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

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Angela Bekesi, Sara Abdellaoui, Natalie Holroyd, Wouter Van Delm, Els Pardon, Jarne Pauwels, Kris Gevaert, Jan Steyaert, Stefaan Derveaux, Antoni Borysik, Peter Tompa

Original languageEnglish
Title of host publicationMethods in Enzymology
Number of pages69
ISBN (Print)9780128156490
Published1 Jan 2018

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

King's Authors


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.

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