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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.
Highlights
Disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology
E. coli thioredoxin, fused to the GAL4 transcriptional activation domain (AD), served as the scaffold to constrain the octa-peptides of randomized sequence in an aptamer loop[13,14]
The thioredoxin scaffold[13], which was used as a negative control, did not show fluorescence quenching upon adding Pup
Summary
Disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology. 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 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-Guerin, BCG, strain of M. bovis This technology can be applied to study the elusive action of IDPs under near physiological conditions. CLIPs technology is an attractive molecular tool to analyze the functional consequences of nascent structures present in free IDPs due to the in vivo nature of target selection and comprehensive coverage of the potential binding surfaces
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