Effect of the substitution of muscle actin-specific subdomain 1 and 2 residues in yeast actin on actin function.
AuthorsMcKane M, Wen KK, Meyer A, Rubenstein PA
JournalJ Biol Chem
PubMed ID16882670
'Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two ... More
Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.
AuthorsQiu H, Edmunds T, Baker-Malcolm J, Karey KP, Estes S, Schwarz C, Hughes H, Van Patten SM
JournalJ Biol Chem
PubMed ID12801930
'One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if ... More
A pathway of structural changes produced by monastrol binding to Eg5.
'Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and ... More
AuthorsSteinert S, Lee E, Tresset G, Zhang D, Hortsch R, Wetzel R, Hebbar S, Sundram JR, Kesavapany S, Boschke E, Kraut R,
JournalPLoS ONE
PubMed ID18716682
'BACKGROUND: The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order ... More
Structure-function relationships in OxlT, the oxalate/formate transporter of Oxalobacter formigenes. Topological features of transmembrane helix 11 as visualized by site-directed fluorescent labeling.
AuthorsFu D, Maloney PC
JournalJ Biol Chem
PubMed ID9651403
'Analysis of hydropathy suggests that in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes, lysine 355 is within transmembrane helix no. 11. To test this idea, we used single-cysteine, histidine-tagged OxlT variants to study the organization of a 30-residue segment (residues 344-373) containing this region. Topology was examined by probing the ... More
Self-assembling light-harvesting systems from synthetically modified tobacco mosaic virus coat proteins.
AuthorsMiller RA, Presley AD, Francis MB
JournalJ Am Chem Soc
PubMed ID17319656
'A new protein-based approach has been developed for the construction of light-harvesting systems through self-assembly. The building blocks were prepared by attaching fluorescent chromophores to cysteine residues introduced on tobacco mosaic virus coat protein monomers. When placed under the appropriate buffer conditions, these conjugates could be assembled into stacks of ... More
A method for site-specific labeling of multiple protein thiols.
'We present a generic method for the site-specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is removed, making available a reactive ... More
Fast and slow voltage sensor movements in HERG potassium channels.
AuthorsSmith PL, Yellen G
JournalJ Gen Physiol
PubMed ID11865022
'HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG ... More
Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study.
AuthorsBoskovic DS, Troxler T, Krishnaswamy S
JournalJ Biol Chem
PubMed ID14988397
'The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal ... More
Fluorescence quenching: A tool for single-molecule protein-folding study.
AuthorsZhuang X, Ha T, Kim HD, Centner T, Labeit S, Chu S
JournalProc Natl Acad Sci U S A
PubMed ID11121030
'By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from ... More
Trigger factor binds to ribosome-signal-recognition particle (SRP) complexes and is excluded by binding of the SRP receptor.
AuthorsBuskiewicz I, Deuerling E, Gu SQ, Jöckel J, Rodnina MV, Bukau B, Wintermeyer W,
JournalProc Natl Acad Sci U S A
PubMed ID15148364
Trigger factor (TF) and signal recognition particle (SRP) bind to the bacterial ribosome and are both crosslinked to protein L23 at the peptide exit, where they interact with emerging nascent peptide chains. It is unclear whether TF and SRP exclude one another from their ribosomal binding site(s). Here we show ... More
Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET.
AuthorsBuskiewicz I, Peske F, Wieden HJ, Gryczynski I, Rodnina MV, Wintermeyer W,
JournalJ Mol Biol
PubMed ID16005894
The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure ... More
Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy.
AuthorsMegens RT, Oude Egbrink MG, Cleutjens JP, Kuijpers MJ, Schiffers PH, Merkx M, Slaaf DW, van Zandvoort MA,
JournalMol Imaging
PubMed ID17711780
We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), ... More
Strong growth polarity of yeast prion fiber revealed by single fiber imaging.
AuthorsInoue Y, Kishimoto A, Hirao J, Yoshida M, Taguchi H
JournalJ Biol Chem
PubMed ID11473105
Using the yeast prion as a model, we have developed a novel system to observe the growth of individual prion fibers directly. NM fragments, the prion-determining region of the yeast protein Sup35p, were labeled by either red or green fluorescent dyes, and the fiber growth was observed under a fluorescence ... More
Manipulation of carrier proteins in antibiotic biosynthesis.
Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here ... More
Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles.
AuthorsDe Keyzer J, Van Der Does C, Driessen AJ
JournalJ Biol Chem
PubMed ID12226104
Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, ... More
Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation.
AuthorsEssex DW, Li M, Miller A, Feinman RD
JournalBiochemistry
PubMed ID11352743
The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation ... More
Translocator proteins in the two-partner secretion family have multiple domains.
AuthorsSurana NK, Buscher AZ, Hardy GG, Grass S, Kehl-Fie T, St Geme JW
JournalJ Biol Chem
PubMed ID16648638
The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, ... More
Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.
AuthorsYe L, Jia Z, Jung T, Maloney PC
JournalJ Bacteriol
PubMed ID11274108
The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 ... More
Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.
AuthorsKrahn KN, Bouten CV, van Tuijl S, van Zandvoort MA, Merkx M
JournalAnal Biochem
PubMed ID16476406
Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains ... More
Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins.
AuthorsGambhir A, Hangyás-Mihályné G, Zaitseva I, Cafiso DS, Wang J, Murray D, Pentyala SN, Smith SO, McLaughlin S
JournalBiophys J
PubMed ID15041659
The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of ... More
Nondisruptive, sequence-specific coupling of fluorochromes to plasmid DNA.
AuthorsHillery E, Munkonge FM, Xenariou S, Dean DA, Alton EW
JournalAnal Biochem
PubMed ID16579951
A method to attach a fluorochrome sequence-specifically to supercoiled plasmid DNA (pDNA) without perturbing transgene expression would provide an invaluable aid in a variety of applications requiring probes for the intracellular tracking of transfected pDNA. Here we report a method to couple commercially available fluorochromes covalently and sequence-specifically to pDNA ... More
Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers.
AuthorsRoss MF, Filipovska A, Smith RA, Gait MJ, Murphy MP
JournalBiochem J
PubMed ID15270716
CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have ... More
Cysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helix.
AuthorsKaratza P, Panos P, Georgopoulou E, Frillingos S
JournalJ Biol Chem
PubMed ID17077086
The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli ... More
In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.
AuthorsLarsen RA, Letain TE, Postle K
JournalMol Microbiol
PubMed ID12823822
Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in ... More