Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2

ABSTRACT Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile. IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

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The highly conserved synapsin domain E mediates synapsin dimerization and phospholipid vesicle clustering

Biochemical Journal ◽

10.1042/bj20090762 ◽

2010 ◽

Vol 426 (1) ◽

pp. 55-64 ◽

Cited By ~ 16

Author(s):

Ilaria Monaldi ◽

Massimo Vassalli ◽

Angela Bachi ◽

Silvia Giovedì ◽

Enrico Millo ◽

...

Keyword(s):

Molecular Mechanisms ◽

Synapse Formation ◽

Phospholipid Composition ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Phospholipid Vesicle ◽

Synapsin I ◽

Protein Partners ◽

And Function ◽

Necessary And Sufficient

Synapsins are abundant SV (synaptic vesicle)-associated phosphoproteins that regulate synapse formation and function. The highly conserved C-terminal domain E was shown to contribute to several synapsin functions, ranging from formation of the SV reserve pool to regulation of the kinetics of exocytosis and SV cycling, although the molecular mechanisms underlying these effects are unknown. In the present study, we used a synthetic 25-mer peptide encompassing the most conserved region of domain E (Pep-E) to analyse the role of domain E in regulating the interactions between synapsin I and liposomes mimicking the phospholipid composition of SVs (SV–liposomes) and other pre-synaptic protein partners. In affinity-chromatography and cross-linking assays, Pep-E bound to endogenous and purified exogenous synapsin I and strongly inhibited synapsin dimerization, indicating a role in synapsin oligomerization. Consistently, Pep-E (but not its scrambled version) counteracted the ability of holo-synapsin I to bind and coat phospholipid membranes, as analysed by AFM (atomic force microscopy) topographical scanning, and significantly decreased the clustering of SV–liposomes induced by holo-synapsin I in FRET (Förster resonance energy transfer) assays, suggesting a causal relationship between synapsin oligomerization and vesicle clustering. Either Pep-E or a peptide derived from domain C was necessary and sufficient to inhibit both dimerization and vesicle clustering, indicating the participation of both domains in these activities of synapsin I. The results provide a molecular explanation for the effects of domain E in nerve terminal physiology and suggest that its effects on the size and integrity of SV pools are contributed by the regulation of synapsin dimerization and SV clustering.

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The Glycine Lipids ofBacteroides thetaiotaomicronAre Important for Fitness during GrowthIn VivoandIn Vitro

Applied and Environmental Microbiology ◽

10.1128/aem.02157-18 ◽

2018 ◽

Vol 85 (10) ◽

Cited By ~ 2

Author(s):

Alli Lynch ◽

Seshu R. Tammireddy ◽

Mary K. Doherty ◽

Phillip D. Whitfield ◽

David J. Clarke

Keyword(s):

Amino Acid ◽

Gut Microbiome ◽

Molecular Mechanisms ◽

Cellular Membrane ◽

Bacteroides Thetaiotaomicron ◽

Human Gut ◽

Acyltransferase Activity ◽

Content Type ◽

Health And Disease ◽

And Function

ABSTRACTAcylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by theN-acylation of the amino acid, and this is followed by subsequentO-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced inBacteroides thetaiotaomicron. We, and others, have previously reported the identification of a gene, namedglsBin this study, that encodes anN-acyltransferase activity responsible for the production of a monoacylated glycine calledN-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of theBacteroidalesgenomes sequenced so far, theglsBgene is located immediately downstream from a gene, namedglsA, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression ofglsBandglsAresults in the production of GL inEscherichia coli. We constructed a deletion mutant of theglsBgene inB. thetaiotaomicron, and we confirm thatglsBis required for the production of GL inB. thetaiotaomicron. Moreover, we show thatglsBis important for the ability ofB. thetaiotaomicronto adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacteriumB. thetaiotaomicron.IMPORTANCEThe gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from theBacteroidales, an order that includesBacteroidesandPrevotella. In this study, we have identified an acylated amino acid, called glycine lipid, produced byBacteroides thetaiotaomicron, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation ofB. thetaiotaomicronto a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut byB. thetaiotaomicron. This work identifies glycine lipids as an important fitness determinant inB. thetaiotaomicronand therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.

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Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

Journal of Lipids ◽

10.1155/2011/208457 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 13

Author(s):

Katie D. Hickey ◽

Mary M. Buhr

Keyword(s):

Lipid Bilayer ◽

Lipid Composition ◽

Protein Function ◽

Transmembrane Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Outer Leaflet ◽

Lipid Environment ◽

Protein Incorporation ◽

And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

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Role of metAB in Methionine Metabolism and Optimal Chicken Colonization in Campylobacter jejuni

Infection and Immunity ◽

10.1128/iai.00542-20 ◽

2020 ◽

Vol 89 (1) ◽

pp. e00542-20

Author(s):

Brandon Ruddell ◽

Alan Hassall ◽

Orhan Sahin ◽

Qijing Zhang ◽

Paul J. Plummer ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Differential Distribution ◽

Time Resolved ◽

Content Type ◽

Reverse Transcription Pcr ◽

Adenosyl Methionine ◽

Strain Type

ABSTRACTCampylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain type NCTC 11168); however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to l-methionine. Comparative genomics identified differential distribution of the genes metA and metB, which function to convert homoserine for downstream production of l-methionine, between IA3902 and W7, which could enable a secondary pathway for l-methionine biosynthesis in a W7 ΔluxS but not in an IA3902 ΔluxS strain. To test the hypothesis that the genes metA and metB contribute to l-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison, the W7 ΔmetAB ΔluxS and IA3902 ΔluxS::metAB mutants. Quantitative reverse transcription-PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in the IA3902 ΔluxS::metAB mutant and absent in the W7 ΔmetAB ΔluxS mutant. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated that l-methionine and S-adenosyl methionine concentrations decreased in the W7 ΔmetAB ΔluxS mutant and increased in the IA3902 ΔluxS::metAB mutant. Assessment of chicken colonization revealed that the IA3902 ΔluxS::metAB strain partially rescued the colonization defect of the IA3902 ΔluxS strain, while the W7 ΔmetAB ΔluxS strain showed significantly decreased colonization compared to that of the wild-type and the W7 ΔluxS strain. These results indicate that the ability to maintain l-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

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Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804560115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 9923-9928 ◽

Cited By ~ 3

Author(s):

Vivian Gonzalez-Perez ◽

Manu Ben Johny ◽

Xiao-Ming Xia ◽

Christopher J. Lingle

Keyword(s):

Single Channel ◽

Regulatory Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bk Channels ◽

Bk Channel ◽

Single Type ◽

Regulatory Subunits ◽

And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

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Abstract 311: Measurements of cAMP Dynamics in the SERCA2 Microdomain in Adult Mouse Cardiomyocytes using a Targeted FRET Biosensor

Circulation Research ◽

10.1161/res.113.suppl_1.a311 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Julia U Sprenger ◽

Viacheslav O Nikolaev

Keyword(s):

Transgenic Mice ◽

Molecular Mechanisms ◽

Ventricular Myocytes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Heart Weight ◽

Cell Fractionation ◽

Adrenergic Stimulation ◽

Cardiac Phenotype ◽

Fractionation Analysis

PURPOSE: cAMP is a central regulator of cardiac function and disease. This global second messenger acts in a compartmentalized fashion, and changes in cAMP dynamics are linked to cardiac diseases. In this project, we visualized cAMP signals directly in such microdomains to gain insights into the molecular mechanisms involved in cAMP compartmentation and its alterations in hypertrophy. Methods: We generated transgenic mice expressing a new Förster resonance energy transfer (FRET)-based cAMP sensor Epac1-camps-PLN to measure cAMP dynamics in the microdomain around the sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2). This sensor is targeted to SERCA2 via phospholamban (PLN). Results: Colocalization and cell fractionation analysis confirmed proper localization of the sensor in transgenic mouse hearts. qPCR analysis revealed a two-fold overexpression of PLN. However, no adverse cardiac phenotype could be detected by histological analysis and heart weight to body weight ratios. Local cAMP dynamics were measured using freshly isolated adult ventricular myocytes and compared to cAMP signals in the bulk cytosol using cardiomyocytes from Epac1-camps mice. We detected the predominant role of phosphodiesterases (PDEs) 4 and 3 in the SERCA2 compartment under basal conditions. These PDEs were responsible for shaping the microdomain and its segregation from the cytosolic compartment. Interestingly, beta1-adrenergic stimulation led to a stronger increase of local cAMP in the SERCA2 compartment compared to the bulk cytosol. 8 weeks after transverse aortic constriction (TAC), PDE4 activity was downregulated in the SERCA2 microdomain compared to sham cardiomyocytes. Conclusion: We successfully generated transgenic mice expressing the targeted Epac1-camps-PLN biosensor to visualize cAMP dynamics in the SERCA2 compartment. We could show distinct cAMP dynamics around the SERCA2 compartment compared to the bulk cytosol and uncovered its alterations in hypertrophied cardiomyocytes

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Association with Coregulators Is the Major Determinant Governing Peroxisome Proliferator-activated Receptor Mobility in Living Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m608172200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4417-4426 ◽

Cited By ~ 32

Author(s):

Cicerone Tudor ◽

Jérôme N. Feige ◽

Harikishore Pingali ◽

Vidya Bhushan Lohray ◽

Walter Wahli ◽

...

Keyword(s):

Ligand Binding ◽

Molecular Mechanisms ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Living Cells ◽

Correlation Spectroscopy ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Peroxisome Proliferator Activated Receptors

The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

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Methods used to study the oligomeric structure of G-protein-coupled receptors

Bioscience Reports ◽

10.1042/bsr20160547 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 32

Author(s):

Hui Guo ◽

Su An ◽

Richard Ward ◽

Yang Yang ◽

Ying Liu ◽

...

Keyword(s):

Energy Transfer ◽

G Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

G Protein Coupled Receptors ◽

Experimental Techniques ◽

Distribution Analysis ◽

Wide Range ◽

And Function ◽

G Protein Coupled

G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.

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Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

10.1101/730465 ◽

2019 ◽

Author(s):

Bijoy J. Desai ◽

Ruben L. Gonzalez

Keyword(s):

Amino Acids ◽

Structural Biology ◽

Structural Dynamics ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Functional Studies ◽

Bioorthogonal Labeling ◽

Biomolecular Complexes ◽

And Function

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

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Actin-binding compounds, discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

10.1101/2020.05.19.104257 ◽

2020 ◽

Cited By ~ 1

Author(s):

Piyali Guhathakurta ◽

Lien A. Phung ◽

Ewa Prochniewicz ◽

Sarah Lichtenberger ◽

Anna Wilson ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Striated Muscle ◽

Muscle Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Structure And Function ◽

Actin Binding ◽

Myosin Isoforms ◽

And Function

AbstractWe have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle α-actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

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