Enzyme-targeted fluorescent small-molecule probes for bacterial imaging

Marshall, Andrew P.; Shirley, Joshua D.; Carlson, Erin E.

doi:10.1016/j.cbpa.2020.05.012

Cited by 27 publications

(16 citation statements)

References 60 publications

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“…While important, these experiments have limitations. For example, previous studies have demonstrated that fusion proteins can perturb a native protein's activity, expression, function, and localization [41][42][43][44]. In Yoshino et al, 2010 [29], Yoshino and Matsunaga, 2006 [31], Quinlan et al, 2011 [32] and Yoshino et al, 2021 [36] an inducible plasmid expression system was used in M. magneticum AMB-1 to overexpress comparatively large non-native proteins (i.e., GFP is~27 kDa; CD81 is~25 kDa; Luciferase is~61kDa; TSHR, thyroid-stimulating hormone receptor is~87 kDa) fused to the much small native Mms13 protein (i.e., Mms13 is~12 kDa).…”

Section: Discussionmentioning

confidence: 99%

“…In Yoshino et al, 2010 [29], Yoshino and Matsunaga, 2006 [31], Quinlan et al, 2011 [32] and Yoshino et al, 2021 [36] an inducible plasmid expression system was used in M. magneticum AMB-1 to overexpress comparatively large non-native proteins (i.e., GFP is~27 kDa; CD81 is~25 kDa; Luciferase is~61kDa; TSHR, thyroid-stimulating hormone receptor is~87 kDa) fused to the much small native Mms13 protein (i.e., Mms13 is~12 kDa). The considerably larger size of the non-native protein (e.g., GFP is twice as large as Mms13, Luciferase is five-times larger than Mms13, TSHR is seven-times larger than Mms13) has steric consequences for protein folding, function and targeting that could alter expression and/or localization of the native protein [41][42][43][44]. In addition, the resolution limit of conventional fluorescence microscopy is approximately 250 nm due to its dependance on the wavelength of the excitation light and microscope optics.…”

Section: Discussionmentioning

confidence: 99%

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Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

et al. 2021

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Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

et al. 2021

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“…For many systems, developing a general probe to a protein is not sufficient. [81] Here one could require a probe that targets a specific protein within a pathway. In such cases, the fact that the substrates are often very similar requires the probe design to specifically target a given enzymatic pocket.…”

Section: Target-guided Probe Discoverymentioning

confidence: 99%

Lessons in Organic Fluorescent Probe Discovery

et al. 2021

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Fluorescent probes have gained profound use in biotechnology, drug discovery, medical diagnostics, molecular and cell biology. The development of methods for the translation of fluorophores into fluorescent probes continues to be a robust field for medicinal chemists and chemical biologists, alike. Access to new experimental designs has enabled molecular diversification and led to the identification of new approaches to probe discovery. This review provides a synopsis of the recent lessons in modern fluorescent probe discovery.

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“…144 The electrophilic nature of the beta-lactam scaffold has also been used to image penicillin binding protein (PBPs) activities in pathogens. 145 Initial approaches were marred by a poor substrate specificity of these probes for the various PBPs. This was recently solved for another facultative intracellular pathogen, Streptococcus pneumoniae.…”

Section: Abpp Clem Of Bacterial Enzymesmentioning

confidence: 99%