Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

Stanley, Will A.; Versluis, Kees; Schultz, Carsten; Heck, Albert J. R.; Wilmanns, Matthias

doi:10.1016/j.abb.2007.02.026

Cited by 9 publications

(11 citation statements)

References 40 publications

(92 reference statements)

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“…Further, our data support the suggestion that mSCP2 has not one but two very long-chain fatty acyl CoA binding sites. 13,20,21 Finally, the direct and high-fidelity binding assay, ITC, strongly supports the data obtained from our competition assay with 1a. The standard deviations from assays with 1a are rather higher than those obtained from ITC but can be considered tolerable for a relatively quick, easy, and more conservative (in terms of reagent consumption) assay.…”

Section: Calorimetric Datasupporting

confidence: 72%

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Probing lipid- and drug-binding domains with fluorescent dyes

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Stanley

Filipp

et al. 2008

Bioorganic & Medicinal Chemistry

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A series of 2-and 3-OH Nile red dyes was prepared in order to generate water-soluble probes that could be used to probe lipid binding to proteins. Various substitutions in positions 2-/3-, 6-, and 7-shifted wavelengths while maintaining the environmental sensitivity of Nile red. In order to increase the solubility of the dyes in aqueous solutions, we attached butyric acid groups to the 2-or 3-OH position. In addition, phenothiazine dyes, which exhibited particularly long excitation properties, were synthesized and tested for the first time. All dyes showed Stoke's shifts of 70-100 nm and changes in excitation and emission of over 100 nm, depending on the hydrophobicity of the environment. Binding studies with bovine serum albumin and the non-specific lipid transfer protein SCP2 revealed emission changes of more than 30 nm upon binding to the protein and a five-fold increase in emission intensity. Titration of the dye-loaded proteins with various lipids or drugs replaced the dye and thereby reversed the shift in wavelength intensity. This allowed us to estimate the lipid binding affinity of the investigated proteins. For SCP2, isothermal calorimetry (ITC) data verified the titration experiments. NMR titration experiments of SCP2 with Nile red 2-O-butyric acid (1a) revealed that the dye is bound within the lipid binding pocket and competes with lipid ligands for this binding site. These results give valuable insight into lipid and drug transport by proteins outside and inside cells.

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Section: Calorimetric Datasupporting

confidence: 72%

“…4B), as has previously been demonstrated for several long-chain fatty acid CoAs. 20,21 Site 1 was estimated to have a higher occupancy (n ~ 0.73) and stronger binding properties (K d ~ 339 nM) than Site 2 (n ~ 0.59, K d ~ 947 nM), as shown in Table 1.…”

Section: Lipid Binding Assaysmentioning

confidence: 99%

Probing lipid- and drug-binding domains with fluorescent dyes

Black

Stanley

Filipp

et al. 2008

Bioorganic & Medicinal Chemistry

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“…6) based on the known three-dimensional structures of human and trypanosome Pex5p (17,50). Besides protein and species-dependent amino acid variations of the PTS1 (9), the structure of the signal sequence seems to be very similar, as shown for SCP2 (sterol carrier protein 2), and the YQSKL peptide (16,17,59,60). Sites of interaction of human PEX5 with the PTS1 of SCP2 and AGT revealed five conserved asparagines at the ␣-helices of TPR3, TPR6, and TPR7 of Pex5p (21,59).…”

Section: Discussionmentioning

confidence: 99%

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor

Hagen

Drepper

Fischer

et al. 2015

Journal of Biological Chemistry

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Background: Peroxisomal proteins are recognized in the cytosol by the import receptor Pex5p. Results: Photo-cross-linking and mass spectrometry reveal the binding interface of Pex5p and its cargo protein Pcs60p. Conclusion: Pex5p-cargo interaction extends beyond signal sequence recognition and exhibits a bivalent binding mode. Significance: These data indicate a two-step concept of peroxisomal cargo recognition with initial tethering and subsequent lock-in.

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“…To date, however, there have been few reported examples of the application of ESI-MS to characterize protein-hydrophobic ligand binding [11][12][13][14] and, to our knowledge, no reports of absolute affinity measurements using the direct ESI-MS assay. The two principal challenges to quantifying interactions between water soluble proteins and hydrophobic ligands using ESI-MS are the low solubility of the ligand in aqueous solution, and the propensity of the desolvated complexes to dissociate during analysis.…”

Section: Introductionmentioning

confidence: 99%

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

Liu

Kitova

Klassen

2011

J. Am. Soc. Mass Spectrom.

104

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The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH 3 (CH 2 ) x COOH, where x=6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants (K a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K a value measured in negative ion mode and using sampling conditions that minimize insource dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.

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Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

Cited by 9 publications

References 40 publications

Probing lipid- and drug-binding domains with fluorescent dyes

Probing lipid- and drug-binding domains with fluorescent dyes

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

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