A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry
"> Figure 1
<p>Determining the binding-site of a combinatorially-selected peptide using a rationally designed photo-crosslinker, which is a bioisostere of the solvatochromic fluoroprobe present in the parent peptide. Irradiation with UV light simultaneously crosslinks the fluorophore to the protein binding site and uncages the fluorescence property by forming an intramolecular charge transfer (ICT) structure. This facilitates the rapid deduction of the binding site of the peptide using SDS-PAGE with fluorescence imaging followed by tandem MS analysis. The dotted line stands for the fluorescent color of each probe.</p> "> Figure 2
<p>Overall scheme for the synthesis of the cysteine-reactive small photo-crosslinker <b>3</b>. Reagents and conditions: (i) K<sub>3</sub>PO<sub>4</sub>, XPhos Pd G2, 1,4-dioxane/H<sub>2</sub>O, reflux, 2 hours; 1.7 M HCl (dichloromethane/ethyl acetate), room temperature, 24 hours, 77%; (ii) NaNO<sub>2</sub>, NaN<sub>3</sub>, 3.4 M aqueous HCl, 0 °C, 3 hours, 84%; (iii) <span class="html-italic">N</span>-Bromosuccinimide (NBS), TsOH monohydrate, MeCN, 35 °C, 24 hours, 33%.</p> "> Figure 3
<p>Specific conjugation between glutathione-S-transferase (GST) and the caged binder, confirmed by 15% SDS-PAGE/fluorescence imaging. GST (blue arrow) was visualized by CBB staining (left panel), and the binder-conjugated GST was visualized by fluorescence in the same gel (right panel). For the fluorescence imaging, the excitation wavelength was 405 nm, and a band-pass filter (605 nm) was used for the detection. Plus (+) and minus (-) stand for presence and absence of the suggested molecules (i.e., GST or the binder), respectively.</p> "> Figure 4
<p>(<b>A</b>) Identification of trypsinized peptide fragments derived from the covalent-binder-conjugated GST by MALDI-TOF-MS analysis (lower panel). As a negative control, pristine GST was also trypsinized, and the resulting fragments were also analyzed under the same conditions (upper panel). A newly appeared fragment in the presence of the covalent binder is highlighted by a blue arrow. (<b>B</b>) MS/MS spectra of the newly appeared fragment. All the detected fragments were consistent with theoretical <span class="html-italic">m</span>/<span class="html-italic">z</span> values of the represented structure; b- and y-ions are highlighted by using blue and red colors, respectively. The peptide fragment of LTQSMAIIR was derived from a constituent of the glutathione binding pocket of GST protein; judging from the peak intensity of the remaining non-crosslinked fragment (lower panel in A), the crosslinking reaction yield was estimated to be a few percent. M* and C* mean conjugated methionine and cysteine, respectively; the conjugation is highlighted by a thick red line.</p> "> Figure 5
<p>Molecular docking simulation of the caged binder (shown as a stick; C, N, O and S atoms are highlighted by cyan, blue, red and yellow colors, respectively) to GST (PDB ID: 1UA5) using the sievgene of myPresto; the best docking model with a lowest binding energy of −12.0 kcal/mol was presented. The azido group (i.e., N<sub>3</sub>) in the caged fluorophore and conjugated methionine in GST were colored in blue (double-lined) and red, respectively. GST was shown as a cartoon with side chains as a line description. Including this lowest model, 23 independent models out of the 30 separate poses resulted that the azido group was also closely located to the methionine.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Synthesis of Cysteine-Reactive Small Photo-Crosslinkers and their Attachment to the Combinatorially-Selected Peptide
2.2. Targeted Covalent Conjugation of Caged Binder with GST
2.3. Molecular Docking Simulation for Rationalizing Cross-Linking Specificity
3. Materials and Methods
3.1. General
3.2. Photo-Crosslinking of the Caged Binder and GST
3.3. In-Gel Trypsinization of Crosslinked GST and Mass Spectrometric Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
NMR | Nuclear magnetic resonance |
SDS-PAGE | Sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
MALDI-TOF-MS/MS | Matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry |
Prodan | 6-Propionyl-2-dimethylaminonaphthalene |
UV | Ultraviolet |
DMSO | Dimethyl sulfoxide |
D-PBS | Dulbecco’s phosphate-buffered saline |
PDB | Protein Data Bank |
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Yatabe, K.; Hisada, M.; Tabuchi, Y.; Taki, M. A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry. Int. J. Mol. Sci. 2018, 19, 3682. https://doi.org/10.3390/ijms19113682
Yatabe K, Hisada M, Tabuchi Y, Taki M. A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry. International Journal of Molecular Sciences. 2018; 19(11):3682. https://doi.org/10.3390/ijms19113682
Chicago/Turabian StyleYatabe, Kazuki, Masaru Hisada, Yudai Tabuchi, and Masumi Taki. 2018. "A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry" International Journal of Molecular Sciences 19, no. 11: 3682. https://doi.org/10.3390/ijms19113682