Rapid measurement of binding constants and heats of binding using a new titration calorimeter

Wiseman, Thomas; Williston, Samuel; Brandts, John F.; Lin, Lung‐Nan

doi:10.1016/0003-2697(89)90213-3

Cited by 2,648 publications

(2,655 citation statements)

References 8 publications

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“…The K d values for binding of (GlcNAc) 6 , (GlcNAc) 5 and (GlcNAc) 4 to Chit-42 from T. harzianum determined by Lienemann et al using surface plasmon resonance are approximately 2-, 10-and 5-fold higher than the values reported for ChiB in this study [18]. For ChiB, six subsites, spanning from À3 to + 3 have been described [24] (Fig.…”

Section: Resultscontrasting

confidence: 47%

“…ITC experiments were performed with a VP-ITC system from Microcal, Inc (Northampton, MA) [18]. Solutions were thoroughly degassed prior to experiments to avoid air bubbles in the calorimeter.…”

Section: Isothermal Titration Calorimetry Experimentsmentioning

confidence: 99%

“…ITC data were collected automatically using the Microcal Origin v.7.0 software accompanying the VP-ITC system [18]. Prior to further analysis, all data were corrected for heat of dilution by subtracting the heat produced by continuing injections of ligand into the reaction cell after completion of the binding reaction.…”

Section: Analysis Of Calorimetric Datamentioning

confidence: 99%

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Determination of substrate binding energies in individual subsites of a family 18 chitinase

Norberg¹,

Karlsen²,

Hoell³

et al. 2010

FEBS Letters

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a b s t r a c tThermodynamic parameters for binding of N-acetylglucosamine (GlcNAc) oligomers to a family 18 chitinase, ChiB of Serratia marcescens, have been determined using isothermal titration calorimetry. Binding studies with oligomers of different lengths showed that binding to subsites À2 and +1 is driven by a favorable enthalpy change, while binding to the two other most important subsites, +2 and +3, is driven by entropy with unfavorable enthalpy. These remarkable unfavorable enthalpy changes are most likely due to favorable enzyme-substrate interactions being offset by unfavorable enthalpic effects of the conformational changes that accompany substrate-binding.

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Section: Resultscontrasting

confidence: 47%

Section: Isothermal Titration Calorimetry Experimentsmentioning

confidence: 99%

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Determination of substrate binding energies in individual subsites of a family 18 chitinase

Norberg¹,

Karlsen²,

Hoell³

et al. 2010

FEBS Letters

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show abstract

“…The peptides generated by tryptic digest were analyzed by two independent means, Mascot identity database searching and BLAST homology searching. Following de novo sequencing the Mascot analysis showed that several digest peptides matched significantly with the hypothetical protein Q5BAR4_EMENI from A. nidulans (Mascot score ¼ 252; threshold score ¼ 25; probability of a random match ¼ 1 in 10 21 ). An independent analysis method using the program DeNovoX was also used to attempt to sequence the peptides de novo (that is without prior reference to any databases) from their MS/MS spectra.…”

Section: Identification Of Pnmbmentioning

confidence: 99%

“…7(C)], however, the calculated stoichiometry of the interaction (approximately 0.6) was lower than the value of 1 known from structural studies 12 and the K D could not be reliably calculated as the 'c' value fell below 1. 21 The reason for the aberrant stoichiometry and low c value is possibly due to the subtle structural changes revealed by the far-UV CD spectrum of FNM1BþC-term. These changes may cause a reduction in the half life of the ability of FNM1BþC-term to bind NAD þ .…”

Section: Production Of the Pnma B And C Protease Activities Is Assocmentioning

confidence: 99%

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

et al. 2010

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The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C-terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site retained the ability to bind NAD 1 and showed a resistance to further digestion that was enhanced by the presence of NAD 1 . This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated.

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The high-resolution crystal structure of a 24-kDa gyrase B fragment fromE. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

et al. 1997

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Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3' position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5'-adenylyl-beta, gamma-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.

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Rapid measurement of binding constants and heats of binding using a new titration calorimeter

Cited by 2,648 publications

References 8 publications

Determination of substrate binding energies in individual subsites of a family 18 chitinase

Determination of substrate binding energies in individual subsites of a family 18 chitinase

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

The high-resolution crystal structure of a 24-kDa gyrase B fragment fromE. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

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