Life Sciences Summer
Undergraduate Research Program

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Enhanced Crabp1 Protein Stability Guided by Differential Scanning Fluorimetry

Cristal I. Hernández-Hernández(1), Shawna Persuad(2), Dr. Li-Na Wei(2)

(1)University of Puerto Rico at Mayagüez, (2)University of Minnesota Department of Pharmacology

LSSURP- Heart, Lung, & Blood Program, University of Minnesota

Background

AtRA exerts its canonical genomic activity by binding to nuclear RA receptors (RARs) to regulate gene expression. Sadly, toxic side effects through RARs binding have drastically limited its clinical use. Cellular retinoic acid binding protein 1 (Crabp1) has a physiological role in mediating the non-genomic activities of atRA, such as the activation of extracellular regulated kinase. Its expression has not been reported in cancerous tumors, which supports its association as a tumor suppressor. Therefore, the conditions that stabilize this protein are of great importance, since they will allow a downstream set of biochemical experiments to understand the proposed tumor suppressor function of Crabp1. In order to achieve this, Differential Scanning Fluorimetry (DSF) can be used as a screening method that relies upon the determination of protein stability. With stabilizing conditions, a shift in the melting temperature (Tm) of Crabp1 should be noticed.

Source: Persuad SP, 2013.

Figure 1. Crabp1 pathway. Crap1-atRA complex activates kinase 1/2 (ERK 1/2). Activated ERK stimulates protein phosphatase 2 A (PP2A). PP2A dephosphorylates nuclear P27, which elevates nuclear p27 protein levels to block G1 progression to S phase.

                                                                                   Source: RSCB Protein Data Bank.
Figure 2. Crabp1-atRA complex viewed by Crystallography.
Research Goals

  • Search for the conditions/buffers that stabilize Crabp1 protein stability using Differential Scanning                   Fluorimetry (DSF) by analyzing the shifts in melting temperature (Tm) produced by each condition.
  • Determine which conditions enhance Crabp1 protein stability for it to be used for future downstream              biochemical experiments.
Justification

All-trans Retinoic acid (atRA) is a potent therapeutic agent for cancer. Unfortunately, off target effects through retinoic acid receptors (RAR) limit the clinical application of this treatment. Independent of RAR’s, the cellular retinoic acid binding protein 1 (Crabp1) can mediate events to slow cancer cell growth. The mechanism through which Crabp1 can slow growth is thought to be through its non-genomic activity. Cellular retinoic acid binding protein 1, Crabp1, mediates the non-genomic activity of atRA since the complex of Crabp1-atRA activates kinase 1/2 (ERK 1/2). Understanding the mechanism through which this protein can act as a tumor suppressor is interesting given that there are several genetic association studies that associate reduced Crabp1 expression in tumors to poorer patient outcomes.

Differential Scanning Fluorimetry (DSF) can be used to visualize conditions that favor protein stability by shifting the melting temperature (Tm). It works as a screening method that can rapidly determine and optimize protein stability conditions. Among the factors that may influence protein stability are buffers, salts, and detergents, which have no specific interaction with the protein. With DSF, the conditions that enhance Crabp1 protein stability can be determined.

Methods & Techniques

Source: Hernández-Hernández C, 2016.

  • The competent cells (E. coli) will be transformed with the plasmid containing the gene that codifies for          Crabp1 protein. They will grow in Ampicillin+LB plates at 37 °C overnight.
  • A colony will be selected to grow in 25 mL of LB+ 25 µl Ampicillin at 25 °C overnight.
  • The culture will be transferred to 500 mL of LB+ 500 µl Ampicillin to grow at 37 °C for three hours.
  • The transcription of the protein will be induced during three hours using 500 µl IPTG compound, which            binds to the repressor of the gene, when the bacteria get to the exponential phase (with an Absorbance     between 0.6-0.7).
  • Cell lysis will be performed and the lysate will be passed through a HisTag column for the protein                   purification. Various elutions of imidazole buffers at increasing concentrations will run through the column.
  • At last, a Differential Scanning Fluorimetry (DSF) stability screen for Crabp1 protein will be performed and       the results will be analyzed. 
Figure 3. HisTrap Column will bind Crabp1 protein in its Histidine tag. As the concentration of imidazole rizes in each buffer, it will fight the binding of the protein to the column and the protein will then elute.

Source: http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11448&catalogId=10101&productId=23408&storeId=11787&langId=-1





Figure 4. DSF stability screen. DSF discriminates between native, unfolded, and misfolded protein states using the Applied BioSystems Real Time PCR machine showed above. Sypro orange dye binds to the hydrophobic patches of the unfolded regions of the protein and fluorescent light is then emitted.




Source:https://de.wikipedia.org/wiki/Thermal_Shift_Assay#/medi/File:Thermal_Shift_Assay_diagram.svg

Results & Discussion

Figure 5. DSF stability screen for Crabp1 protein under 0.1M HEPES pH 7.5 and ionic concentrations of 0.10-1.0M. The melting temperature (Tm) of the protein was shifted under 0.1M HEPES pH 7.5 buffer and 0.30M-1.0M ionic concentrations. Ionic concentrations outside of this range made the protein unstable by lowering its Tm. The significant shifts were between 0.30-0.50M ionic concentrations.
Figure 6. The lowest value of the melt curve represents the Tm of the protein under each buffer.

Figure 7. DSF stability screen for Crabp1 protein under Osmolytes. The melting temperature (Tm) of the protein was shifted under Xylitol 1M, D-Sorbitol 1M, and Sucrose 1M.

Figure 8. DSF stability screen for Crabp1 protein under 0.1M BIS-TRIS pH 6.5 and ionic concentrations of 0.10-1.0M. The melting temperature (Tm) of the protein was shifted under 0.1M BIS-TRIS pH 8.5 buffer and 0.20M-1M ionic concentrations. Ionic concentrations outside of this range made the protein unstable by lowering its Tm. The significant shifts were between 0.30-0.40M ionic concentrations.

Figure 9. DSF stability screen for Crabp1 protein under 0.1M BIS-TRIS propane pH 8.5 and ionic concentrations of 0.10-1.0M. The melting temperature (Tm) of the protein was shifted under 0.1M BIS-TRIS propane pH 8.5 buffer and 0.10M-0.50M ionic concentrations. Ionic concentrations outside of this range made the protein unstable by lowering its Tm. The significant shifts were between 0.10-0.30M ionic concentrations.

Conclusions

Cellular retinoic acid binding protein 1 (Crabp1) stability was enhanced under 0.1M HEPES pH 7.5 buffer with specific ionic concentrations. The screen suggested Crabp1 protein was also stabilized in BIS-TRIS buffer systems as well. These results coincides with a previous research project in which Crabp1 was crystallized in BIS-TRIS buffer (James Thompson et al., 1995). Still, protein stability was further increased by HEPES buffer than with BIS-TRIS/BIS-TRIS propane buffer. Crabp1 protein stability was also increased by adding osmolytes such as 1M Xylitol, 1M D-Sorbitol, and 1M Sucrose.
Previous Research

This research coincides with a previous research project in which the crystal structure of Crabp1 was obtained using BIST-TRIS buffer, since it provided stabilizing conditions for Crabp1 protein (James Thomson et al., 1995). Therefore, it can be concluded that the results obtained were validated twice using Crystallography technique and Differential Scanning Fluorimetry stability screens.
                                                                     Source: RCSB Protein Data Bank.
Figure 10. Crabp1 protein structure obtained using Crystallography.
Future Works

  • Stabilized Crabp1 protein will be used for in vitro protein-protein interaction studies.
  • Expand the use of Differential Scanning Fluorimetry by screening for the compounds that bind to Crabp1. 
References
Biter AB, de la Pena AH, Thapar R, Lin JZ, Phillips KJ. DSF Guided   Refolding As A Novel Method Of Protein Production. Sci   Rep. 2016;6:18906.   doi: 10.1038/srep18906. PubMed   PMID: 26783150; PMCID: PMC4726114.
 
Niesen FH, Berglund H, Vedadi M. The use of differential scanning   fluorimetry to detect ligand interactions that promote protein stability. Nat   Protoc. 2007;2(9):2212-21. doi:   10.1038/nprot.2007.321. PubMed PMID: 17853878.

Persaud SD, Lin YW, Wu CY, Kagechika H, Wei LN. Cellular retinoic  acid binding protein I mediates rapid non-canonical activation of   ERK1/2 by all-  trans retinoic acid. Cell   Signal. 2013;25(1):19-25. doi: 10.1016/j.cellsig.2012.09.002. PubMed PMID:   22982089;   PMCID: PMC3508141.

Persaud SD, Park SW, Ishigami-Yuasa M, Koyano-Nakagawa N,   Kagechika H, Wei LN. All trans-retinoic acid analogs promote cancer cell   apoptosis through non-genomic   Crabp1 mediating   ERK1/2 phosphorylation. Sci Rep. 2016;6:22396.   doi:0 10.1038/srep22396.   PubMed   PMID: 26935534; PMCID: PMC4776112.

Thompson JR, Bratt JM, Banaszak LJ. Crystal structure of cellular retinoic acid binding protein I shows increased access to the binding cavity due to formation of an intermolecular beta-sheet. J Mol Biol. 1995;252(4):433-46. Epub 1995/09/29. doi: 10.1006/jmbi.1995.0509. PubMed PMID: 7563063.
Acknowledgements

I would like to thank to Dr. Li-Na Wei for the opportunity of being part of her laboratory, as well as Shawna Persuad for her mentoring during the summer. This work was supported by DK54733 and DK60521 to LNW.
I also thank the support of NIH for sponsoring the Heart, Lung, & Blood Program at the University of Minnesota.