Shan Huang

All Publications

• Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids PROTEIN ENGINEERING DESIGN & SELECTION Haloi, N., Huang, S., Nichols, A. L., Fine, E. J., Friesenhahn, N. J., Marotta, C. B., Dougherty, D. A., Lindahl, E., Howard, R. J., Mayo, S. L., Lester, H. A. 2024; 37

  Abstract

  We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 μM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

  View details for DOI 10.1093/protein/gzae003

  View details for Web of Science ID 001173155400001

  View details for PubMedID 38302088

  View details for PubMedCentralID PMC10896302

• Genome manipulation by guide-directed Argonaute cleavage NUCLEIC ACIDS RESEARCH Huang, S., Wang, K., Mayo, S. L. 2023

  Abstract

  Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses. However, whether such a CbAgo-mediated DNA cleavage is mutagenic remains unexplored. Here we demonstrate that CbAgo, directed by plasmid-encoded guide sequences, can cleave genome target sites and induce chromosome recombination between downstream homologous sequences in Escherichia coli. The recombination rate correlates well with pAgo DNA cleavage activity and the mechanistic study suggests the recombination involves DSBs and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. We anticipate this novel DNA-guided interference to be useful in broader genetic manipulation. Our study also provides an in vivo assay to characterize or engineer pAgo DNA cleavage activity.

  View details for DOI 10.1093/nar/gkad188

  View details for Web of Science ID 000949298300001

  View details for PubMedID 36928676

• Tri(<i>o</i>-tolyl)phosphine for highly efficient Suzuki coupling of propargylic carbonates with boronic acids CHEMICAL COMMUNICATIONS Xiao, J., Luo, H., Huang, S., Qian, H., Ma, S. 2018; 54 (74): 10451-10454

  Abstract

  A highly efficient catalytic system consisting of Pd2(dba)3·CHCl3 and tri(o-tolyl)phosphine has been identified for the coupling of propargylic carbonates with different types of organo boronic acids at room temperature. Excellent central-to-axial chirality transfer was also demonstrated.

  View details for DOI 10.1039/c8cc04186e

  View details for Web of Science ID 000444483700012

  View details for PubMedID 30155547

• Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis SCIENCE Hammer, S. C., Kubik, G., Watkins, E., Huang, S., Minges, H., Arnold, F. H. 2017; 358 (6360): 215-+

  Abstract

  Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.

  View details for DOI 10.1126/science.aao1482

  View details for Web of Science ID 000412841500046

  View details for PubMedID 29026041