In Vivo Imaging of a Bacterial Cell Division Protein Using a Protease‐Assisted Small‐Molecule Labeling Approach

Chattopadhaya, Souvik; Bakar, Farhana B. Abu; Srinivasan, Rajavel; Yao, Shao Q.

doi:10.1002/cbic.200700647

Cited by 4 publications

(3 citation statements)

References 40 publications

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“…Coiled coils can drive specific protein association in living cells but have limited stability (34). Native chemical ligation is an elegant way to link proteins (35,36) but specificity and yield appear to be limited in cells (36). Sortase can be used to link together two proteins in vitro or to link a peptide with a small molecule on the surface of living cells, but requires high [Ca 2þ ] (15), which is damaging in the cytosol or nucleus.…”

Section: Discussionmentioning

confidence: 99%

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Zakeri

Fierer

Çelik

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

1,261

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Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures. agging with peptides (e.g., HA, myc, FLAG, His 6 ) is one of the most common ways to detect, purify, or immobilize proteins (1-4). Peptides are very useful minimally disruptive probes (5) but they are also "slippery"-antibodies or other proteins typically bind peptides with low affinity and poor mechanical strength (6-9). We sought to form a rapid covalent bond to a peptide tag without the use of chemical modification, artificial amino acids, or cysteines (disulfide bond formation is reversible and restricted to particular cellular locations).It has recently been found that Streptococcus pyogenes, like many other Gram-positive bacteria, contains extracellular proteins stabilized by spontaneous intramolecular isopeptide bonds (10). Here we explored the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein FbaB, found in invasive strains of S. pyogenes (11,12) and essential for phagocytosis-like uptake of the bacteria by endothelial cells (13). CnaB2 contains a single isopeptide bond conferring exceptional stability: CnaB2 remains folded even at pH 2 or up to 100°C (12). By splitting CnaB2 into peptide and protein fragments, followed by rational modification of the parts, we developed a peptide tag of 13 amino acids that rapidly formed a covalent bond with its protein partner (138 amino acids, 15 kDa) and characterized the conditions for reaction, cellular specificity of bond formation, and resilience of the reacted product.

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Section: Discussionmentioning

confidence: 99%

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Zakeri

Fierer

Çelik

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

1,261

1,525

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“…Furthermore, fluorescent probes that can be efficiently controlled with wavelengths of light longer than the commonly used 405 nm will extend the time‐lapse, live‐cell imaging capability of localization‐based methods. Finally, labelling strategies involving unnatural amino acids (Plass et al ., ; ; Milles et al ., ) or protease cleavage (Chattopadhaya et al ., ), which entail single amino acid substitutions, may provide ways to perform superresolution imaging with minimal perturbations to protein function.…”

Section: Future Directionsmentioning

confidence: 99%

Superresolution microscopy for microbiology

2012

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Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments.

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“…As a bimolecular reaction the ligation requires high concentrations of the peptides/proteins (ideally millimolar or high micromolar), which can be difficult to achieve. Furthermore, because of the latter issue and the instability of the thioester moiety, NCL and EPL are rather unsuitable for chemical manipulation of proteins in living cells, although a few examples have been reported 29…”

Section: Biochemical Characteristics Of Inteinsmentioning

confidence: 99%