ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Many biotechniques use complementary split-fluorescent protein (sFPs) fragments to visualize protein-protein interactions, image cells by ensemble or single molecule fluorescence microscopy, or assemble nanomaterials and protein superstructures. Yet, the reassembly mechanisms of sFPs, including fragment binding rates, folding, chromophore maturation and overall photophysics remain poorly characterized. Here, we evolved asymmetric and self-complementing green, yellow and cyan sFPs together with their full-length equivalents (flFPs) and described their biochemical and photophysical properties in vitro and in cells. While re-assembled sFPs have spectral properties similar to flFPs, they display slightly reduced quantum yields and fluorescence lifetimes due to a less sturdy ß-barrel structure. The complementation of recombinant sFPs expressed in vitro follows a conformational selection mechanism whereby the larger sFP fragments exist in a monomer-dimer equilibrium and only monomers are competent for fluorescence complementation. This bimolecular fragment interaction involves a slow and irreversible binding step, followed by chromophore maturation at a rate similar to that of flFPs. When expressed as fusion tags in cells, sFPs behave as monomers directly activated with synthetic complementary fragments. This study resulted in the development of sFP color variants having improved maturation kinetics, brightness, and photophysics for fluorescence microscopy imaging of cellular processes, including single molecule detection.
Subject(s)
Luminescent Proteins/chemistry , Luminescent Proteins/metabolism , Mutant Proteins , Protein Multimerization , Fluorescence , Gene Expression , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinetics , Luminescent Proteins/genetics , Microscopy, Fluorescence , Protein Binding , Protein Folding , Recombinant Proteins , Spectrometry, FluorescenceABSTRACT
The in cellulo assembly of plasmonic nanomaterials into photo-responsive probes is of great interest for many bioimaging and nanophotonic applications but remains challenging with traditional nucleic acid scaffolds-based bottom-up methods. Here, we address this quandary using split-fluorescent protein (FP) fragments as molecular glue and switchable Raman reporters to assemble gold or silver plasmonic nanoparticles (NPs) into photonic clusters directly in live cells. When targeted to diffusing surface biomarkers in cancer cells, the NPs self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters having hot spots homogenously seeded by the reconstruction of full-length FPs. Within plasmonic hot spots, autocatalytic activation of the FP chromophore and near-field amplification of its Raman fingerprints enable selective and sensitive SERS imaging of targeted cells. This FP-driven assembly of metal colloids also yields enhanced photoacoustic signals, allowing the hybrid FP/NP nanoclusters to serve as contrast agents for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.
Subject(s)
Green Fluorescent Proteins/chemistry , Metal Nanoparticles/chemistry , Molecular Probes , Photoacoustic Techniques/methods , Spectrum Analysis, Raman/methods , Acoustics , Biomarkers, Tumor/analysis , Catalysis , Colloids/chemistry , Diffusion , Fluorescence , Gold/chemistry , Humans , Microscopy/methods , Microscopy, Electron, Transmission , Silver/chemistry , Tumor Cells, CulturedABSTRACT
The assembly of plasmonic metal nanoparticles into hot spot surface-enhanced Raman scattering (SERS) nanocluster probes is a powerful, yet challenging approach for ultrasensitive biosensing. Scaffolding strategies based on self-complementary peptides and proteins are of increasing interest for these assemblies, but the electronic and the photonic properties of such hybrid nanoclusters remain difficult to predict and optimize. Here, split-green fluorescence protein (sGFP) fragments are used as molecular glue and the GFP chromophore is used as a Raman reporter to assemble a variety of gold nanoparticle (AuNP) clusters and explore their plasmonic properties by numerical modeling. It is shown that GFP seeding of plasmonic nanogaps in AuNP/GFP hybrid nanoclusters increases near-field dipolar couplings between AuNPs and provides SERS enhancement factors above 108 . Among the different nanoclusters studied, AuNP/GFP chains allow near-infrared SERS detection of the GFP chromophore imidazolinone/exocyclic CC vibrational mode with theoretical enhancement factors of 108 -109 . For larger AuNP/GFP assemblies, the presence of non-GFP seeded nanogaps between tightly packed nanoparticles reduces near-field enhancements at Raman active hot spots, indicating that excessive clustering can decrease SERS amplifications. This study provides rationales to optimize the controlled assembly of hot spot SERS nanoprobes for remote biosensing using Raman reporters that act as molecular glue between plasmonic nanoparticles.
