A noncommutative combinatorial protein logic circuit controls cell orientation in nanoenvironments

Chen, Jiaxing; Vishweshwaraiah, Yashavantha; Mailman, Richard B.; Tabdanov, Erdem D.; Dokholyan, Nikolay V.

doi:10.1126/sciadv.adg1062

Cited by 7 publications

(3 citation statements)

References 89 publications

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“…Optogenetic control of the POI inputs is compatible with continuous directed evolution since administering light, even for weeks, is degrons, and kinases, acting on the POI. [117][118][119][120][121][122] Chemical-controlled POIs can in some instances be converted to light control by swapping the relevant domains 121,[123][124][125][126] . Light control has also been built into POIs specifically to be used for screening for improved protein functionality.…”

Section: Practical Requirements On the Input And Output Of The Poimentioning

confidence: 99%

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

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Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require applying selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the dynamics, transient states, and input-output relations of the POI in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution isin vivo, continuous, self-selecting, and genetically robust. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 25 new variants of the widely used LOV transcription factor El222. These mutants were stronger, less leaky, or green- and red-responsive. The latter was conjectured to be impossible for LOV domains but is needed for multiplexing and lowering phototoxicity. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss ofYOR1makes supplementing the expensive and unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by creating and evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities evolvable.

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Section: Practical Requirements On the Input And Output Of The Poimentioning

confidence: 99%

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

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“…NCAs were introduced in 2018 as a conceptual prototype based on a body of switch-like designs for regulating protein functions. In the past years, we have demonstrated that NCAs can also serve as a functional, logical gate and even as a small circuit . The conceptual design of NCA is based on a single polypeptide sequence that incorporates the target protein with the desired function, sensor, or regulatory units (RUs), and the logic of wiring. , Nikolay Dokholyan’s group at Pennsylvania State University is interested in target protein regulation to control a specific cellular pathway and, therefore, phenotype.…”

Section: Introductionmentioning

confidence: 99%

Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology

Avila,

Rebolledo,

Skelly

et al. 2024

ACS Appl. Bio Mater.

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Nature continually refines its processes for optimal efficiency, especially within biological systems. This article explores the collaborative efforts of researchers worldwide, aiming to mimic nature's efficiency by developing smarter and more effective nanoscale technologies and biomaterials. Recent advancements highlight progress and prospects in leveraging engineered nucleic acids and proteins for specific tasks, drawing inspiration from natural functions. The focus is developing improved methods for characterizing, understanding, and reprogramming these materials to perform user-defined functions, including personalized therapeutics, targeted drug delivery approaches, engineered scaffolds, and reconfigurable nanodevices. Contributions from academia, government agencies, biotech, and medical settings offer diverse perspectives, promising a comprehensive approach to broad nanobiotechnology objectives. Encompassing topics from mRNA vaccine design to programmable protein-based nanocomputing agents, this work provides insightful perspectives on the trajectory of nanobiotechnology toward a future of enhanced biomimicry and technological innovation.

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“…With rapamycin as the first signal, followed by blue light as the second. 46 Along with computational methods such as MSA (multiple sequence alignments), MD (molecular dynamics), and DMD simulations (discreet molecular dynamics); molecular docking and in silico modeling 47 paired with mass spectroscopy have also been used to define protein structures and map allosteric sites in proteins. Through microscopic techniques, such as cryo-EM, two-dimensional structures of proteins can be acquired and morphed into three-dimensional models using artificial intelligence 48 .…”

Section: Introductionmentioning

confidence: 99%

Allosteric regulation of kinase activity in living cells

Godbole

Dokholyan

2023

Preprint

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The dysregulation of protein kinases is associated with multiple diseases due to the kinases' involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities making it difficult to specifically target one kinase, allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or "sensors" are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation.

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A noncommutative combinatorial protein logic circuit controls cell orientation in nanoenvironments

Cited by 7 publications

References 89 publications

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology

Allosteric regulation of kinase activity in living cells

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