Abstract:
:Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mori MX,Imai Y,Itsuki K,Inoue Rdoi
10.1021/bi200287xsubject
Has Abstractpub_date
2011-05-31 00:00:00pages
4685-96issue
21eissn
0006-2960issn
1520-4995journal_volume
50pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::The Raman spectra of the nonbridging V--O bonds in the myosin S1.MgADP.Vi complex, often believed to be a transition-state analogue for the phosphotransfer reaction catalyzed by myosin, and in a vanadate solution model compound have been obtained using Raman difference spectroscopic techniques. A symmetric/asymmetric ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi980556n
更新日期:1998-08-04 00:00:00
abstract::Escherichia coli dihydrofolate reductase contains five tryptophan residues that are spatially distributed throughout the protein and located in different secondary structural elements. When these tryptophans are replaced with [6-19F]tryptophan, distinct native and unfolded resonances can be resolved in the 1-D 19F NMR...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971962u
更新日期:1998-01-06 00:00:00
abstract::Human alpha-lactalbumin (alpha-LA), a 123-residue calcium-binding protein, has been studied using (15)N NMR relaxation methods in order to characterize backbone dynamics of the native state at the level of individual residues. Relaxation data were collected at three magnetic field strengths and analyzed using the mode...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi900023m
更新日期:2009-05-19 00:00:00
abstract::The expression of apoE mRNA in the adrenal gland is inversely correlated to steroidogenesis and directly correlated to the level of cholesteryl ester stores. To further investigate the relationship between apoE and cellular cholesterol homeostasis, several parameters of cholesterol metabolism in the murine Y1 adrenal ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00183a007
更新日期:1994-05-03 00:00:00
abstract::Amyloid diseases make up a set of fatal disorders in which proteins aggregate to form fibrils that deposit in tissues throughout the body. Amyloid-associated diseases are challenging to study because amyloid formation occurs on time scales that span several orders of magnitude and involve heterogeneous, interconvertin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00769
更新日期:2019-11-05 00:00:00
abstract::The globular domain of chicken histone H1 (GH1) has been studied by 1H homonuclear and 1H-15N heteronuclear 2D NMR spectroscopy. After the full assignment of the proton and 15N resonances, the tertiary structure of GH1 was determined by an iterative procedure using distance geometry and restrained simulated annealing....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00203a004
更新日期:1994-09-20 00:00:00
abstract::The complex of tropomyosin and troponin binds to actin and inhibits activation of myosin ATPase activity and force production of striated muscles at low free Ca(2+) concentrations. Ca(2+) stimulates ATP activity, and at subsaturating actin concentrations, the binding of NEM-modified S1 to actin-tropomyosin-troponin in...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi048646h
更新日期:2004-12-07 00:00:00
abstract::The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus harbors two paralogous genes, acmI and acmL, encoding methyltransferases. To unveil their suspected role in the formation of 3-hydroxy-4-methyl-anthranilic acid (4-MHA), the building block of the actinomycin chromophore, each gene was expressed in ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi101422r
更新日期:2010-11-16 00:00:00
abstract::The dynamics of the enthalpy and volume changes found in the photodissociation of CO from sperm whale carboxymyoglobin and two site-directed mutants in which arginine-45 is replaced by glycine and asparagine are examined by photoacoustic calorimetry. An intermediate is observed whose lifetime at 20 degrees C is 700 ns...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00480a026
更新日期:1990-07-17 00:00:00
abstract::The ligand-binding dynamics and the reaction with O(2) of the fully (five-electron) reduced cytochrome caa(3) from the thermohalophilic bacterium Rhodothermus (R.) marinus were investigated. The enzyme is a proton pump which has all the residues of the proton-transfer pathways found in the mitochondrial-like enzymes c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi010344h
更新日期:2001-09-04 00:00:00
abstract::(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Esc...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi047432x
更新日期:2005-03-22 00:00:00
abstract::Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00458a025
更新日期:1990-02-13 00:00:00
abstract::Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b00012
更新日期:2017-04-18 00:00:00
abstract::Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi001976r
更新日期:2000-12-26 00:00:00
abstract::Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high man...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00101a028
更新日期:1991-09-17 00:00:00
abstract::The conformations of melittin, an amphipathic polypeptide consisting of 26 amino acid residues, and its hydrophobic (residues 1--19) and hydrophilic (residues 20--26) fragments were examined in various solvent systems, including H2O, 2H2O, 2-chloroethanol, and 1,2-dimyristoylphosphatidylcholine (DMPC) multilayers, by ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00539a006
更新日期:1982-05-11 00:00:00
abstract::To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. A...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi026100u
更新日期:2002-11-19 00:00:00
abstract::For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involv...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0255322
更新日期:2002-04-09 00:00:00
abstract::The question of the possible identity of catalytic and regulatory proton pathways in the chloroplast FoF1 ATPase has been studied using different energy-transfer inhibitors. Venturicidin, a reversible inhibitor of Fo, affects neither the delta mu H(+)-dependent thiol reduction of the membrane-bound chloroplast ATPase ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00132a013
更新日期:1992-05-05 00:00:00
abstract::Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cle...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00402a016
更新日期:1988-01-26 00:00:00
abstract::Base excision repair (BER) is the major corrective pathway for most spontaneous, oxidative, and alkylation DNA base and sugar damage. X-ray cross-complementing 1 (XRCC1) has been suggested to function at nearly every step of this repair process, primarily through direct protein-protein interactions. Using whole cell e...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi051161o
更新日期:2005-11-01 00:00:00
abstract::Phospholipase Cβ (PLCβ) enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate to produce second messengers that regulate intracellular Ca2+, cell proliferation, and survival. Their activity is dependent upon interfacial activation that occurs upon localization to cell membranes. However, the molecular basis for how ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b00547
更新日期:2017-10-17 00:00:00
abstract::S100A6 is a member of the S100 subfamily of EF-hand Ca (2+) binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCF-like E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this com...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi801233z
更新日期:2008-10-14 00:00:00
abstract::Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9002265
更新日期:2009-06-09 00:00:00
abstract::The present study was undertaken for the purpose of evaluating the solvent perturbation technique as a method to identify enzyme catalytic residues. For establishment of expected directions and sizes of pKa perturbations for different types of acids in different classes of solvents, a study of the pKa of a series of a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00287a013
更新日期:1983-08-30 00:00:00
abstract::Tyr71 is an invariant residue in all known sequences of tyrosine phenol-lyase (TPL). The substitution of Tyr71 in TPL by phenylalanine results in a mutant Y71F TPL with no detectable activity (greater than 3 x 10(5)-fold reduction) for beta-elimination of L-tyrosine. Y71F TPL can react with S-alkylcysteines, but these...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00038a023
更新日期:1995-09-26 00:00:00
abstract::The enhancement of the longitudinal proton relaxation rate of solvent water protons which occurs when Mn(II) is bound to the "tight" metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes. The binary enhancement for ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00648a014
更新日期:1976-02-10 00:00:00
abstract::Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)(2+) clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)(2+) to (4Fe-4S)(2+,+). However, until now, no detai...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi034426c
更新日期:2003-07-29 00:00:00
abstract::A specific requirement for lipids, particularly cardiolipin (CL), in cytochrome c oxidase (CcO) has been reported in many previous studies using mainly in vitro lipid removal approaches in mammalian systems. Our accompanying paper shows that CcO produced in markedly CL-depleted Rhodobacter sphaeroides displays wild-ty...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi1017039
更新日期:2011-05-17 00:00:00
abstract::5-HT(2A) serotonin receptors represent the principal molecular targets for LSD-like hallucinogens and atypical antipsychotic drugs. It has been proposed that a dysregulation of 5-HT(2A) receptor-mediated signaling may contribute to the pathogenesis of schizophrenia and related diseases. A major mechanism for the atten...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035061z
更新日期:2003-09-16 00:00:00