An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

Kraunsoe, James A.E.; Aplin, Robin T.; Green, Brian; Lowe, Gordoǹ

doi:10.1016/0014-5793(96)01081-2

Cited by 28 publications

(20 citation statements)

References 20 publications

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“…In contrast, the charge state distribution corresponding to more folded molecules centered on the n ϭ ϩ9 charge state changes little with increasing cone voltage until 190 V, and only then does this particular charge state distribution widen, consistent with partial unfolding at the higher cone voltage. This indicates a remarkable stability of this subset of molecules to cone voltage compared with other native proteins (49). The resistance of this distribution to increases in sampling cone voltage supports the notion that these molecules are natively folded in a stable structure.…”

Section: Resultssupporting

confidence: 54%

Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain

Ashcroft

Brinker

Coyle

et al. 2002

Journal of Biological Chemistry

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Section: Resultssupporting

confidence: 54%

Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain

Ashcroft

Brinker

Coyle

et al. 2002

Journal of Biological Chemistry

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“…Although these studies have been extended to quantitative measurements of binding affinities of noncovalent complexes [2][3][4], it is still not clear to what extent the relative yield of gas-phase ions produced by ESI represents their relative solution concentrations. If electrospray ionization mass spectrometry (ESI-MS) is to be used to determine binding constants of noncovalent complexes, this question is of profound significance.…”

mentioning

confidence: 99%

Shrinking droplets in electrospray ionization and their influence on chemical equilibria

Wortmann

Kistler-Momotova

Zenobi

et al. 2007

J. Am. Soc. Mass Spectrom.

117

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We investigated how chemical equilibria are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing number of publications in which electrospray ionization mass spectrometry is used for binding constant determination. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, we found an average initial droplet diameter of 10 m in the electrospray. The PDA allowed us to follow the evolution of these droplets down to a size of 1 m. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equilibrium of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equilibrium. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concentration increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the average pair distance. ( An important aspect of the highly dynamic electrospray process concerns the shrinking droplets. After the droplet creation, solvent evaporation and Coulomb explosions lead to a decrease of droplet size until all solvent has evaporated and the final desolvated and ionized analytes are produced. A decrease of droplet size resulting from solvent evaporation will lead to a concentration increase of nonvolatile analytes within the droplets. For dissolved species that participate in a chemical equilibrium, such as free and bound states of partners that can form a noncovalent complex, the question arises whether the increasing concentrations affect the equilibrium position. Given that binding constant determination with ESI-MS usually relies on measuring the relative amounts of free and bound species, a perturbation of the equilibrium introduced by the shrinking droplets would influence the results of such an experiment. Recent studies show that the time between droplet creation and production of desolvated ions in ESI is within milliseconds [5]. In other words, if the association and dissociation reactions are fast, an equilibrium should be able to track a concentration increase within the droplets.The shrinking droplet volume also leads to Coulomb explosions/droplet fission, when the mutual repulsion of charges is high enough to overcome the surface tension of the droplet. The parent droplet ejects several smaller offspring droplets, which together carry 10 -25% of the charge, but only about 5% of the mass of the original droplet [9]. These offspring droplets then segregate within the electrospray, as shown theoretically by Wilhelm et al. [10]. Analytes will not generally be evenly distributed between the parent and offspring droplets. Partitioning processes, which have been investigated by several research g...

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“…Detailed biophysical and biochemical studies have provided a greater insight into the structural basis for the association of BPTI, or its homologues, to proteinases. Moreover, using semisynthetic (20,21) or recombinant approaches (22,23), it has been possible to change or enhance the inhibitory activity and spectrum of BPTI, as well as its homologues. Kunitz-type inhibitors possess a compact pear-shaped structure stabilized by three disulfide bonds containing a reactive site region featuring the principal determinant P 1 residue in a rigid conformation.…”

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confidence: 99%

Structure-Function Analysis of the Reactive Site in the First Kunitz-type Domain of Human Tissue Factor Pathway Inhibitor-2

Chand

Schmidt

Bajaj

et al. 2004

Journal of Biological Chemistry

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Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P 1 residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P 1 Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P 6 ) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P 1 ) with Asp-735 in plasmin, and Arg-29 (P 5 ) with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P 2 -P 4 ) in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P 6 -P 5 region of KD1, mutation of a highly conserved Phe at the P 18 position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P 1 residue, the nature of other residues flanking the P 1 residue, particularly at P 6 and P 5 , strongly influences the inhibitory activity and specificity of human TFPI-2.

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An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

Cited by 28 publications

References 20 publications

Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain

Structural Plasticity and Noncovalent Substrate Binding in the GroEL Apical Domain

Shrinking droplets in electrospray ionization and their influence on chemical equilibria

Structure-Function Analysis of the Reactive Site in the First Kunitz-type Domain of Human Tissue Factor Pathway Inhibitor-2

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