research Center (T.C.S.K., J.T.B., G.B.B.-F., C.M., L.J.D., X.S., A.K.B., M.E.G., B.E.I.), Department of Pharmacology (L.J.D.), Division of Nephrology, Department of Medicine (S.C., T.H.L.), and Division of Endocrinology, Department of Medicine (E.B.), University of Virginia School of Medicine, Charlottesville; Department of Physiological Sciences, Federal University of Espirito Santa, Brazil (G.B.B.-F., A.S.P.); Departments of Pediatrics and Biochemistry, Washington University in Saint Louis, MO (S.R., A.D.); Department of Biomedical Engineering (B.N., S.M.P., S.H.) and Department of Chemistry (J.N.M., L.C.), University of Virginia, Charlottesville; and College of Pharmacy, Dalian Medical University, Dalian, China (X.S.).

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, HbαX administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II-induced hypertensive states. HbαX acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O binding in red blood cells. In human vasculature, HbαX blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, HbαX modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.116.08171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159279PMC
December 2016

Neisserial Opa Protein-CEACAM Interactions: Competition for Receptors as a Means of Bacterial Invasion and Pathogenesis.

Biochemistry 2016 08 1;55(31):4286-94. Epub 2016 Aug 1.

Department of Chemistry and ‡Department of Microbiology, Immunology, and Cancer Biology, University of Virginia , Charlottesville, Virginia 22903, United States.

Carcino-embryonic antigen-like cellular adhesion molecules (CEACAMs), members of the immunoglobulin superfamily, are responsible for cell-cell interactions and cellular signaling events. Extracellular interactions with CEACAMs have the potential to induce phagocytosis, as is the case with pathogenic Neisseria bacteria. Pathogenic Neisseria species express opacity-associated (Opa) proteins, which interact with a subset of CEACAMs on human cells, and initiate the engulfment of the bacterium. We demonstrate that recombinant Opa proteins reconstituted into liposomes retain the ability to recognize and interact with CEACAMs in vitro but do not maintain receptor specificity compared to that of Opa proteins natively expressed by Neisseria gonorrhoeae. We report that two Opa proteins interact with CEACAMs with nanomolar affinity, and we hypothesize that this high affinity is necessary to compete with the native CEACAM homo- and heterotypic interactions in the host. Understanding the mechanisms of Opa protein-receptor recognition and engulfment enhances our understanding of Neisserial pathogenesis. Additionally, these mechanisms provide insight into how human cells that are typically nonphagocytic can utilize CEACAM receptors to internalize exogenous matter, with implications for the targeted delivery of therapeutics and development of imaging agents.
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http://dx.doi.org/10.1021/acs.biochem.6b00124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980159PMC
August 2016

Endothelial nitric oxide synthase in the microcirculation.

Cell Mol Life Sci 2015 Dec 25;72(23):4561-75. Epub 2015 Aug 25.

Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, P.O. Box 801394, Charlottesville, VA, 22908, USA.

Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.
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http://dx.doi.org/10.1007/s00018-015-2021-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628887PMC
December 2015

Known structure, unknown function: An inquiry-based undergraduate biochemistry laboratory course.

Biochem Mol Biol Educ 2015 Jul-Aug;43(4):245-62. Epub 2015 Jul 6.

Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22904.

Undergraduate biochemistry laboratory courses often do not provide students with an authentic research experience, particularly when the express purpose of the laboratory is purely instructional. However, an instructional laboratory course that is inquiry- and research-based could simultaneously impart scientific knowledge and foster a student's research expertise and confidence. We have developed a year-long undergraduate biochemistry laboratory curriculum wherein students determine, via experiment and computation, the function of a protein of known three-dimensional structure. The first half of the course is inquiry-based and modular in design; students learn general biochemical techniques while gaining preparation for research experiments in the second semester. Having learned standard biochemical methods in the first semester, students independently pursue their own (original) research projects in the second semester. This new curriculum has yielded an improvement in student performance and confidence as assessed by various metrics. To disseminate teaching resources to students and instructors alike, a freely accessible Biochemistry Laboratory Education resource is available at http://biochemlab.org.
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http://dx.doi.org/10.1002/bmb.20873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758391PMC
April 2016

Post-expression strategies for structural investigations of membrane proteins.

Authors:
Linda Columbus

Curr Opin Struct Biol 2015 Jun 16;32:131-8. Epub 2015 May 16.

Department of Chemistry, University of Virginia, Charlottesville, VA 22902, United States. Electronic address:

Currently, membrane proteins only comprise 1.5% of the protein data bank and, thus, still remain a challenge for structural biologists. Expression, stabilization in membrane mimics (e.g. detergent), heterogeneity (conformational and chemical), and crystallization in the presence of a membrane mimic are four major bottlenecks encountered. In response, several post-expression protein modifications have been utilized to facilitate structure determination of membrane proteins. This review highlights four approaches: limited proteolysis, deglycosylation, cysteine alkylation, and lysine methylation. Combined these approaches have facilitated the structure determination of more than 40 membrane proteins and, therefore, are a useful addition to the membrane protein structural biologist's toolkit.
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http://dx.doi.org/10.1016/j.sbi.2015.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512879PMC
June 2015

Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins: A Guide to Spin Label Paramagnetic Relaxation Enhancement Restraints.

Methods Enzymol 2015 17;557:329-48. Epub 2015 Mar 17.

Department of Biochemistry and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

Solution nuclear magnetic resonance structures of polytopic α-helical membrane proteins require additional restraints beyond the traditional Nuclear Overhauser Effect (NOE) restraints. Several methods have been developed and this review focuses on paramagnetic relaxation enhancement (PRE). Important aspects of spin labeling, PRE measurements, structure calculations, and structural quality are discussed.
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http://dx.doi.org/10.1016/bs.mie.2014.12.005DOI Listing
February 2016

Opa+ Neisseria gonorrhoeae exhibits reduced survival in human neutrophils via Src family kinase-mediated bacterial trafficking into mature phagolysosomes.

Cell Microbiol 2015 May 25;17(5):648-65. Epub 2014 Nov 25.

Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA.

During gonorrhoeal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial contents. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signalling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signalling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils' full antimicrobial arsenal.
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http://dx.doi.org/10.1111/cmi.12389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402142PMC
May 2015

Tuning micelle dimensions and properties with binary surfactant mixtures.

Langmuir 2014 Nov 28;30(44):13353-61. Epub 2014 Oct 28.

Department of Chemistry, University of Virginia , Charlottesville, Virginia 22904, United States.

Detergent micelles are used in many areas of research and technology, in particular, as mimics of the cellular membranes in the purification and biochemical and structural characterization of membrane proteins. Applications of detergent micelles are often hindered by the limited set of properties of commercially available detergents. Mixtures of micelle-forming detergents provide a means to systematically obtain additional micellar properties and expand the repertoire of micelle features available; however, our understanding of the properties of detergent mixtures is still limited. In this study, the shape and size of binary mixtures of seven different detergents commonly used in molecular host-guest systems and membrane protein research were investigated. The data suggests that the detergents form ideally mixed micelles with sizes and shapes different from those of pure individual micelles. For most measurements of size, the mixtures varied linearly with detergent mole fraction and therefore can be calculated from the values of the pure detergents. We propose that properties such as the geometry, size, and surface charge can be systematically and predictably tuned for specific applications.
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http://dx.doi.org/10.1021/la503458nDOI Listing
November 2014

Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

Biophys J 2014 Oct;107(7):1697-702

Department of Chemistry, University of Virginia, Charlottesville, Virginia. Electronic address:

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.
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http://dx.doi.org/10.1016/j.bpj.2014.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190660PMC
October 2014

Hemoglobin α/eNOS coupling at myoendothelial junctions is required for nitric oxide scavenging during vasoconstriction.

Arterioscler Thromb Vasc Biol 2014 Dec 2;34(12):2594-600. Epub 2014 Oct 2.

From the Department of Pharmacology and Chemical Biology (A.C.S.) and Heart, Lung, Blood and Vascular Medicine Institute (A.C.S., A.T.N., M.P.M.), University of Pittsburgh, PA; Robert M. Berne Cardiovascular Research Center (J.T.B., M.B., S.M.M., T.J., A.K.B., B.E.I.), Department of Molecular Physiology and Biophysics (M.B., M.V.A., A.V.S., B.E.I.), Deparment of Pediatrics (L.A.P.), Department of Chemistry (L.C.), and Department of Medicine (T.H.L.), University of Virginia, Charlottesville.

Objective: Hemoglobin α (Hb α) and endothelial nitric oxide synthase (eNOS) form a macromolecular complex at myoendothelial junctions; the functional role of this interaction remains undefined. To test if coupling of eNOS and Hb α regulates nitric oxide signaling, vascular reactivity, and blood pressure using a mimetic peptide of Hb α to disrupt this interaction.

Approach And Results: In silico modeling of Hb α and eNOS identified a conserved sequence of interaction. By mutating portions of Hb α, we identified a specific sequence that binds eNOS. A mimetic peptide of the Hb α sequence (Hb α X) was generated to disrupt this complex. Using in vitro binding assays with purified Hb α and eNOS and ex vivo proximity ligation assays on resistance arteries, we have demonstrated that Hb α X significantly decreased interaction between eNOS and Hb α. Fluorescein isothiocyanate labeling of Hb α X revealed localization to holes in the internal elastic lamina (ie, myoendothelial junctions). To test the functional effects of Hb α X, we measured cyclic guanosine monophosphate and vascular reactivity. Our results reveal augmented cyclic guanosine monophosphate production and altered vasoconstriction with Hb α X. To test the in vivo effects of these peptides on blood pressure, normotensive and hypertensive mice were injected with Hb α X, which caused a significant decrease in blood pressure; injection of Hb α X into eNOS(-/-) mice had no effect.

Conclusions: These results identify a novel sequence on Hb α that is important for Hb α/eNOS complex formation and is critical for nitric oxide signaling at myoendothelial junctions.
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http://dx.doi.org/10.1161/ATVBAHA.114.303974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239174PMC
December 2014

Hemoglobin α in the blood vessel wall.

Free Radic Biol Med 2014 Aug 14;73:136-42. Epub 2014 May 14.

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA; Department of Molecular Physiology and Biophysics, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

Hemoglobin has been studied and well characterized in red blood cells for over 100 years. However, new work has indicated that the hemoglobin α subunit (Hbα) is also found within the blood vessel wall, where it appears to localize at the myoendothelial junction (MEJ) and plays a role in regulating nitric oxide (NO) signaling between endothelium and smooth muscle. This discovery has created a new paradigm for the control of endothelial nitric oxide synthase activity, nitric oxide diffusion, and, ultimately, vascular tone and blood pressure. This review discusses the current knowledge of hemoglobin׳s properties as a gas exchange molecule in the bloodstream and extrapolates the properties of Hbα biology to the MEJ signaling domain. Specifically, we propose that Hbα is present at the MEJ to regulate NO release and diffusion in a restricted physical space, which would have powerful implications for the regulation of blood flow in peripheral resistance arteries.
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135531PMC
August 2014

Structure of the Neisserial outer membrane protein Opa₆₀: loop flexibility essential to receptor recognition and bacterial engulfment.

J Am Chem Soc 2014 Jul 19;136(28):9938-46. Epub 2014 May 19.

Department of Chemistry, University of Virginia , Charlottesville, Virginia 22904, United States.

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen-host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.
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http://dx.doi.org/10.1021/ja503093yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105060PMC
July 2014

Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane.

Mol Metab 2014 Apr 28;3(2):114-23. Epub 2013 Nov 28.

Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA ; Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA ; Department of Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA ; Emily Couric Clinical Cancer Center, University of Virginia, Charlottesville, VA 22908, USA.

Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
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http://dx.doi.org/10.1016/j.molmet.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3953706PMC
April 2014

Label-free method for cell counting in crude biological samples via paramagnetic bead aggregation.

Anal Chem 2013 Dec 14;85(23):11233-9. Epub 2013 Nov 14.

Department of Chemistry, ‡Department of Pathology, §Department of Biomedical Engineering, ⊥Department of Mechanical and Aerospace Engineering, ∥Center for Microsystems for the Life Sciences, University of Virginia , Charlottesville, VA 22904.

Under chaotropic conditions, DNA released from lysed cells causes the aggregation of paramagnetic beads in a rotating magnetic field in a manner that is independent of the presence of other cellular components. The extent of aggregation correlates with the mass of DNA in a quantitative manner (Leslie, D. C. et al., J. Am. Chem. Soc. 2012, 134, 5689-96), and from this, the number of DNA-containing cells in the sample can be enumerated. Microbial growth testing is demonstrated by monitoring bead aggregation with E. coli in the presence of ampicillin. Without the need for fluorescent labeling or Coulter counting, the white blood cell count can be defined directly from a microliter of crude whole blood. Specificity is brought to the process by coupling bead-based immunocapture with DNA-bead aggregation allowing for the enumeration of CD4+ T cells from human blood samples. The results of DNA-induced bead aggregation had a 95% correlation with those generated by flow cytometry. With the process requiring only inexpensive, widely available benchtop laboratory hardware, a digital camera, and a simple algorithm, this provided a highly accessible alternative to more expensive cell-counting techniques.
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http://dx.doi.org/10.1021/ac401402hDOI Listing
December 2013

Solution NMR resonance assignment strategies for β-barrel membrane proteins.

Protein Sci 2013 Aug 27;22(8):1133-40. Epub 2013 Jun 27.

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β-barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight-stranded β-barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β-barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β-barrel region (97% of the resonances corresponding to the β-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β-barrel membrane proteins.
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http://dx.doi.org/10.1002/pro.2291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832050PMC
August 2013

Dependence of micelle size and shape on detergent alkyl chain length and head group.

PLoS One 2013 8;8(5):e62488. Epub 2013 May 8.

Department of Chemistry, University of Virginia, Charlottesville, Virginia, United States of America.

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062488PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648574PMC
December 2013

A broad specificity nucleoside kinase from Thermoplasma acidophilum.

Proteins 2013 Apr 17;81(4):568-82. Epub 2013 Jan 17.

Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA.

The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, fructokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase-like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB-like family substrates; activity was not observed for ribose, fructose-1-phosphate, or fructose-6-phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49, and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent KM for guanosine of 0.21 μM and catalytic efficiency of 345,000 M(-1) s(-1) . These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK is a member of a new sub-family with broad nucleoside specificities. Proteins 2013. © 2012 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.24212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595323PMC
April 2013

Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

Nature 2012 Nov 31;491(7424):473-7. Epub 2012 Oct 31.

Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908, USA.

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.
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http://dx.doi.org/10.1038/nature11626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531883PMC
November 2012

Backbone ¹H, ¹³C and ¹⁵N resonance assignments of the α-helical membrane protein TM0026 from Thermotoga maritima.

Biomol NMR Assign 2013 Oct 28;7(2):203-6. Epub 2012 Jul 28.

Department of Chemistry, University of Virginia, McCormick Rd, Charlottesville, VA 22904, USA.

Critical to the use of solution NMR to describe the structure and flexibility of membrane proteins is the thorough understanding of the degree of perturbation induced by the detergent or other membrane mimetic. To develop a deeper understanding of the interaction between membrane proteins and micelles or bicelles, we will investigate the differences in structure and flexibility of a model membrane protein TM0026 from Thermotoga maritima using solution NMR. A comparison of the structural differences between TM0026 solubilized in different detergent combinations will provide important insight into the degree of modulation of membrane proteins by detergent physical properties. Here we report the nearly complete backbone and Cβ resonance assignments of the two transmembrane helical model protein TM0026. These assignments are the first step to using TM0026 to elucidate the interaction between membrane proteins and membrane mimetics.
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http://dx.doi.org/10.1007/s12104-012-9410-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543498PMC
October 2013

MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation.

Circ Res 2012 Jul 31;111(2):201-11. Epub 2012 May 31.

Robert M. Berne Cardiovascular Research Center, Charlottesville, VA 22908, USA.

Rationale: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis.

Objective: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.

Methods And Results: We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury.

Conclusions: We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.
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http://dx.doi.org/10.1161/CIRCRESAHA.112.272302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405546PMC
July 2012
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