Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding

Llano-Sotelo, Beatriz; Hickerson, Robyn P.; Lancaster, Laura; Noller, Harry F.; Mankin, Alexander S.

doi:10.1261/rna.1681609

Cited by 25 publications

(24 citation statements)

References 59 publications

(77 reference statements)

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“…Streptomycin, also an antibiotic, inhibits protein synthesis by binding the S12 protein of the 30S subunit of the bacterial ribosome with a K D in the range of 1 μM (23-25). Streptomycin was among the top 20 assay positives for protein S836, a mediocre binder for S824, and non-binder to protein S23 (Table S1).…”

Section: Resultsmentioning

confidence: 99%

Proteins from an Unevolved Library of de novo Designed Sequences Bind a Range of Small Molecules

et al. 2012

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The availability of large collections of de novo designed proteins presents new opportunities to harness novel macromolecules for synthetic biological functions. Many of these new functions will require binding to small molecules. Is the ability to bind small molecules a property that arises only in response to biological selection or computational design? Or alternatively, is small molecule binding a property of folded proteins that occurs readily amidst collections of unevolved sequences? These questions can be addressed by assessing the binding potential of de novo proteins that are designed to fold into stable structures, but are “naïve” in the sense that they (i) share no significant sequence similarity with natural proteins, and (ii) were neither selected nor designed to bind small molecules. We chose three naïve proteins from a library of sequences designed to fold into 4-helix bundles, and screened for binding to 10,000 compounds displayed on small molecule microarrays. Several binders were identified, and binding was characterized by a series of biophysical assays. Surprisingly, despite the similarity of the three de novo proteins to one another, they exhibit selective ligand binding. These findings demonstrate the potential of novel proteins for molecular recognition, and have significant implications for a range of applications in synthetic biology.

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Section: Resultsmentioning

confidence: 99%

Proteins from an Unevolved Library of de novo Designed Sequences Bind a Range of Small Molecules

et al. 2012

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“…To further probe their mechanism of action, different approaches have been developed and employed in the past decades, including fluorescence-based assays, 3, 53–56 computational simulations, 57, 58 microarray assays, 59–63 and X-ray crystallography. 4, 64, 65 As a result, AGs have been demonstrated to bind not only to the ribosomal decoding A-site on the 16S rRNA, causing miscoding in the nascent polypeptide, but also to the helix 69 (h69) in the large 50S ribosomal subunit, which is critical in the processes of mRNA/tRNA translocation and ribosome recycling.…”

Section: Aminoglycosides Mode Of Action: Binding To the Ribosomementioning

confidence: 99%

New trends in the use of aminoglycosides

Fosso

2014

Med. Chem. Commun.

100

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Despite their inherent toxicity and the acquired bacterial resistance that continuously threaten their long-term clinical use, aminoglycosides (AGs) still remain valuable components of the antibiotic armamentarium. Recent literature shows that the AGs’ role has been further expanded as multi-tasking players in different areas of study. This review aims at presenting some of the new trends observed in the use of AGs in the past decade, along with the current understanding of their mechanisms of action in various bacterial and eukaryotic cellular processes.

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“…Therefore, in order to investigate binding of the inhibitory peptide to its likely target, we used a recently developed method 25 which employs fluorescently-labeled 30S subunits. The ribosomal protein S12 was mutagenized to carry a single cysteine residue at position 44 and then conjugated with the environment-sensitive fluorophore 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS).…”

Section: Binding Of the Agaamsh Peptide To The 30s Subunitmentioning

confidence: 99%

Selection of Small Peptides, Inhibitors of Translation

Llano-Sotelo

Klepacki

Mankin

2009

Journal of Molecular Biology

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Identification of small molecular weight compounds targeting specific sites in the ribosome can accelerate development of new antibiotics and provide new tools for ribosomal research. We demonstrate here that antibiotic-size short peptides capable of inhibiting protein synthesis can be selected by using specific elements of ribosomal RNA as a target. The ‘h18’ pseudoknot encompassing residues 500-545 of the small ribosomal subunit RNA was used as a target in screening a heptapeptide phage display library. Two of the selected peptides could efficiently interfere with both bacterial and eukaryotic translation. One of these inhibitory peptides exhibited a high-affinity binding to the isolated small ribosomal subunit (Kd of 1.1 μM). Identification of inhibitory peptides which likely target a specific rRNA structure may pave new ways for validating new antibiotic sites in the ribosome. The selected peptides can be used as a tool in search of novel site-specific inhibitors of translation.

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Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding

Cited by 25 publications

References 59 publications

Proteins from an Unevolved Library of de novo Designed Sequences Bind a Range of Small Molecules

Proteins from an Unevolved Library of de novo Designed Sequences Bind a Range of Small Molecules

New trends in the use of aminoglycosides

Selection of Small Peptides, Inhibitors of Translation

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