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Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.

Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.

The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.

Background: In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra., Results: Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins., Conclusion: Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.

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 cis-trans 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.

We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs.

Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both E. coli and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness. [ABSTRACT FROM AUTHOR]

Peroxynitrite is a reactive nitrogen species (RNS) that plays critical roles in signal transduction, stress response, and numerous human diseases. Advanced molecular tools that permit the selective, sensitive, and noninvasive detection of peroxynitrite are essential for understanding its pathophysiological functions. Here, we present pnGFP-Ultra, a high-performance, reaction-based, genetically encodable biosensor for imaging peroxynitrite in live cells. pnGFP-Ultra features a p -boronophenylalanine-modified chromophore as the sensing moiety and exhibits a remarkable ~110-fold fluorescence turn-on response toward peroxynitrite while displaying virtually no cross-reaction with other reactive oxygen/nitrogen species. To facilitate the expression of pnGFP-Ultra in mammalian cells, we engineered an efficient noncanonical amino acid (ncAA) expression system that is broadly applicable to the mammalian expression of ncAA-containing proteins. pnGFP-Ultra robustly detected peroxynitrite production in activated macrophages and primary glial cells. pnGFP-Ultra fills an important technical gap and represents a valuable addition to the molecular toolbox for probing RNS biology. [Display omitted] • pnGFP-Ultra is a genetically encoded biosensor with an ~110-fold turn-on response • pnGFP-Ultra exhibits high selectivity and sensitivity toward peroxynitrite • An optimized plasmid-based system increases noncanonical amino acid incorporation • pnGFP-Ultra robustly detects peroxynitrite in macrophages and primary glial cells Advanced molecular tools that permit the selective, sensitive, and noninvasive detection of peroxynitrite are essential for understanding its pathophysiological functions. Chen et al. report on pnGFP-Ultra, a high-performance fluorescent turn-on biosensor for minimally invasive and selective imaging of peroxynitrite production in live cells. [ABSTRACT FROM AUTHOR]