Molecular biosensing system based on intrinsically disordered proteins

Kyle A. Cissell, Suresh Shrestha, Jennifer Purdie, Derrick Kroodsma, Sapna K. Deo

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219-498) was utilized in the detection of p53 (residues 311-393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311-393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1- sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219-498). Dansyl-X, SE-conjugated BRCA1 (219-498) detected p53 (311-393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change.

Original languageEnglish (US)
Pages (from-to)1721-1729
Number of pages9
JournalAnalytical and bioanalytical chemistry
Volume391
Issue number5
DOIs
StatePublished - Jul 1 2008
Externally publishedYes

Keywords

  • Bioanalytical methods
  • Biosensors
  • Breast cancer protein (BRCA1)
  • Fluorescence
  • Intrinsically disordered protein (IDP)
  • P53

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry

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