Xin Zhang

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Associate Professor, Department of Biochemistry and Molecular Biology, Department of Chemistry
Penn State University


Scopus Publications


Scholar Citations


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Scopus Publications

  • AggFluor: Fluorogenic Toolbox Enables Direct Visualization of the Multi-Step Protein Aggregation Process in Live Cells
    Charles H. Wolstenholme, Hang Hu, Songtao Ye, Brian E. Funk, Divya Jain, Chia-Heng Hsiung, Gang Ning, Yu Liu, Xiaosong Li, and Xin Zhang

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 142, Pages: 17515-17523, Published: 14 October 2020 American Chemical Society (ACS)
    Aberrantly processed or mutant proteins misfold and assemble into a variety of soluble oligomers and insoluble aggregates, a process that is associated with an increasing number of diseases that are not curable or manageable. Herein, we present a chemical toolbox, AggFluor, that allows for live cell imaging and differentiation of complex aggregated conformations in live cells. Based on the chromophore core of green fluorescent proteins, AggFluor is comprised of a series of molecular rotor fluorophores that span a wide range of viscosity sensitivity. As a result, these compounds exhibit differential turn-on fluorescence when incorporated in either soluble oligomers or insoluble aggregates. This feature allows us to develop, for the first time, a dual-color imaging strategy to distinguish unfolded protein oligomers from insoluble aggregates in live cells. Furthermore, we have demonstrated how small molecule proteostasis regulators can drive formation and disassembly of protein aggregates in both conformational states. In summary, AggFluor is the first set of rationally designed molecular rotor fluorophores that evenly cover a wide range of viscosity sensitivities. This set of fluorescent probes not only change the status quo of current imaging methods to visualize protein aggregation in live cells, but also can be generally applied to study other biological processes that involve local viscosity changes with temporal and spatial resolutions.

  • Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies
    Conner A. Hoelzel and Xin Zhang

    ChemBioChem, ISSN: 14394227, eISSN: 14397633, Pages: 1935-1946, Published: 16 July 2020 Wiley
    Visualizing and manipulating the behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labeling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self‐labeling protein tags, focusing mainly on the application of HaloTag and SNAP‐tag systems. We describe the latest developments in small‐molecule probes that enable fluorogenic (no wash) imaging and super‐resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, current technical advances in the HaloTag and SNAP‐tag systems means that they are becoming powerful tools to enable the visualization and manipulation of biological processes, providing invaluable scientific insights that are difficult to obtain by traditional methodologies. As the multiplex of self‐labeling protein tag systems continues to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.

  • Fluorogenic detection of protein aggregates in live cells using the AggTag method
    Kwan Ho Jung and Xin Zhang

    Methods in Enzymology, ISSN: 00766879, eISSN: 15577988, Volume: 639, Pages: 1-22, Published: 2020 Elsevier
    Protein aggregation is a process that occurs through the self-assembly of misfolded proteins to form soluble oligomers and insoluble aggregates. While there has been significant interest in protein aggregation for neurodegenerative diseases, progress in this field of research has been limited by the lack of effective methods to detect and interrogate these species in live cells. To resolve this issue, we have developed a new imaging method named the AggTag to report on protein aggregation in live cells with fluorescence microscopy. The AggTag method utilizes a genetic fusion of a protein of interest (POI) to a protein tag to conjugate with the AggTag probe, which contains a fluorophore that turns on its fluorescence upon interaction with protein aggregates. Unlike the conventional methods, this method enables one to detect soluble misfolded oligomers that were previously invisible. Furthermore, the AggTag method has been applied for the simultaneous detection of co-aggregation between two different POIs by a dual-color and orthogonal tagging system. This chapter aims to provide step-by-step procedures of the AggTag method for researchers who intend to study aggregation of POIs in mammalian cell lines.

  • Super-Resolution Optical Lithography with DNA
    Shi Ho Kim, Yu Liu, Conner Hoelzel, Xin Zhang, and Tae-Hee Lee

    Nano Letters, ISSN: 15306984, eISSN: 15306992, Pages: 6035-6042, Published: 11 September 2019 American Chemical Society (ACS)
    We developed an efficient, versatile, and accessible super-resolution microscopy method to construct a nanoparticle assembly at a spatial resolution below the optical diffraction limit. The method utilizes DNA and a photo-activated DNA crosslinker. Super-resolution optical techniques have been used only as a means to make measurements below the light diffraction limit. Furthermore, no optical technique is currently available to construct nanoparticle assemblies with a precisely designed shape and internal structure at a resolution of a few tens of nanometers (nm). Here we demonstrate that we can fulfill this deficiency by utilizing spontaneous structural dynamics of DNA hairpins combined with single-molecule fluorescence resonance energy transfer (smFRET) microscopy and a photo-activated DNA crosslinker. The stochastic fluorescence blinking due to the spontaneous folding and unfolding motions of DNA hairpins enables us to precisely localize a folded hairpin and solidify it only when it is within a pre-designed target area whose size is below the diffraction limit. As the method is based on an optical microscope and an easily clickable DNA crosslinking reagent, it will provide an efficient means to create large nanoparticle assemblies with a shape and internal structure at an optical super-resolution, opening a wide window of opportunities toward investigating their photophysical and optoelectronic properties and developing novel devices.

  • A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells
    Kwan Ho Jung, Sojung F. Kim, Yu Liu, and Xin Zhang

    ChemBioChem, ISSN: 14394227, eISSN: 14397633, Pages: 1078-1087, Published: 15 April 2019 Wiley
    Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher‐order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo‐tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo‐tag‐based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP‐tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe—P2, based on a SNAP‐tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP‐tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.

  • Monitoring proteome stress in live cells using halotag-based fluorogenic sensor
    Yu Liu, Matthew Fares, and Xin Zhang

    Methods in Molecular Biology, ISSN: 10643745, Volume: 1873, Pages: 171-182, Published: 2019 Springer New York
    Fluorescent folding sensor is a powerful tool to detect proteome stresses, including heat, osmotic, oxidative, and drug induced stresses. Monitoring proteome stress using these sensors allows us to dissect the mechanism of cellular stress and find therapeutics that ameliorate stress related diseases. Here we present a HaloTag-based fluorogenic proteome stress sensor (AgHalo) to robustly detect and quantify proteome stresses in live cells. We describe how proteome stresses are monitored in both bacterial and mammalian live cells using fluorescence confocal microscope and fluorescence plate reader.

  • A HaloTag-Based Multicolor Fluorogenic Sensor Visualizes and Quantifies Proteome Stress in Live Cells Using Solvatochromic and Molecular Rotor-Based Fluorophores
    Yu Liu, Kun Miao, Yinghao Li, Matthew Fares, Shuyuan Chen, and Xin Zhang

    Biochemistry, ISSN: 00062960, eISSN: 15204995, Pages: 4663-4674, Published: 7 August 2018 American Chemical Society (ACS)
    Protein homeostasis, or proteostasis, is essential for cellular fitness and viability. Many environmental factors compromise proteostasis, induce global proteome stress, and cause diseases. The proteome stress sensor is a powerful tool for dissecting the mechanism of cellular stress and finding therapeutics that ameliorate these diseases. In this work, we present a multicolor HaloTag-based sensor (named AgHalo) to visualize and quantify proteome stresses in live cells. The current AgHalo sensor is equipped with three fluorogenic probes that turn on fluorescence when the sensor forms either soluble oligomers or insoluble aggregates upon exposure to stress conditions, both in vitro and in cellulo. In addition, AgHalo probes can be combined with commercially available always-fluorescent HaloTag ligands to enable two-color imaging, allowing for direct visualization of the AgHalo sensor both before and after cells are subjected to stress conditions. Finally, pulse-chase experiments can be performed to discern changes in the cellular proteome in live cells by first forming the AgHalo conjugate and then either applying or removing stress at any desired time point. In summary, the AgHalo sensor can be used to visualize and quantify proteome stress in live cells, a task that is difficult to accomplish using previous always-fluorescent methods. This sensor should be suited to evaluating cellular proteostasis under various exogenous stresses, including chemical toxins, drugs, and environmental factors.

  • NMR Measurements Reveal the Structural Basis of Transthyretin Destabilization by Pathogenic Mutations
    Benjamin I. Leach, Xin Zhang, Jeffery W. Kelly, H. Jane Dyson, and Peter E. Wright

    Biochemistry, ISSN: 00062960, eISSN: 15204995, Pages: 4421-4430, Published: 31 July 2018 American Chemical Society (ACS)
    Inherited mutations of transthyretin (TTR) destabilize its structure, leading to aggregation and familial amyloid disease. Although numerous crystal structures of wild-type (WT) and mutant TTRs have been determined, they have failed to yield a comprehensive structural explanation for destabilization by pathogenic mutations. To identify structural and dynamic variations that are not readily observed in the crystal structures, we used NMR to study WT TTR and three kinetically and/or thermodynamically destabilized pathogenic variants (V30M, L55P, and V122I). Sequence-corrected chemical shifts reveal important structural differences between WT and mutant TTR. The L55P mutation linked to aggressive early onset cardiomyopathy and polyneuropathy induces substantial structural perturbations in both the DAGH and CBEF β-sheets, whereas the V30M polyneuropathy-linked substitution perturbs primarily the CBEF sheet. In both variants, the structural perturbations propagate across the entire width of the β-sheets from the site of mutation. Structural changes caused by the V122I cardiomyopathy-associated mutation are restricted to the immediate vicinity of the mutation site, directly perturbing the subunit interfaces. NMR relaxation dispersion measurements show that WT TTR and the three pathogenic variants undergo millisecond time scale conformational fluctuations to populate a common excited state with an altered structure in the subunit interfaces. The excited state is most highly populated in L55P. The combined application of chemical shift analysis and relaxation dispersion to these pathogenic variants reveals differences in ground state structure and in the population of a transient excited state that potentially facilitates tetramer dissociation, providing new insights into the molecular mechanism by which mutations promote TTR amyloidosis.

  • Modulation of Fluorescent Protein Chromophores to Detect Protein Aggregation with Turn-On Fluorescence
    Yu Liu, Charles H. Wolstenholme, Gregory C. Carter, Hongbin Liu, Hang Hu, Leeann S. Grainger, Kun Miao, Matthew Fares, Conner A. Hoelzel, Hemant P. Yennawar, Gang Ning, Manyu Du, Lu Bai, Xiaosong Li, and Xin Zhang

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 140, Pages: 7381-7384, Published: 20 June 2018 American Chemical Society (ACS)
    We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

  • Role of HSP90 in the Regulation of de Novo Purine Biosynthesis
    Anthony M. Pedley, Georgios I. Karras, Xin Zhang, Susan Lindquist, and Stephen J. Benkovic

    Biochemistry, ISSN: 00062960, eISSN: 15204995, Pages: 3217-3221, Published: 12 June 2018 American Chemical Society (ACS)
    Despite purines making up one of the largest classes of metabolites in a cell, little is known about the regulatory mechanisms that facilitate efficient purine production. Under conditions resulting in high purine demand, enzymes within the de novo purine biosynthetic pathway cluster into multienzyme assemblies called purinosomes. Purinosome formation has been linked to molecular chaperones HSP70 and HSP90; however, the involvement of these molecular chaperones in purinosome formation remains largely unknown. Here, we present a new-found biochemical mechanism for the regulation of de novo purine biosynthetic enzymes mediated through HSP90. HSP90-client protein interaction assays were employed to identify two enzymes within the de novo purine biosynthetic pathway, PPAT and FGAMS, as client proteins of HSP90. Inhibition of HSP90 by STA9090 abrogated these interactions and resulted in a decrease in the level of available soluble client protein while having no significant effect on their interactions with HSP70. These findings provide a mechanism to explain the dependence of purinosome assembly on HSP90 activity. The combined efforts of molecular chaperones in the maturation of PPAT and FGAMS result in purinosome formation and are likely essential for enhancing the rate of purine production to meet intracellular purine demand.

  • Carbon nitride nanothread crystals derived from pyridine
    Xiang Li, Tao Wang, Pu Duan, Maria Baldini, Haw-Tyng Huang, Bo Chen, Stephen J. Juhl, Daniel Koeplinger, Vincent H. Crespi, Klaus Schmidt-Rohr, Roald Hoffmann, Nasim Alem, Malcolm Guthrie, Xin Zhang, and John V. Badding

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 140, Pages: 4969-4972, Published: 18 April 2018 American Chemical Society (ACS)
    Carbon nanothreads are a new one-dimensional sp3 carbon nanomaterial. They assemble into hexagonal crystals in a room temperature, nontopochemical solid-state reaction induced by slow compression of benzene to 23 GPa. Here we show that pyridine also reacts under compression to form a well-ordered sp3 product: C5NH5 carbon nitride nanothreads. Solid pyridine has a different crystal structure from solid benzene, so the nontopochemical formation of low-dimensional crystalline solids by slow compression of small aromatics may be a general phenomenon that enables chemical design of properties. The nitrogen in the carbon nitride nanothreads may improve processability, alters photoluminescence, and is predicted to reduce the bandgap.

  • Heat Shock Protein Reports on Proteome Stress
    Yu Liu and Xin Zhang

    Biotechnology Journal, ISSN: 18606768, eISSN: 18607314, Published: April 2018 Wiley
    Proper regulation of protein homeostasis (proteostasis) is essential to maintain cellular fitness. Proteome stress causes imbalance of the proteostasis, leading to various diseases represented by neurodegenerative diseases, cancers, and metabolic disorders. The biosensor community recently embarked on the development of proteome stress sensors to report on the integrity of proteostasis in live cells. While most of these sensors are based on metastable mutants of specific client proteins, a recent sensor takes advantage of the specific association of heat shock protein 27 with protein aggregates and exhibits a diffusive to punctate fluorescent change in cells that are subjected to stress conditions. Thus, heat shock proteins can be also used as a family of sensors to monitor proteome stress.

  • A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome
    Matthew Fares, Yinghao Li, Yu Liu, Kun Miao, Zi Gao, Yufeng Zhai, and Xin Zhang

    Bioconjugate Chemistry, ISSN: 10431802, eISSN: 15204812, Pages: 215-224, Published: 17 January 2018 American Chemical Society (ACS)
    Cellular stress leads to disruption of protein homeostasis (proteostasis) that is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced proteostasis deficiency remains one of the major technical challenges facing established sensors of this process. Available sensors use solvatochromic fluorophores to detect protein aggregation in forms of soluble oligomers or insoluble aggregates when cells are subjected to severe stress conditions. Misfolded monomers induced by mild stresses, however, remain largely invisible to these sensors. Here, we describe a fluorogenic proteome stress sensor by conjugating a fluorescent molecular rotor with a metastable Halo-tag protein domain that contains a K73T mutation (named AgHalo hereinafter). In nonstressed cells, the AaHalo sensor remains largely folded and the AgHalo•ligand conjugate is fluorescent dark in the folded state. Under various stress conditions, the AgHalo sensor has been established to form both soluble and insoluble aggregates along with metastable proteins of the endogenous cellular proteome. Thus, the AgHalo•ligand conjugate fluoresces strongly when the sensor forms misfolded monomers (a 16-fold increase) or aggregates in both soluble and insoluble forms (a 20-fold increase). Compared to the solvatochromic fluorophore-based sensor, we demonstrate that the molecular rotor-based sensor not only is more effective in detecting mild proteome stress that induces primarily misfolding conformations, but also exhibits a higher fluorescence signal in detecting more severe proteome stress that involves protein aggregates. Thus, the conjugation of a fluorescent molecular rotor to AgHalo further improves the capacity of this sensor to detect conditions of proteome stress. This work highlights the utility of molecular rotor-based fluorophores in direct visualization of the protein aggregation cascade in live cells, providing new methodologies for real-time analyses of cellular proteostasis upon exposure to different types of stress conditions.

  • The Cation−π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification
    Yu Liu, Kun Miao, Noah P. Dunham, Hongbin Liu, Matthew Fares, Amie K. Boal, Xiaosong Li, and Xin Zhang

    Biochemistry, ISSN: 00062960, eISSN: 15204995, Pages: 1585-1595, Published: 21 March 2017 American Chemical Society (ACS)
    The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a "channel dye" into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation-π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation-π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation-π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.

  • AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress
    Yu Liu, Matthew Fares, Noah P. Dunham, Zi Gao, Kun Miao, Xueyuan Jiang, Samuel S. Bollinger, Amie K. Boal, and Xin Zhang

    Angewandte Chemie - International Edition, ISSN: 14337851, eISSN: 15213773, Pages: 8672-8676, Published: 2017 Wiley
    Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.

  • Fluorescence Turn-On Folding Sensor to Monitor Proteome Stress in Live Cells
    Yu Liu, Xin Zhang, Wentao Chen, Yun Lei Tan, and Jeffery W. Kelly

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 137, Pages: 11303-11311, Published: 9 September 2015 American Chemical Society (ACS)
    Proteome misfolding and/or aggregation, caused by a thermal perturbation or a related stress, transiently challenges the cellular protein homeostasis (proteostasis) network capacity of cells by consuming chaperone/chaperonin pathway and degradation pathway capacity. Developing protein client-based probes to quantify the cellular proteostasis network capacity in real time is highly desirable. Herein we introduce a small-molecule-regulated fluorescent protein folding sensor based on a thermo-labile mutant of the de novo designed retroaldolase (RA) enzyme. Since RA enzyme activity is not present in any cell, the protein folding sensor is bioorthogonal. The fluorogenic small molecule was designed to become fluorescent when it binds to and covalently reacts with folded and functional RA. Thus, in the first experimental paradigm, cellular proteostasis network capacity and its dynamics are reflected by RA–small molecule conjugate fluorescence, which correlates with the amount of folded and functional RA present, provided that pharmacologic chaperoning is minimized. In the second experimental scenario, the RA–fluorogenic probe conjugate is pre-formed in a cell by simply adding the fluorogenic probe to the cell culture media. Unreacted probe is then washed away before a proteome misfolding stress is applied in a pulse-chase-type experiment. Insufficient proteostasis network capacity is reflected by aggregate formation of the fluorescent RA–fluorogenic probe conjugate. Removal of the stress results in apparent RA–fluorogenic probe conjugate re-folding, mediated in part by the heat-shock response transcriptional program augmenting cytosolic proteostasis network capacity, and in part by time-dependent RA–fluorogenic probe conjugate degradation by cellular proteolysis.

  • Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli
    Younhee Cho, Xin Zhang, Kristine Faye R. Pobre, Yu Liu, David L. Powers, Jeffery W. Kelly, Lila M. Gierasch, and Evan T. Powers

    Cell Reports, eISSN: 22111247, Pages: 321-333, Published: 2015 Elsevier BV
    The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon.

  • De Novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters
    Yu Liu, Xin Zhang, Yun Lei Tan, Gira Bhabha, Damian C. Ekiert, Yakov Kipnis, Sinisa Bjelic, David Baker, and Jeffery W. Kelly

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 136, Pages: 13102-13105, Published: 24 September 2014 American Chemical Society (ACS)
    Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme–small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.

  • Heat-shock response transcriptional program enables high-yield and high-quality recombinant protein production in Escherichia coli
    Xin Zhang, Yu Liu, Joseph C. Genereux, Chandler Nolan, Meha Singh, and Jeffery W. Kelly

    ACS Chemical Biology, ISSN: 15548929, eISSN: 15548937, Pages: 1945-1949, Published: 19 September 2014 American Chemical Society (ACS)
    The biosynthesis of soluble, properly folded recombinant proteins in large quantities from Escherichia coli is desirable for academic research and industrial protein production. The basal E. coli protein homeostasis (proteostasis) network capacity is often insufficient to efficiently fold overexpressed proteins. Herein we demonstrate that a transcriptionally reprogrammed E. coli proteostasis network is generally superior for producing soluble, folded, and functional recombinant proteins. Reprogramming is accomplished by overexpressing a negative feedback deficient heat-shock response transcription factor before and during overexpression of the protein-of-interest. The advantage of transcriptional reprogramming versus simply overexpressing select proteostasis network components (e.g., chaperones and co-chaperones, which has been explored previously) is that a large number of proteostasis network components are upregulated at their evolved stoichiometry, thus maintaining the system capabilities of the proteostasis network that are currently incompletely understood. Transcriptional proteostasis network reprogramming mediated by stress-responsive signaling in the absence of stress should also be useful for protein production in other cells.

  • Chaperonins resculpt folding free energy landscapes to avoid kinetic traps and accelerate protein folding
    Xin Zhang and Jeffery W. Kelly

    Journal of Molecular Biology, ISSN: 00222836, eISSN: 10898638, Volume: 426, Pages: 2736-2738, Published: 29 July 2014 Elsevier BV

  • Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts
    Y. Liu, Y. L. Tan, X. Zhang, G. Bhabha, D. C. Ekiert, J. C. Genereux, Y. Cho, Y. Kipnis, S. Bjelic, D. Baker, and J. W. Kelly

    Proceedings of the National Academy of Sciences of the United States of America, ISSN: 00278424, eISSN: 10916490, Volume: 111, Pages: 4449-4454, Published: 25 March 2014 Proceedings of the National Academy of Sciences
    Significance Historically, the folding of individual proteins in buffers has been studied spectroscopically. The majority of spectroscopic methods (NMR and fluorescence excluded) cannot be used in a cell, because the protein of interest (POI) cannot be distinguished from the background proteome. Herein, we introduce folding probes, which when used in cell lysates with sufficient holdase activity, faithfully quantify the folded and functional fraction of a POI at a time point of interest in a cell by selectively reacting with that state to afford a fluorescent signal. This work provides a blueprint for how to convert enzyme inhibitors, ligands for nonenzyme proteins, etc. into folding probes to efficiently and specifically investigate how intracellular function is controlled by the proteostasis network as a function of cellular perturbations. Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein’s functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.

  • Fidelity of cotranslational protein targeting by the signal recognition particle
    Xin Zhang and Shu-ou Shan

    Annual Review of Biophysics, ISSN: 1936122X, eISSN: 19361238, Pages: 381-408, Published: May 2014 Annual Reviews
    Accurate folding, assembly, localization, and maturation of newly synthesized proteins are essential to all cells and require high fidelity in the protein biogenesis machineries that mediate these processes. Here, we review our current understanding of how high fidelity is achieved in one of these processes, the cotranslational targeting of nascent membrane and secretory proteins by the signal recognition particle (SRP). Recent biochemical, biophysical, and structural studies have elucidated how the correct substrates drive a series of elaborate conformational rearrangements in the SRP and SRP receptor GTPases; these rearrangements provide effective fidelity checkpoints to reject incorrect substrates and enhance the fidelity of this essential cellular pathway. The mechanisms used by SRP to ensure fidelity share important conceptual analogies with those used by cellular machineries involved in DNA replication, transcription, and translation, and these mechanisms likely represent general principles for other complex cellular pathways.

  • Mechanisms of transthyretin inhibition of β-amyloid aggregation in vitro
    X. Li, X. Zhang, A. R. A. Ladiwala, D. Du, J. K. Yadav, P. M. Tessier, P. E. Wright, J. W. Kelly, and J. N. Buxbaum

    Journal of Neuroscience, ISSN: 02706474, eISSN: 15292401, Pages: 19423-19433, Published: 2013 Society for Neuroscience
    Tissue-specific overexpression of the human systemic amyloid precursor transthyretin (TTR) ameliorates Alzheimer's disease (AD) phenotypes in APP23 mice. TTR–β-amyloid (Aβ) complexes have been isolated from APP23 and some human AD brains. We now show that substoichiometric concentrations of TTR tetramers suppress Aβ aggregation in vitro via an interaction between the thyroxine binding pocket of the TTR tetramer and Aβ residues 18–21 (nuclear magnetic resonance and epitope mapping). The KD is micromolar, and the stoichiometry is <1 for the interaction (isothermal titration calorimetry). Similar experiments show that engineered monomeric TTR, the best inhibitor of Aβ fibril formation in vitro, did not bind Aβ monomers in liquid phase, suggesting that inhibition of fibrillogenesis is mediated by TTR tetramer binding to Aβ monomer and both tetramer and monomer binding of Aβ oligomers. The thousand-fold greater concentration of tetramer relative to monomer in vivo makes it the likely suppressor of Aβ aggregation and disease in the APP23 mice.

  • Bio-inspired protein-gold nanoconstruct with core-void-shell structure: Beyond a chemo drug carrier
    Xiangyou Liu, Wei Wei, Shijiao Huang, Shrong-Shi Lin, Xin Zhang, Chuanmao Zhang, Yuguang Du, Guanghui Ma, Mei Li, Stephen Mann, and Ding Ma

    Journal of Materials Chemistry B, ISSN: 20507518, eISSN: 2050750X, Pages: 3136-3143, Published: 7 July 2013 Royal Society of Chemistry (RSC)
    Chemotherapy has been widely used in clinical practice for cancer treatment. A major challenge for a successful chemotherapy is to potentiate the anticancer activity, whilst reducing the severe side effects. In this context, we design a bio-inspired protein-gold nanoconstruct (denoted as AFt-Au hereafter) with a core-void-shell structure which exhibits a high selectivity towards carcinoma cells. Anticancer drug 5-fluorouracil (5-FU) can be sequestered into the void space of the construct to produce an integrated nanoscale hybrid AFt-AuFU that exhibits an increased cellular uptake of 5-FU. More importantly, AFt-Au, serving as a bio-nano-chemosensitizer, renders carcinoma cells more susceptible to 5-FU by cell-cycle regulation, and thus, leads to a dramatic decrease of the IC50 value (i.e. the drug concentration required to kill 50% of the cell population) of 5-FU in HepG2 cells from 138.3 μM to 9.2 μM. Besides HepG2 cells, a remarkably enhanced anticancer efficacy and potentially reduced side effects are also achieved in other cell lines. Our further work reveals that the drug 5-FU is internalized into cells with AFt-Au primarily via receptor-mediated endocytosis (RME). After internalization, AFt-AuFU colocalizes with lysosomes which trigger the release of 5-FU under acidic conditions. Overall, our approach provides a novel procedure in nanoscience that promises an optimal chemotherapeutic outcome.

  • Signal recognition particle: An essential protein-targeting machine
    David Akopian, Kuang Shen, Xin Zhang, and Shu-ou Shan

    Annual Review of Biochemistry, ISSN: 00664154, eISSN: 15454509, Pages: 693-721, Published: June 2013 Annual Reviews
    The signal recognition particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.


  • A General Strategy to Control Viscosity Sensitivity of Molecular Rotor‐Based Fluorophores
    S Ye, H Zhang, J Fei, CH Wolstenholme, X Zhang
    Angewandte Chemie International Edition 2020

  • AggFluor: Fluorogenic Toolbox Enables Direct Visualization of the Multi-Step Protein Aggregation Process in Live Cells
    CH Wolstenholme, H Hu, S Ye, BE Funk, D Jain, CH Hsiung, G Ning, ...
    Journal of the American Chemical Society 142 (41), 17515-17523 2020

  • A General Strategy to Enhance Donor‐Acceptor Molecules Using Solvent‐Excluding Substituents
    CA Hoelzel, H Hu, CH Wolstenholme, BA Karim, KT Munson, KH Jung, ...
    Angewandte Chemie International Edition 59 (12), 4785-4792 2020

  • Visualizing and Manipulating Biological Processes Using HaloTag and SNAP‐Tag Technologies
    CA Hoelzel, X Zhang
    ChemBioChem 2020

  • A Fluorogenic AggTag Method Based on Halo‐ and SNAP‐Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells
    KH Jung, SF Kim, Y Liu, X Zhang
    ChemBioChem 20 (8), 1078-1087 2019

  • Detecting protein aggregation in live cells with turn-on fluorescence using chemically modulated fluorescent protein chromophores
    X Zhang, Y Liu, C Wolstenholme

  • A SNAP-tag fluorogenic probe mimicking the chromophore of the red fluorescent protein Kaede
    KH Jung, M Fares, LS Grainger, CH Wolstenholme, A Hou, Y Liu, X Zhang
    Organic & biomolecular chemistry 17 (7), 1906-1915 2019

  • Inverted solvatochromic Stokes shift in GFP-like chromophores with extended conjugation
    H Hu, CH Wolstenholme, X Zhang, X Li
    Chinese Journal of Chemical Physics 31 (4), 599 2018

  • 具有延长共轭的绿色发光蛋白生色团的反向溶剂化斯托克斯 (Stokes) 位移
    胡航, 张鑫, 李晓松
    化学物理学报 31 (4), 599-607 2018

  • Modulation of fluorescent protein chromophores to detect protein aggregation with turn-on fluorescence
    Y Liu, CH Wolstenholme, GC Carter, H Liu, H Hu, LS Grainger, K Miao, ...
    Journal of the American Chemical Society 140 (24), 7381-7384 2018

  • Carbon nitride nanothread crystals derived from pyridine
    X Li, T Wang, P Duan, M Baldini, HT Huang, B Chen, SJ Juhl, ...
    Journal of the American Chemical Society 140 (15), 4969-4972 2018

  • A HaloTag-based multicolor fluorogenic sensor visualizes and quantifies proteome stress in live cells using solvatochromic and molecular rotor-based fluorophores
    Y Liu, K Miao, Y Li, M Fares, S Chen, X Zhang
    Biochemistry 57 (31), 4663-4674 2018

  • A molecular rotor-based Halo-Tag ligand enables a fluorogenic proteome stress sensor to detect protein misfolding in mildly stressed proteome
    M Fares, Y Li, Y Liu, K Miao, Z Gao, Y Zhai, X Zhang
    Bioconjugate chemistry 29 (1), 215-224 2018

  • AgHalo: A Facile Fluorogenic Sensor to Detect Drug‐Induced Proteome Stress
    Y Liu, M Fares, NP Dunham, Z Gao, K Miao, X Jiang, SS Bollinger, ...
    Angewandte Chemie 129 (30), 8798-8802 2017

  • The cation− π interaction enables a halo-tag fluorogenic probe for fast no-wash live cell imaging and gel-free protein quantification
    Y Liu, K Miao, NP Dunham, H Liu, M Fares, AK Boal, X Li, X Zhang
    Biochemistry 56 (11), 1585-1595 2017

  • Fluorescence turn-on folding sensor to monitor proteome stress in live cells
    Y Liu, X Zhang, W Chen, YL Tan, JW Kelly
    Journal of the American Chemical Society 137 (35), 11303-11311 2015

  • Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E.coli
    Y Cho, X Zhang, KFR Pobre, Y Liu, DL Powers, JW Kelly, LM Gierasch, ...
    Cell reports 11 (2), 321-333 2015

  • De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters
    Y Liu, X Zhang, YL Tan, G Bhabha, DC Ekiert, Y Kipnis, S Bjelic, D Baker, ...
    Journal of the American Chemical Society 136 (38), 13102-13105 2014

  • Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli
    X Zhang, Y Liu, JC Genereux, C Nolan, M Singh, JW Kelly
    ACS chemical biology 9 (9), 1945-1949 2014

  • Chaperonins resculpt folding free energy landscapes to avoid kinetic traps and accelerate protein folding.
    X Zhang, JW Kelly
    Journal of molecular biology 426 (15), 2736 2014


  • Signal recognition particle: an essential protein-targeting machine
    D Akopian, K Shen, X Zhang, S Shan
    Annual review of biochemistry 82, 693-721 2013
    Citations: 291

  • High‐order symplectic integration in quasi‐classical trajectory simulation: Case study for O(1D) + H2
    X Zhang, KL Han
    International journal of quantum chemistry 106 (8), 1815-1819 2006
    Citations: 148

  • Multiple conformational switches in a GTPase complex control co-translational protein targeting
    X Zhang, C Schaffitzel, N Ban, S Shan
    Proceedings of the National Academy of Sciences 106 (6), 1754-1759 2009
    Citations: 115

  • A quantum wave-packet study of intersystem crossing effects in the reaction
    TS Chu, X Zhang, KL Han
    The Journal of chemical physics 122 (21), 214301 2005
    Citations: 107

  • Sequential checkpoints govern substrate selection during cotranslational protein targeting
    X Zhang, R Rashid, K Wang, S Shan
    Science 328 (5979), 757-760 2010
    Citations: 99

  • Mechanisms of transthyretin inhibition of β-amyloid aggregation in vitro
    X Li, X Zhang, ARA Ladiwala, D Du, JK Yadav, PM Tessier, PE Wright, ...
    Journal of Neuroscience 33 (50), 19423-19433 2013
    Citations: 92

  • Demonstration of a multistep mechanism for assembly of the SRP SRP receptor complex: Implications for the catalytic role of SRP RNA
    X Zhang, S Kung, S Shan
    Journal of molecular biology 381 (3), 581-593 2008
    Citations: 84

  • Time-dependent quantum wave packet calculation for nonadiabatic reaction
    Y Zhang, TX Xie, KL Han, JZH Zhang
    The Journal of chemical physics 119 (24), 12921-12925 2003
    Citations: 78

  • Donor/acceptor vinyl monomers and their polymers: synthesis, photochemical and photophysical behavior
    X Zhang, ZC Li, KB Li, S Lin, FS Du, FM Li
    Progress in polymer science 31 (10), 893-948 2006
    Citations: 64

  • Synthesis of bismaleimides bearing electron-donating chromophores and their fluorescence behavior during copolymerization
    X Zhang, YH Jin, HX Diao, FS Du, ZC Li, FM Li
    Macromolecules 36 (9), 3115-3127 2003
    Citations: 54

  • Fidelity of cotranslational protein targeting by the signal recognition particle
    X Zhang, S Shan
    Annual review of biophysics 43, 381-408 2014
    Citations: 50

  • MRCI potential curve and analytical potential energy function of the ground state of H2
    CL Yang, YJ Huang, X Zhang, KL Han
    Journal of Molecular Structure: THEOCHEM 625 (1-3), 289-293 2003
    Citations: 49

  • Modulation of fluorescent protein chromophores to detect protein aggregation with turn-on fluorescence
    Y Liu, CH Wolstenholme, GC Carter, H Liu, H Hu, LS Grainger, K Miao, ...
    Journal of the American Chemical Society 140 (24), 7381-7384 2018
    Citations: 45

  • Molecular dynamics simulation reveals preorganization of the chloroplast FtsY towards complex formation induced by GTP binding
    MJ Yang, XQ Pang, X Zhang, KL Han
    Journal of structural biology 173 (1), 57-66 2011
    Citations: 38

  • Signal recognition particle (SRP) and SRP receptor: a new paradigm for multistate regulatory GTPases
    S Shan, SL Schmid, X Zhang
    Biochemistry 48 (29), 6696-6704 2009
    Citations: 38

  • AgHalo: A Facile Fluorogenic Sensor to Detect Drug‐Induced Proteome Stress
    Y Liu, M Fares, NP Dunham, Z Gao, K Miao, X Jiang, SS Bollinger, ...
    Angewandte Chemie 129 (30), 8798-8802 2017
    Citations: 36

  • Carbon nitride nanothread crystals derived from pyridine
    X Li, T Wang, P Duan, M Baldini, HT Huang, B Chen, SJ Juhl, ...
    Journal of the American Chemical Society 140 (15), 4969-4972 2018
    Citations: 35

  • Analytical potential energy function and spectroscopic parameters for the ground and excited states of NaH
    CL Yang, X Zhang, KL Han
    Journal of Molecular Structure: THEOCHEM 676 (1-3), 209-213 2004
    Citations: 35

  • The cation− π interaction enables a halo-tag fluorogenic probe for fast no-wash live cell imaging and gel-free protein quantification
    Y Liu, K Miao, NP Dunham, H Liu, M Fares, AK Boal, X Li, X Zhang
    Biochemistry 56 (11), 1585-1595 2017
    Citations: 34

  • Direct visualization reveals dynamics of a transient intermediate during protein assembly
    X Zhang
    Multistate GTPase Control Co-translational Protein Targeting, 27-48 2012
    Citations: 32