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.
Bibliographical noteFunding Information:
This work was supported by NIH grants GM085006 to A.S. and R01AI063499 to K.M. Naked Plasmid pVV16 for Expression in Mycobacterium smegmatis, NR-13402, was obtained through BEI Resources, NIAID, NIH.
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