The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

Fluorescence resonance energy transfer (FRET) with fluorescent proteins is a powerful method for detection of protein-protein interactions, enzyme activities and small molecules in the intracellular milieu. Aided by a new violet-excitable yellow-fluorescing variant of Aequorea victoria GFP, we developed dual FRET-based caspase-3 biosensors. Owing to their distinct excitation profiles, each FRET biosensor can be ratiometrically imaged in the presence of the other.

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".

Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cistrans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.crystallography ͉ fluorescence ͉ photoswitching ͉ protein structure G reen fluorescent protein (GFP) from the jellyfish Aequorea victoria (1) and more recently, cyan, yellow, and red fluorescent proteins (FPs), isolated from coral reef organisms (2), have become standard research tools in cellular biology. Structural studies have revealed a universally conserved fold for FPs: an 11-stranded ␤-barrel with a central and axial ␣-helix (3, 4). A sequence of three amino acid residues on this central ␣-helix (Ser-65-Tyr-66-Gly-67 in Aequorea GFP) undergoes a series of autocatalytic posttranslational modifications to form the chromophore. Despite overall structural similarity across FPs from a variety of species and a variety of hues, members of the GFP family are quite diverse with respect to their photophysical properties. For example, properties such as emission color, extinction coefficient, quantum yield, and fluorescence lifetime can vary dramatically among FP variants. These properties are determined by both the influence of the protein matrix on the chromophore environment and the particular covalent chemical structure of the chromophore (5).Another photophysical property common to all FPs is wavelength-dependent change in their f luorescence emission properties upon illumination. In most cases, these changes appear to result from irreversible covalent modification of the chromophore structure or its local protein environment. Examples of such permanent changes include light-induced transition from a f luorescent to a nonf luorescent state (photobleaching), transition from a nonf luorescent to a f luorescent state (photoactivation), or a change in excitation or emission wavelength maxima (photoconversion). Photobleaching, photoactivation, and photoconversion are well documented and can be advantageous for f luorescence imaging applications such as in vivo tracking of fusion protein movements (6 -9). A less well documented process, occasionally described as ''re...

Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular shows superior sensitivity over other commonly used bioluminescence reporters in vitro and in vivo. This pair was adapted to develop a bioluminescence resonance energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.

The detection of hydrogen sulfide (H(2)S), a toxic gas and an important biological signaling molecule, has been a long-time challenge. Here we report genetically encoded fluorescent protein (FP)-based probes that can selectively detect H(2)S. By expanding the genetic codes of E. coli and mammalian cells, FP chromophores were modified with the sulfide-reactive azide functional group. These structurally modified chromophores were selectively reduced by H(2)S, resulting in sensitive fluorescence enhancement detectable by spectroscopic and microscopic techniques. Exploration of a circularly permuted FP led to an improved sensor with faster responses, and the feasibility of using such a genetically encoded probe to monitor H(2)S in living mammalian cells has also been demonstrated.

Although fluorescent reporters and biosensors have become indispensable tools in biological and biomedical fields, fluorescence measurements require external excitation light, thereby limiting their use in thick tissues and live animals. Bioluminescent reporters and biosensors may potentially overcome this hurdle because they use enzyme-catalyzed exothermic biochemical reactions to generate excited-state emitters. This review first introduces the development of bioluminescent reporters, and next, their applications in sensing biological changes in vitro and in vivo as biosensors. Lastly, we discuss chemiluminescent sensors that produce photons in the absence of luciferases. This review aims to explore fundamentals and experimental insights and to emphasize the yet-to-be-reached potential of next-generation luminescent reporters and biosensors.

Peroxynitrite is a highly reactive molecule involved in cell signaling and pathological processes. We hereby report a novel genetically encoded probe, pnGFP, which can selectively sense peroxynitrite. A boronic acid moiety was site-specifically introduced into circularly permuted fluorescent proteins. By examining different protein templates followed with site-targeted random mutagenesis, we identified a selective peroxynitrite sensor, which is essentially unresponsive to other common cellular redox signaling molecules. The new probe has been genetically introduced into mammalian cells to image peroxynitrite at physiologically relevant concentrations.