Project description:Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.
Project description:Many biomedical fields rely on proteins that are selectively modified. These can be attached using reactive or catalytic moieties, but the position where these moieties are attached is often poorly controlled. We assessed how catalyst position affects the efficiency and selectivity of protein modification. For this, we anchored a template DNA strand to three different proteins, which were subsequently hybridized to DNA strands that contained catalysts at different positions. We found a strong correlation between the catalyst-to-protein distance and the efficiency of protein modification for acyl transfer catalysts, which operate via a covalently bound reactant intermediate. Additionally, we found that the catalyst's distance and orientation with respect to the protein surface, also influences its site-selectivity. A catalyst operating with unbound reactant intermediates showed only enhanced efficiency. Our results are rationalized using computational simulations, showing that one-point anchoring of the DNA construct leads to notable differences in the site of modification.
Project description:We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca(2+) ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (?2000-fold) and the change in the apparent pKa of the active lysine residue.
Project description:Environmental problems are among the most pressing issues in the modern world, including the shortage of clean drinking water partially caused by contamination from various industries and the excessive emission of CO2 primarily from the massive use of fossil fuels. Consequently, it is crucial to develop inexpensive, effective, and environmentally friendly methods for wastewater treatment and CO2 reduction, turning them into useful feedstocks. This study explores a unique method that addresses both challenges by utilizing ZnO, which is recognized as one of the most active semiconductors for photocatalysis, as well as a cost-effective electrocatalyst for the CO2 reduction reaction (CO2RR). Specifically, we investigate the influence of the morphology of various ZnO nanostructures synthesized via different low-cost routes on their photocatalytic properties for degrading the rhodamine-B dye (RhB) and on their electrocatalytic performance for the CO2RR. Our results show that the ZnO lamella morphology achieves the best performance compared to the nanorod and nanoparticle structures. This outcome is likely attributed to the lamella's higher aspect ratio, which plays a critical role in determining the structural, optical, and electrical properties of ZnO.
Project description:Future smart nanostructures will have to rely on molecular assembly for unique or advanced desired functions. For example, the evolved ribosome in nature is one example of functional self-assembly of nucleic acids and proteins employed in nature to perform specific tasks. Artificial self-assembled nanodevices have also been developed to mimic key biofunctions, and various nucleic acid- and protein-based functional nanoassemblies have been reported. However, functionally regulating these nanostructures is still a major challenge. Here we report a general approach to fine-tune the catalytic function of DNA-enzymatic nanosized assemblies by taking advantage of the trans-cis isomerization of azobenzene molecules. To the best of our knowledge, this is the first study to precisely modulate the structures and functions of an enzymatic assembly based on light-induced DNA scaffold switching. Via photocontrolled DNA conformational switching, the proximity of multiple enzyme catalytic centers can be adjusted, as well as the catalytic efficiency of cofactor-mediated DNAzymes. We expect that this approach will lead to the advancement of DNA-enzymatic functional nanostructures in future biomedical and analytical applications.
Project description:Molecular dynamics simulations are often used to provide feedback in the design workflow of DNA nanostructures. However, even with coarse-grained models, the convergence of distributions from unbiased simulation is slow, limiting applications to equilibrium structural properties. Given the increasing interest in dynamic, reconfigurable, and deformable devices, methods that enable efficient quantification of large ranges of motion, conformational transitions, and mechanical deformation are critically needed. Metadynamics is an automated biasing technique that enables the rapid acquisition of molecular conformational distributions by flattening free energy landscapes. Here we leveraged this approach to sample the free energy landscapes of DNA nanostructures whose unbiased dynamics are nonergodic, including bistable Holliday junctions and part of a bistable DNA origami structure. Taking a DNA origami-compliant joint as a case study, we further demonstrate that metadynamics can predict the mechanical response of a full DNA origami device to an applied force, showing good agreement with experiments. Our results exemplify the efficient computation of free energy landscapes and force response in DNA nanodevices, which could be applied for rapid feedback in iterative design workflows and generally facilitate the integration of simulation and experiments. Metadynamics will be particularly useful to guide the design of dynamic devices for nanorobotics, biosensing, or nanomanufacturing applications.
Project description:The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated β-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.
Project description:Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID-19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS-CoV-2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme-DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi-enzyme catalysis; through system-wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single-copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS-CoV-2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID-19 patients.
Project description:A biological reaction network may serve multiple purposes, processing more than one input and impacting downstream processes via more than one output. These networks operate in a dynamic cellular environment in which the levels of network components may change within cells and across cells. Recent evidence suggests that protein concentration variability could explain cell fate decisions. However, systems with multiple inputs, multiple outputs, and changing input concentrations have not been studied in detail due to their complexity. Here, we take a systems biochemistry approach, combining physiochemical modeling and information theory, to investigate how cyclooxygenase-2 (COX-2) processes simultaneous input signals within a complex interaction network. We find that changes in input levels affect the amount of information transmitted by the network, as does the correlation between those inputs. This, and the allosteric regulation of COX-2 by its substrates, allows it to act as a signal integrator that is most sensitive to changes in relative input levels.
Project description:Precisely tailoring the structure and fully making use of the components of nanoparticles are effective to enhance their catalytic performance for a given reaction. We herein demonstrate the design of cage-bell structured Pt-Pd nanoparticles, where a Pd shell is deliberately selected to enhance the catalytic property and methanol tolerance of Pt for oxygen reduction reaction. This strategy starts with the synthesis of core-shell Pt@Ag nanoparticles, followed by galvanic replacement reaction between the Ag shell and Pd(2+) ions to form core-shell-shell Pt@Ag@Ag-Pd nanoparticles with a Pt core and double shells composed of Ag at inner and alloy Ag-Pd at outer, respectively. Then, the core-shell-shell templates are agitated with saturated NaCl solution to eliminate the Ag component from the double shells, leading to the formation of bimetallic Pt-Pd nanoparticles with a cage-bell structure, defined as a movable Pt core enclosed by a porous Pd shell, which show enhanced catalytic activity for oxygen reduction compared with that of the Pt seeds due to the additional catalysis from Pd shell. In addition, owing to the different diffusion behavior of methanol and oxygen molecules in the porous Pd shell, the Pt-Pd cage-bell nanostructures also exhibit superior methanol tolerant property in catalyzing the oxygen reduction.