Chemical Tools for Studying the Impact of cis/trans Prolyl Isomerization on Signaling: A Case Study on RNA Polymerase II Phosphatase Activity and Specificity

Nathaniel Tate Burkholder, Brenda Medellin, Seema Irani, Wendy Matthews, Scott A. Showalter, Yan Jessie Zhang

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Proline isomerization is ubiquitous in proteins and is important for regulating important processes such as folding, recognition, and enzymatic activity. In humans, peptidyl-prolyl isomerase cis–trans isomerase NIMA interacting 1 (Pin1) is responsible for mediating fast conversion between cis- and trans-conformations of serine/threonine–proline (S/T–P) motifs in a large number of cellular pathways, many of which are involved in normal development as well as progression of several cancers and diseases. One of the major processes that Pin1 regulates is phosphatase activity against the RNA polymerase II C-terminal domain (RNAPII CTD). However, molecular tools capable of distinguishing the effects of proline conformation on phosphatase function have been lacking. A key tool that allows us to understand isomeric specificity of proteins toward their substrates is the usage of proline mimicking isosteres that are locked to prevent cis/trans-proline conversion. These locked isosteres can be incorporated into standard peptide synthesis and then used in replacement of native substrates in various experimental techniques such as kinetic and thermodynamic assays as well as X-ray crystallography. We will describe the application of these chemical tools in detail using CTD phosphatases as an example. We will also discuss alternative methods for analyzing the effect of proline conformation such as 13C NMR and the biological implications of proline isomeric specificity of proteins. The chemical and analytical tools presented in this chapter are widely applicable and should help elucidate many questions on the role of proline isomerization in biology.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsKaren N. Allen
PublisherAcademic Press Inc.
Pages269-297
Number of pages29
ISBN (Print)9780128138816
DOIs
Publication statusPublished - Jan 1 2018

Publication series

NameMethods in Enzymology
Volume607
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Burkholder, N. T., Medellin, B., Irani, S., Matthews, W., Showalter, S. A., & Zhang, Y. J. (2018). Chemical Tools for Studying the Impact of cis/trans Prolyl Isomerization on Signaling: A Case Study on RNA Polymerase II Phosphatase Activity and Specificity. In K. N. Allen (Ed.), Methods in Enzymology (pp. 269-297). (Methods in Enzymology; Vol. 607). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.04.020