•Photoswitchable fluorescent proteins cycle between on and off states in response to light.•Structural studies reveal diversity in the details of photoswitching mechanisms.•These proteins are ...extensively used for protein tracking and superresolution imaging.•Newer uses include information storage and optical control of protein activity.
Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. In the last 10 years, many new RSFPs have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties. This review will describe research on the mechanisms of reversible photoswitching and recent applications using RSFPs. While cis–trans isomerization of the chromophore is believed to be the general mechanism for most RSFPs, structural studies reveal diversity in the details of photoswitching mechanisms, including different effects of protonation, chromophore planarity, and pocket flexibility. Applications of RSFPs include new types of live-cell superresolution imaging, tracking of protein movements and interactions, information storage, and optical control of protein activity.
Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or ...manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.
An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein ...binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.
Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe ...light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.
This study presents an adaptive coupling peridynamic least-square minimization with the finite element method (PDLSM-FEM) for fracture analysis. The presented method utilizes the PDLSM modeling ...discontinuities while maximizing the FEM region for computational efficiency. Within the presented adaptive PDLSM-FEM, only elements intersecting with the crack path and their neighboring elements are defined as PD elements, whose stiffness matrices are derived based on PDLSM equations. The remaining elements are conventional finite elements. Numerical integration of interaction integral is proposed and implemented to evaluate the stress intensity factors (SIFs) for 2-D problems. The criterion of maximum hoop tensile stress is employed for failure prediction. New contributions of this work include the adaptive coupling of PDLSM with FEM for minimizing the PD region and the application of the adaptive PDLSM-FEM to quasi-static crack propagation analysis. Simulations of three 2-D plane stress plates and one 3-D block with static or quasi-static cracks propagation are performed. Results show the proposed method improves computational efficiency substantially and has reasonable accuracy and good capability of crack propagation prediction.
Abstract Objective We examined the surface characteristics and corrosion properties of selective laser melted (SLM) cobalt–chromium (Co–Cr) dental alloys before and after porcelain-fused-to-metal ...(PFM) firing. Methods Samples were manufactured utilizing SLM techniques and control specimens were fabricated using traditional casting methods. The microstructure and surface composition were examined using metallographic microscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Corrosion properties were evaluated using electrochemical impedance spectroscopy. Student's t -test was used to evaluate differences in numerical results of electrochemical corrosion tests between SLM and cast specimens before or after PFM firing. The results of electrochemical corrosion tests of the SLM and cast samples before and after firing were analyzed using one-way ANOVA. Results Although PFM firing altered the microstructure of the SLM specimens, they still exhibited a compact and homogeneous structure, and XPS analysis indicated that there were no significant differences in the surface composition of the specimens after firing. In artificial saliva at pH 5, the Rp value of the SLM specimens was 6.21 MΩ cm−2 before firing and 2.84 MΩ cm−2 after firing, suggesting there was no significant difference in electrochemical corrosion properties ( P > 0.05). In artificial saliva at pH 2.5, the Rp value of the SLM group was 4.80 MΩ cm−2 before firing and 2.88 MΩ cm−2 after firing, again indicating no significant difference in electrochemical corrosion properties ( P > 0.05). At pH 2.5, there was a significant difference in corrosion behavior between the cast and SLM groups, with the Rp value of the cast group being 0.78 MΩ cm−2 vs. 2.88 MΩ cm−2 for the SLM group. Significance The improved post-firing corrosion resistance of SLM specimens provides further support for their use in prosthodontic applications, as the oral environment may become temporarily acidic following meals.
A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and ...engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been ...achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
We present optical observations of an SN 2002cx-like supernova SN 2013en in UGC 11369, spanning from a phase near maximum light (t = + 1 d) to t = + 60 d with respect to the R-band maximum. Adopting ...a distance modulus of μ = 34.11 ± 0.15 mag and a total extinction (host galaxy+Milky Way) of A
V ≈ 1.5 mag, we found that SN 2013en peaked at MR
≈ −18.6 mag, which is underluminous compared to the normal SNe Ia. The near maximum spectra show lines of Si ii, Fe ii, Fe iii, Cr ii, Ca ii and other intermediate-mass and iron group elements which all have lower expansion velocities (i.e. ∼ 6000 km s− 1). The photometric and spectroscopic evolution of SN 2013en is remarkably similar to those of SN 2002cx and SN 2005hk, suggesting that they are likely to be generated from a similar progenitor scenario or explosion mechanism.
Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle ...immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.
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