The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the “optical window” above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.
Red fluorescent protein (RFP) derived indicators are popular due to advantages such as increased imaging depth and reduced autofluorescence and cytotoxicity. However, most RFP-based indicators have low brightness and are susceptible to blue-light-induced photoactivation. In this study, we aimed to overcome the limitations of existing red fluorescent indicators. We utilized mScarlet-I, a highly bright and robust monomeric RFP, to develop a circularly permuted variant called cpmScarlet. We further engineered cpmScarlet into a novel red fluorescent indicator specifically for hydrogen peroxide (H2O2), a crucial reactive oxygen species (ROS) involved in redox signaling and oxidative stress. The resultant indicator, SHRIMP (mScarlet-derived H2O2 Redox Indicator with Minimal Photoactivation), exhibited excitation and emission peaks at ~570 and 595 nm, respectively, and demonstrated a maximum five-fold fluorescence turn-off response to H2O2. Importantly, SHRIMP was not susceptible to blue-light-induced photoactivation and showed high brightness both in its purified protein form and when expressed in mammalian cells. We successfully employed SHRIMP to visualize H2O2 dynamics in mammalian cells with exogenously added H2O2 and in activated macrophages. Additionally, we demonstrated its utility for multiparameter imaging by co-expressing SHRIMP with GCaMP6m, a green fluorescent calcium indicator, enabling simultaneous monitoring of H2O2 and calcium dynamics in mammalian cells in response to thapsigargin (TG) and epidermal growth factor (EGF) stimulation. Furthermore, we expressed SHRIMP in isolated primary mouse islet tissue, and SHRIMP exhibited excellent brightness and capability for effective detection of H2O2 production during streptozotocin (STZ)-induced β-cell damage. This study successfully transformed mScarlet-I, a bright and robust monomeric RFP, into a circularly permuted variant (cpmScarlet) and developed the first cpmScarlet-based genetically encoded fluorescent indicator called SHRIMP. SHRIMP exhibits high brightness and insensitivity to photoactivation and is a valuable tool for real-time monitoring of H2O2 dynamics in various biological systems. Further research may yield an expanded family of cpmScarlet-based red fluorescent indicators with enhanced photophysical properties.
Redox-active molecules play essential roles in cell homeostasis, signaling, and other biological processes. Dysregulation of redox signaling can lead to toxic effects and subsequently cause diseases. Therefore, real-time tracking of specific redox-signaling molecules in live cells would be critical for deciphering their functional roles in pathophysiology. Fluorescent protein (FP)-based genetically encoded redox indicators (GERIs) have emerged as valuable tools for monitoring the redox states of various redox-active molecules from subcellular compartments to live organisms. In the first section of this review, we overview the background, focusing on the sensing mechanisms of various GERIs. Next, we review a list of selected GERIs according to their analytical targets and discuss their key biophysical and biochemical properties. In the third section, we provide several examples which applied GERIs to understanding redox signaling and oxidative toxicology in pathophysiological processes. Lastly, a summary and outlook section is included.
There is great interest in developing boronolectins, which are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin, which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid-and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.
There is great interest in developing boronolectins that are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.
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