Molecular biosensing system based on intrinsically disordered proteins

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.

Turncoat Polypeptides: We Adapt to Our Environment

“…The specific interaction between the intrinsically disordered polypeptide regions of the BRCA1 and p53 proteins induces the folding of both. This structural change can be used to detect p53 by quantitative quenching by using both intrinsic tryptophan residues and extrinsic fluorescent probes; the latter are strategically inserted into environmentally sensitive amino acid positions and detected upon binding on the visible region …”

“…This structuralc hange can be used to detect p53 by quantitative quenching by using both intrinsic tryptophan residues and extrinsic fluorescent probes; the latter are strategically inserted into environmentally sensitive amino acid positions and detected upon binding on the visible region. [89] Ac omplete unfolding of the protein is nota lwaysn ecessary for ap owerful biosensor based on turncoatp roteins.S tratton and Loh developeda ne legant, general method, designated AFF (Figure 4), that may be appliedt on umerous proteins and by which protein conformationalc hanges can be engineered in responset oas pecific signal and coupled to ar eport output. [90] In essence, the general approach consists of the duplication of as egment located in the Cterminus, which is appendedt ot he Nterminus, or vice versa (the segment chosen depends on the location of key functional residues).…”

Protein Conformational Switches: From Nature to Design

“…Cissell et al. utilized the 219–498 fragment of BRCA1 as an intrinsically disordered binding element to detect p53 93. Binding of p53 induced the BRCA1 fragment to fold, which was then detected by strategic placement of environmentally sensitive tetramethylrhodamine or dansyl dyes.…”

Converting a protein into a switch for biosensing and functional regulation

“…Impressively, their sensor worked even when tested in the complex, contaminant‐ridden environment of blood serum. Others have taken advantage of intrinsically disordered regions in p5374 and BRCA175 that fold upon binding to create fluorescent sensors for their respective ligands.…”