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Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology
In This Article
Summary
Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.
Abstract
Biomolecular interactions play versatile roles in numerous cellular processes by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 µM, 0.053 µM, and 0.072 µM in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.
Introduction
Biomolecular interactions:
Proteins are essential parts of organisms and participate in numerous molecular pathways such as cell metabolism, cell structure, cell signaling, immune responses, cell adhesion, and more. While some proteins perform their function(s) independently, most proteins interact with other proteins using a binding interface to coordinate proper biological activity1.
Biomolecular interactions can mainly be classified based on the distinct structural and functional characteristics of proteins involved2, for example, based on the protein surfaces, the co....
Protocol
NOTE: The recombinant proteins used in this study, Hsp90 and Cdc37, were commercially obtained (see Table of Materials).
1. Chip activation
NOTE: All materials to be used in the experiment are listed in the Table of Materials. Filter all prepared solutions through a sterile 0.2 μm filter.
- Prepare 1-Ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) solution by adding 2 mg of EDC to 2.5 mL of 1 M 2-.......
Representative Results
Results from experiment 1:
The target protein Hsp90 (500 nM) was immobilized to the chip following the target immobilization protocol as described above. For the first experiment, 10 concentrations of the analyte protein, Cdc37, ranging from 25 nM to 5,000 nM, were prepared based on the data available in the literature (see Table 1).
The steps of the experiment can be monitored in real-time by following the alterations occurring in the I-Response (
Discussion
In this study, the feasibility of using the FEB technology (a real-time kinetic characterization approach) was evaluated to determine the biomolecular interaction between Hsp90 and Cdc37. The initial exploratory experiment (first experiment) suggested that choosing the proper analyte concentrations is a critical part of the experiment and that the experiment should be designed by including concentration points above and below the KD value, which were predicted based on the data available in the literature.
.......Acknowledgements
This research was supported by a grant from the Binational Science Foundation (BSF) to S.K.S. and N.Q.
....Materials
| Name | Company | Catalog Number | Comments |
| Automated analysis software | Agile plus software, Cardea (Nanomed) | NA CAS number: NA | Referred to in the text as the automated analysis software supplied with the instrument. Generates automated analysis. |
| COOH-BPU (Biosensing Processing Unit) | Agile plus software, Cardea (Nanomed) | NA CAS number: NA | biosensor chip |
| Data review software | Datalign 1.0, Cardea (Nanomed) | NA CAS number: NA | Referred to as the supplied data review software in the text. Supplied with the instrument and allows to review and export the information data points. |
| Dialysis bag | CelluSep, Membrane filtration products | T2-10-15 CAS number: NA | T2 tubings (6,000-8,000 MWCO), (10 mm fw, 6.4mm Ø, 0.32ml/cm, 15m) |
| EDC (1-Ethyl-3-(3-dimethylamino propyl) carbodiimide) | Cardea (Nanomed) | EDC160322-02 CAS number: 25952-53-8 | White powder |
| ITC (Isothermal titration calorimetry) system | Microcal-PEAQ-ITC (Malvern, United Kingdom) | NA CAS number: NA | |
| MES (2-(N-morpholino) ethane sulfonic acid) buffer | Merck | M3671-50G CAS number: 4432-31-9 | White powder |
| NHS (N-Hydroxysulfosuccinimide) chips | Cardea (Nanomed) | NA CAS number: NA | Graphene-based chip |
| PBS (Phosphate-buffered saline) X 10 | Bio-Lab | 001623237500 CAS number: 7758-11-4 | Liquid transparent solution |
| Pipete | Thermo Scientific | 11855231 CAS number: NA | Finnpipette F3 5-50 µL, yellow |
| Quench 1 (3.9 mM amino-PEG5-alcohol in 1 X PBS) | Cardea (Nanomed) | 0105-001-002-001 CAS number: NA | Liquid, transparent solution |
| Quench 2 (1 M ethanolamine (pH=8.5)) | Cardea (Nanomed) | 0105-001-003-001 CAS number: NA | Liquid, transparent solution |
| Recombinant protein Cdc37 | Abcam | ab256157 CAS number: NA | |
| Recombinant protein Hsp90 beta | Abcam | ab80033 CAS number: NA | |
| Spreadsheet | Excel, Microsoft office | NA CAS number: NA | |
| Statistical software | GraphPad, Prism | NA CAS number: NA | Referred to as the other statistical software. Sigma plot, phyton or other statistical programes may also be used |
| Sulfo-NHS | Cardea (Nanomed) | NHS160321-07 CAS number: 106627-54-7 | White powder |
| Tips | Alex red | LC 1093-800-000 CAS number: NA | Tip 1-200 µl, in bulk, 1,000 pcs |
References
- Tuncbag, N., Gursoy, A., Guney, E., Nussinov, R., Keskin, O. Architectures and functional coverage of protein-protein interfaces. Journal of Molecular Biology. 381 (3), 785-802 (2008).
- Berggård, T., Linse, S., James, P.
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