These authors contributed equally to this publication.
Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein–protein interaction
Version of Record online: 23 JAN 2014
Copyright © 2013 Wiley Periodicals, Inc.
Volume 101, Issue 4, pages 391–397, April 2014
How to Cite
Lancia, J. K., Nwokoye, A., Dugan, A., Joiner, C., Pricer, R., Mapp, A. K. (2014), Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein–protein interaction. Biopolymers, 101: 391–397. doi: 10.1002/bip.22395
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at firstname.lastname@example.org
- Issue online: 23 JAN 2014
- Version of Record online: 23 JAN 2014
- Accepted manuscript online: 27 AUG 2013 09:50AM EST
- Manuscript Accepted: 22 JUL 2013
- Manuscript Received: 9 JUL 2013
- NIH . Grant Number: GM 2RO106553
- protein–protein interactions;
- unnatural amino acid;
Protein–protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids (UAAs) in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two UAAs utilized, (p-benzoylphenylalanine (pBpa) and p-azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo-crosslinking to capture novel PPIs and to characterize the interfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 391–397, 2014.