title: Caught in action: selecting peptide aptamers against intrinsically disordered proteins in live cells.
abstract: Intrinsically disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology. Low cellular concentration, multiple binding partners, frequent post-translational modifications and the presence of multiple conformations make it difficult to characterize IDP interactions in intact cells. We used peptide aptamers selected by using the yeast-two-hybrid scheme and in-cell NMR to identify high affinity binders to transiently structured IDP and unstructured segments at atomic resolution. Since both the selection and characterization of peptide aptamers take place inside the cell, only physiologically relevant conformations of IDPs are targeted. The method is validated by using peptide aptamers selected against the prokaryotic ubiquitin-like protein, Pup, of the mycobacterium proteasome. The selected aptamers bind to distinct sites on Pup and have vastly different effects on rescuing mycobacterial proteasome substrate and on the survival of the Bacille-Calmette-Guèrin, BCG, strain of M. bovis. This technology can be applied to study the elusive action of IDPs under near physiological conditions.
authors: Department of Chemistry, University at Albany, Albany, NY.
authors: Jacqueline D
authors: JD
authors: Cobbert
authors: Department of Chemistry, University at Albany, Albany, NY.
authors: Christopher
authors: C
authors: DeMott
authors: Department of Chemistry, University at Albany, Albany, NY.
authors: Subhabrata
authors: S
authors: Majumder
authors: Wadsworth Center, NY State Department of Health, Albany, NY.
authors: Eric A
authors: EA
authors: Smith
authors: Department of Chemistry, University at Albany, Albany, NY.
authors: Sergey
authors: S
authors: Reverdatto
authors: Department of Chemistry, University at Albany, Albany, NY.
authors: David S
authors: DS
authors: Burz
authors: Wadsworth Center, NY State Department of Health, Albany, NY.
authors: Kathleen A
authors: KA
authors: McDonough
authors: Department of Chemistry, University at Albany, Albany, NY.
authors: Alexander
authors: A
authors: Shekhtman
mesh_terms: D000595:Amino Acid Sequence; D052158:Aptamers, Peptide / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D052158:Aptamers, Peptide; D001426:Bacterial Proteins; D064267:Intrinsically Disordered Proteins; C533288:Pup protein, Mycobacterium tuberculosis; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex