Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT 2 and ReAsH-EDT 2 . This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.The physical phenomenon known as fluorescence has revolutionized cell biology, and its positive impact on molecular medicine will continue to develop with time. Driving this revolution is the green fluorescent protein (GFP) 1-3 and a rainbow of natural and engineered fluorescent protein (FP) variants that can be fused at the genetic level to proteins expressed in cell cultures and even whole animals 3 . Methodologies based on Förster resonance energy transfer (FRET) that use two or more FPs with overlapping absorption and emission spectra are used frequently as sensors to probe dynamic and complex processes including protein association, metal ion binding, conformational changes and post-translational modifications 4 . However, virtually all sensors based on FRET between FP color variants show dynamic changes in fluorescence intensity (typically 20-50%) that can be smaller than normal variations in cell-to-cell intensity due to differential FP expression 5 and that may be influenced significantly by Mg 2+ -ATP fluctuation 6 . Moreover, the steric bulk and slow folding of FPs limits their spatial and kinetic resolution 3,7 . We selected four structurally characterized polypeptides and protein domains to evaluate whether the Pro-Gly sequence within the linear tetracysteine motif could be replaced with one or more folded proteins while maintaining biarsenical affinity and fluorescence intensity. The feasibility of intramolecular bipartite tetracysteine display was evaluated using avian pancreatic polypeptide (aPP) 20 and Zip4 (ref. 21)-two well-folded polypeptides that were modified to contain one half of the linear tetracysteine motif at each terminus. Intermolecular bipartite tetracysteine display was evaluated using the protein-protein dimerization domains from the basic region leucine zipper (bZIP) proteins GCN4 (ref. 22) and Jun (ref. 23), which were modified to conta...
A luciferase immunoprecipitation systems (LIPS) assay was developed to define the antigenic specificity and titer of antibodies directed against human norovirus (HuNoV). Recombinant proteins, expressed by plasmid constructs encoding Renilla luciferase (Ruc) fused to the full-length HuNoV major capsid protein (VP1) (Ruc-antigen), were generated for ten HuNoV strains. In addition, subdomain constructs Ruc-Shell (S) and Ruc-Protruding (P) were engineered for a representative GII.4 norovirus (strain GII.4/2006b). The LIPS assay measured antibody levels in a well-defined panel of HuNoV-specific sera, and the results were compared to an ELISA standard. In hyperimmune sera, the LIPS produced titers similar to or higher than those measured by the ELISA of HuNoV-specific antibodies. The specificity of antibodies in various sera was profiled by LIPS with a panel of diverse Ruc-antigens containing full-length HuNoV VP1 proteins or VP1 subdomains, and the assay detected both specific and cross-reactive antibodies. Competition assays, in which antibodies were pre-incubated with one or more intact VLPs representing different genotypes, proved useful in further assessment of the antibody specificity detected by LIPS in complex polyclonal sera. The profiling of HuNoV-specific antibodies in the high-throughput LIPS format may prove useful in defining the strength or specificity of the adaptive immune response following natural infection or vaccination.
Good partnership: A novel technique called complex‐edited electron microscopy (CE‐EM) combines bipartite tetracysteine display with EM and permits high‐resolution imaging of discrete protein complexes (A and B in the picture). The strategy facilitates the direct and selective labeling of discrete protein complexes in living cells, followed by imaging with the extraordinary resolution of EM.
Interactions between and among proteins regulate most cell functions, yet detecting these interactions in living cells, especially at high resolution, remains a challenge. Protein complementation[1] proximity-induced biotinylation[2], FRET[3] and bipartite tetracysteine display[4] can all detect interactions between proteins, but only at the moderate resolution provided by epifluorescent microscopy-approximately 200 nm. Super-resolution imaging has begun to overcome this diffraction limit[5], but it cannot detect protein complexes at the near-atomic-level resolution achievable using electron microscopy (EM).[6] Individual tetracysteine-containing proteins can be visualized using EM by use of the biarsenical dye 4,5-bis(1,3,2-dithiarsolan-2-yl)resorufin (ReAsH).[7-9] Irradiation of a protein•ReAsH complex at 585 nm in the presence of oxygen and 3,3′-diaminobenzidine (DAB) catalyzes the formation of an osmophilic DAB polymer that is opaque to electron beams and appears in the electron microscope as a fine granular precipitate.[7-9] An analogous method able to detect a discrete protein complex within a living cell, followed by fixation and sectioning as required by EM, would provide a powerful tool for visualizing at high resolution the interactions between proteins in their native environments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.