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A Dynamic Interaction of Coomassie Dye with the Glycine Transporters N-termini

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Abstract

Coomassie Brilliant Blue interacts with proteins and even though the interactions exhibit variation due to the amino acid content, reported dye interactions with individual proteins appear to be relatively stable. Here we report an atypical dynamic interaction of glycine transporters 1 and 2 N-termini with Coomassie dye, resulting in intramolecular interference with their Bradford assay. These proteins exhibit classic protein-Coomassie G-250 complex with absorption maximum at 595 nm, which within minutes starts to decrease and parallel increase of absorbance shoulders above 300 and 700 nm is observed. Interestingly, these effects are eliminated upon fusion of glycine transporters N-termini with glutathione S-transferase protein or by the presence of glutathione S-transferase or bovine serum albumin in the same solution. Circular dichroism data revealed largely unstructured character of glycine transporters N-termini, which suggests that dynamic properties of these protein- Coomassie complexes might be a signature of high flexibility and protein disorder. The assay might potentially reveal similar domains in other proteins and help to associate them with particular functions.

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Abbreviations

GlyT1:

Glycine transporter 1

GlyT2:

Glycine transporter 2

GST:

Glutathione S-transferase

BSA:

Bovine serum albumin

CBB:

Coomassie Brilliant Blue

SDS:

Sodium dodecyl sulfate

TEV:

Tobacco etch virus

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

References

  1. Jursky F, Nelson N (1996) Developmental expression of the glycine transporters GLYT1 and GLYT2 in mouse brain. J Neurochem 67:336–344

    Article  CAS  Google Scholar 

  2. Kristensen AS, Andersen J, Jorgensen TM, Sorensen L, Eriksen J, Loland CJ, Stromgaard K, Gether U (2011) SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev 63:585–640

    Article  CAS  Google Scholar 

  3. Betz H (1992) Structure and function of inhibitory glycine receptors. Q Rev Biophys 25:381–394

    Article  CAS  Google Scholar 

  4. Johnson JW, Ascher P (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529–531

    Article  CAS  Google Scholar 

  5. Eulenburg V, Retiounskaia M, Papadopoulos T, Gomeza J, Betz H (2010) Glial glycine transporter 1 function is essential for early postnatal survival but dispensable in adult mice. Glia 58:1066–1073

    Article  Google Scholar 

  6. Gomeza J, Ohno K, Hulsmann S, Armsen W, Eulenburg V, Richter DW, Laube B, Betz H (2003) Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron 40:797–806

    Article  CAS  Google Scholar 

  7. Rees MI, Harvey K, Pearce BR, Chung SK, Duguid IC, Thomas P, Beatty S, Graham GE, Armstrong L, Shiang R, Abbott KJ, Zuberi SM, Stephenson JB, Owen MJ, Tijssen MA, van den Maagdenberg AM, Smart TG, Supplisson S, Harvey RJ (2006) Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease. Nat Genet 38:801–806

    Article  CAS  Google Scholar 

  8. Singer P, Dubroqua S, Yee BK (2015) Inhibition of glycine transporter 1: the yellow brick road to new schizophrenia therapy? Curr Pharm Des 21:3771–3787

    Article  CAS  Google Scholar 

  9. Dohi T, Morita K, Kitayama T, Motoyama N, Morioka N (2009) Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol Ther 123:54–79

    Article  CAS  Google Scholar 

  10. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E (2005) Crystal structure of a bacterial homologue of Na+/Cl–dependent neurotransmitter transporters. Nature 437:215–223

    Article  CAS  Google Scholar 

  11. Coleman JA, Green EM, Gouaux E (2016) X-ray structures and mechanism of the human serotonin transporter. Nature 532:334–339

    Article  CAS  Google Scholar 

  12. Jursky F, Baliova M, Mihalikova A (2012) Molecular basis for differential glycine transporters sensitivity to sanguinarine. Toxicol Lett 212:262–267

    Article  CAS  Google Scholar 

  13. Jursky F, Baliova M (2013) Expression and purification of recombinant calpain-derived N-terminal peptides from glycine transporter GlyT2. Protein Expr Purif 88:143–149

    Article  CAS  Google Scholar 

  14. Tompa P, Fuxreiter M (2008) Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions. Trends Biochem Sci 33:2–8

    Article  CAS  Google Scholar 

  15. Oldfield C, Dunker A, Kornberg R (2014) Intrinsically disordered proteins and intrinsically disordered protein regions. Annu Rev Biochem 83:553–584

    Article  CAS  Google Scholar 

  16. Bradford MA (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  17. Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, Takano T, Tian GF (2009) Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. Proc Natl Acad Sci USA 106:12489–12493

    Article  CAS  Google Scholar 

  18. Mennel S, Meyer CH, Schmidt JC, Kaempf S, Thumann G (2008) Trityl dyes patent blue V and brilliant blue G—clinical relevance and in vitro analysis of the function of the outer blood-retinal barrier. Dev Ophthalmol 42:101–114

    Article  CAS  Google Scholar 

  19. Baliova M, Betz H, Jursky F (2004) Calpain-mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2. J Neurochem 88:227–232

    Article  CAS  Google Scholar 

  20. Lawrence AM, Besir HU (2009) Staining of proteins in gels with Coomassie G-250 without organic solvent and acetic acid. J Vis Exp 30:e1350

  21. Zhou H-X (2012) Intrinsic disorder: signaling via highly specific but short-lived association. Trends Biochem Sci 37:43–48

    Article  CAS  Google Scholar 

  22. Umezawa K, Ohnuki J, Higo J, Takano M (2016) Intrinsic disorder accelerates dissociation rather than association. Proteins 84:1124–1133

    Article  CAS  Google Scholar 

  23. DeForte S, Uversky VN (2016) Order, disorder, and everything in between. Molecules 21:1090. doi:10.3390/molecules21081090

    Article  Google Scholar 

  24. Chemes LB, Alonso LG, Noval MG, de Prat-Gay G (2012) Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains. Methods Mol Biol 895:387–404

    Article  CAS  Google Scholar 

  25. Chial HJ, Thompson HB, Splittgerber AG (1993) A spectral study of the charge forms of Coomassie blue G. Anal Biochem 209:258–266

    Article  CAS  Google Scholar 

  26. Aminian M, Nabatchian F, Vaisi-Raygani A, Torabi M (2013) Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: an interference with the Bradford assay. Anal Biochem 434:287–291

    Article  CAS  Google Scholar 

  27. Marshall T, Williams KM (1992) Coomassie blue protein dye-binding assays measure formation of an insoluble protein–dye complex. Anal Biochem 204:107–109

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Slovak grant agency VEGA, Grant 2/0086/13. The authors would like to thank Josef Houser PhD at the Biomolecular Interactions and Crystallization Core Facility of CEITEC for his expert technical assistance with obtaining CD spectra of proteins presented in this paper.

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Authors and Affiliations

  1. Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovakia

    Anna Juhasova, Martina Baliova & Frantisek Jursky

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  1. Anna Juhasova
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  2. Martina Baliova
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  3. Frantisek Jursky
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Correspondence to Frantisek Jursky.

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Juhasova, A., Baliova, M. & Jursky, F. A Dynamic Interaction of Coomassie Dye with the Glycine Transporters N-termini. Protein J 35, 371–378 (2016). https://doi.org/10.1007/s10930-016-9682-x

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  • DOI: https://doi.org/10.1007/s10930-016-9682-x

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