Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.

Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose-binding protein, a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing that Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method that also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ∼1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.

Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose binding protein (MBP), a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method which also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ~1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 Å to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

OptoPI3K, genetic code expansion, and click chemistry reveal mechanisms underlying reciprocal regulation between TRPV1 and PI3K.

Receptor tyrosine kinase signaling is characterized by complex webs of interconnected pathways that regulate diverse cellular functions. The complexity of signaling is a barrier to understanding the pathways that control any particular function. In this work, we use a novel combination of approaches and a new click chemistry probe to determine the role of one pathway in regulating cell surface expression of an ion channel and a receptor tyrosine kinase. We applied an optogenetic approach to uncouple activation of the PI3K pathway from other pathways downstream of RTK activation. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the PM in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

Site-directed spin-labeling (SDSL)─in combination with double electron-electron resonance (DEER) spectroscopy─has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides─including a gem-diethyl-substituted nitroxide with enhanced stability in cells─with rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

Ultra-Fast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

Studying protein structures and dynamics directly in the cellular environments in which they function is essential to fully understand the molecular mechanisms underlying cellular processes. Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. In-cell DEER spectroscopy on proteins in mammalian cells has thus far not been possible due to the notable challenges of spin-labeling in live cells. In-cell SDSL requires exquisite biorthogonality, high labeling reaction rates and low background signal from unreacted residual spin label. While the bioorthogonal reaction must be highly specific and proceed under physiological conditions, many spin labels display time-dependent instability in the reducing cellular environment. Additionally, high concentrations of spin label can be toxic. Thus, an exceptionally fast bioorthogonal reaction is required that can allow for complete labeling with low concentrations of spin-label prior to loss of signal. Here we utilized genetic code expansion to site-specifically encode a novel family of small, tetrazine-bearing non-canonical amino acids (Tet-v4.0) at multiple sites in green fluorescent protein (GFP) and maltose binding protein (MBP) expressed both in E. coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans -cyclooctene (sTCO)-functionalized nitroxides-including a gem -diethyl-substituted nitroxide with enhanced stability in cells-with rate constants that can exceed 10 6 M -1 s -1 . The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live HEK293T cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide added directly to the culture medium. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro . Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and successfully discerned the conformational state of MBP within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET.

With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

TRPV1, TRPA1, and TRPM8 are expressed in axon terminals in the cornea: TRPV1 axons contain CGRP and secretogranin II; TRPA1 axons contain secretogranin 3.

Purpose: The cornea is highly enriched in sensory neurons expressing the thermal TRP channels TRPV1, TRPA1, and TRPM8, and is an accessible tissue for study and experimental manipulation. The aim of this work was to provide a concise characterization of the expression patterns of various TRP channels and vesicular proteins in the mammalian cornea. Methods: Immunohistochemistry (IHC) was performed using wholemount and cryostat tissue preparations of mouse and monkey corneas. The expression patterns of TRPV1 and TRPA1 were determined using specific antisera, and further colocalization was performed with antibodies directed against calcitonin-related gene protein (CGRP), neurofilament protein NF200, and the secretogranins ScgII and SCG3. The expression of TRPM8 was determined using corneas from mice expressing EGFP under the direction of a TRPM8 promoter (TRPM8EGFP mice). Laser scanning confocal microscopy and image analysis were performed. Results: In the mouse cornea, TRPV1 and TRPM8 were expressed in distinct populations of small diameter C fibers extending to the corneal surface and ending either as simple or ramifying terminals, or in the case of TRPM8, as complex terminals. TRPA1 was expressed in large-diameter NF200-positive Aδ axons. TRPV1 and TRPA1 appeared to localize to separate intracellular vesicular structures and were primarily found in axons containing components of large dense vesicles with TRPV1 colocalizing with CGRP and ScgII, and TRPA1 colocalizing with SCG3. Monkey corneas showed similar colocalization of CGRP and TRPV1 on small-diameter axons extending to the epithelial surface. Conclusions: The mouse cornea is abundant in sensory neurons expressing TRPV1, TRPM8, and TRPA1, and provides an accessible tissue source for implementing a live tissue preparation useful for further exploration of the molecular mechanisms of hyperalgesia. This study showed that surprisingly, these TRP channels localize to separate neurons in the mouse cornea and likely have unique physiological functions. The similar TRPV1 expression pattern we observed in the mouse and monkey corneas suggests that mice provide a reasonable initial model for understanding the role of these ion channels in higher mammalian corneal physiology.

Multimerization of Homo sapiens TRPA1 ion channel cytoplasmic domains.

The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modulated by myriad noxious stimuli that interact with multiple regions of the channel, including cysteine-reactive natural extracts from onion and garlic which modify residues in the cytoplasmic domains. The way in which TRPA1 cytoplasmic domain modification is coupled to opening of the ion-conducting pore has yet to be elucidated. The cryo-EM structure of TRPA1 revealed a tetrameric C-terminal coiled-coil surrounded by N-terminal ankyrin repeat domains (ARDs), an architecture shared with the canonical transient receptor potential (TRPC) ion channel family. Similarly, structures of the TRP melastatin (TRPM) ion channel family also showed a C-terminal coiled-coil surrounded by N-terminal cytoplasmic domains. This conserved architecture may indicate a common gating mechanism by which modification of cytoplasmic domains can transduce conformational changes to open the ion-conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal coiled-coil domains can be expressed in bacteria and maintain the ability to interact. We tested three gating regulators: temperature; the polyphosphate compound IP6; and the covalent modifier allyl isothiocyanate to determine whether they alter N- and C-terminal interactions. We found that none of the modifiers tested abolished ARD-coiled-coil interactions, though there was a significant reduction at 37˚C. We found that coiled-coils tetramerize in a concentration dependent manner, with monomers and trimers observed at lower concentrations. Our system provides a method for examining the mechanism of oligomerization of TRPA1 cytoplasmic domains as well as a system to study the transmission of conformational changes resulting from covalent modification.

Reciprocal regulation among TRPV1 channels and phosphoinositide 3-kinase in response to nerve growth factor.

Although it has been known for over a decade that the inflammatory mediator NGF sensitizes pain-receptor neurons through increased trafficking of TRPV1 channels to the plasma membrane, the mechanism by which this occurs remains mysterious. NGF activates phosphoinositide 3-kinase (PI3K), the enzyme that generates PI(3,4)P2 and PIP3, and PI3K activity is required for sensitization. One tantalizing hint came from the finding that the N-terminal region of TRPV1 interacts directly with PI3K. Using two-color total internal reflection fluorescence microscopy, we show that TRPV1 potentiates NGF-induced PI3K activity. A soluble TRPV1 fragment corresponding to the N-terminal Ankyrin repeats domain (ARD) was sufficient to produce this potentiation, indicating that allosteric regulation was involved. Further, other TRPV channels with conserved ARDs also potentiated NGF-induced PI3K activity. Our data demonstrate a novel reciprocal regulation of PI3K signaling by the ARD of TRPV channels.

Visualizing conformational dynamics of proteins in solution and at the cell membrane.

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

Competing Discourses of Scientific Identity among Postdoctoral Scholars in the Biomedical Sciences.

The postdoctoral period is generally one of low pay, long hours, and uncertainty about future career options. To better understand how postdocs conceive of their present and future goals, we asked researchers about their scientific identities while they were in their postdoctoral appointments. We used discourse analysis to analyze interviews with 30 scholars from a research-intensive university or nearby research institutions to better understand how their scientific identities influenced their career goals. We identified two primary discourses: bench scientist and principal investigator (PI). The bench scientist discourse is characterized by implementing other people's scientific visions through work in the laboratory and expertise in experimental design and troubleshooting. The PI discourse is characterized by a focus on formulating scientific visions, obtaining funding, and disseminating results through publishing papers and at invited talks. Because these discourses represent beliefs, they can-and do-limit postdocs' understandings of what career opportunities exist and the transferability of skills to different careers. Understanding the bench scientist and PI discourses, and how they interact, is essential for developing and implementing better professional development programs for postdocs.