Browsing by Author "Wang, Shichao"

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    Oxime as a general photocage for the design of visible light photo-activatable fluorophores
    (Royal Society of Chemistry, 2021) Wang, Lushun; Wang, Shichao; Tang, Juan; Espinoza, Vanessa B.; Loredo, Axel; Tian, Zeru; Weisman, R. Bruce; Xiao, Han; Bioengineering; Biosciences; Chemistry
    Photoactivatable fluorophores have been widely used for tracking molecular and cellular dynamics with subdiffraction resolution. In this work, we have prepared a series of photoactivatable probes using the oxime moiety as a new class of photolabile caging group in which the photoactivation process is mediated by a highly efficient photodeoximation reaction. Incorporation of the oxime caging group into fluorophores results in loss of fluorescence. Upon light irradiation in the presence of air, the oxime-caged fluorophores are oxidized to their carbonyl derivatives, restoring strong fluorophore fluorescence. To demonstrate the utility of these oxime-caged fluorophores, we have created probes that target different organelles for live-cell confocal imaging. We also carried out photoactivated localization microscopy (PALM) imaging under physiological conditions using low-power light activation in the absence of cytotoxic additives. Our studies show that oximes represent a new class of visible-light photocages that can be widely used for cellular imaging, sensing, and photo-controlled molecular release.
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    Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation
    (Springer Nature, 2022) Chen, Yuda; Jin, Shikai; Zhang, Mengxi; Hu, Yu; Wu, Kuan-Lin; Chung, Anna; Wang, Shichao; Tian, Zeru; Wang, Yixian; Wolynes, Peter G.; Xiao, Han; Bioengineering; Biosciences; Chemistry; Physics and Astronomy; Center for Theoretical Biological Physics
    Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.
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    Xanthone-based solvatochromic fluorophores for quantifying micropolarity of protein aggregates
    (Royal Society of Chemistry, 2022) Wang, Lushun; Hsiung, Chia-Heng; Liu, Xiaojing; Wang, Shichao; Loredo, Axel; Zhang, Xin; Xiao, Han; Bioengineering; Biosciences; Chemistry
    Proper three-dimensional structures are essential for maintaining the functionality of proteins and for avoiding pathological consequences of improper folding. Misfolding and aggregation of proteins have been both associated with neurodegenerative disease. Therefore, a variety of fluorogenic tools that respond to both polarity and viscosity have been developed to detect protein aggregation. However, the rational design of highly sensitive fluorophores that respond solely to polarity has remained elusive. In this work, we demonstrate that electron-withdrawing heteroatoms with (d–p)–π* conjugation can stabilize lowest unoccupied molecular orbital (LUMO) energy levels and promote bathochromic shifts. Guided by computational analyses, we have devised a novel series of xanthone-based solvatochromic fluorophores that have rarely been systematically studied. The resulting probes exhibit superior sensitivity to polarity but are insensitive to viscosity. As proof of concept, we have synthesized protein targeting probes for live-cell confocal imaging intended to quantify the polarity of misfolded and aggregated proteins. Interestingly, our results reveal several layers of protein aggregates in a way that we had not anticipated. First, microenvironments with reduced polarity were validated in the misfolding and aggregation of folded globular proteins. Second, granular aggregates of AgHalo displayed a less polar environment than aggregates formed by folded globular protein represented by Htt–polyQ. Third, our studies reveal that granular protein aggregates formed in response to different types of stressors exhibit significant polarity differences. These results show that the solvatochromic fluorophores solely responsive to polarity represent a new class of indicators that can be widely used for detecting protein aggregation in live cells, thus paving the way for elucidating cellular mechanisms of protein aggregation as well as therapeutic approaches to managing intracellular aggregates.