Challenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.

TitleChallenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.
Publication TypeJournal Article
Year of Publication2018
AuthorsBekesi, A., S. Abdellaoui, N. Holroyd, W. Van Delm, E. Pardon, J. Pauwels, K. Gevaert, J. Steyaert, S. Derveaux, A. Borysik, and P. Tompa
JournalMethods Enzymol
Volume611
Pagination607-675
Date Published2018
ISSN1557-7988
KeywordsAmino Acid Sequence, Animals, Camelids, New World, Cell Line, Chromatography, Affinity, Chromatography, Gel, Cloning, Molecular, CREB-Binding Protein, E1A-Associated p300 Protein, Humans, Immunization, Intrinsically Disordered Proteins, Protein Domains, Single-Domain Antibodies, Transfection
Abstract

The structural and functional characterization of large multidomain signaling proteins containing long disordered linker regions represents special methodological and conceptual challenges. These proteins show extreme structural heterogeneity and have complex posttranslational modification patterns, due to which traditional structural biology techniques provide results that are often difficult to interpret. As demonstrated through the example of two such multidomain proteins, CREB-binding protein (CBP) and its paralogue, p300, even the expression and purification of such proteins are compromised by their extreme proteolytic sensitivity and structural heterogeneity. In this chapter, we describe the effective expression of CBP and p300 in a eukaryotic host, Sf9 insect cells, followed by their tandem affinity purification based on two terminal tags to ensure their structural integrity. The major focus of this chapter is on the development of novel accessory tools, single-domain camelid antibodies (nanobodies), for structural-functional characterization. Specific nanobodies against full-length CBP and p300 can specifically target their different regions and can be used for their marking, labeling, and structural stabilization in a broad range of in vitro and in vivo studies. Here, we describe four high-affinity nanobodies binding to the KIX and the HAT domains, either mimicking known interacting partners or revealing new functionally relevant conformations. As immunization of llamas results in nanobody libraries with a great sequence variation, deep sequencing and interaction analysis with different regions of the proteins provide a novel approach toward developing a panel of specific nanobodies.

DOI10.1016/bs.mie.2018.09.032
Alternate JournalMeth. Enzymol.
PubMed ID30471702
Grant ListP41 GM103311 / GM / NIGMS NIH HHS / United States
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