6 research outputs found
Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a proteinâprotein interaction
Proteinâprotein 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, ( pâ benzoylphenylalanine (pBpa) and p âazidophenylalanine (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 photoâcrosslinking to capture novel PPIs and to characterize the interfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 391â397, 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102672/1/bip22395.pd
Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture
Discovery of Enzymatic Targets of Transcriptional Activators via <i>in Vivo</i> Covalent Chemical Capture
The
network of activator protein-protein interactions (PPIs) that
underpin transcription initiation is poorly defined, particularly
in the cellular context. The transient nature of these contacts and
the often low abundance of the participants present significant experimental
hurdles. Through the coupling of <i>in vivo</i> covalent
chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap
the PPIs of transcriptional activators in a cellular setting and identify
the binding partners in an unbiased fashion. Using this approach,
we discover that the prototypical activators Gal4 and VP16 target
the Snf1 (AMPK) kinase complex via direct interactions with both the
core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further,
we use a tandem reversible formaldehyde and irreversible covalent
chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction
at the Gal1 promoter in live yeast. Together, these data support a
critical role for activator PPIs in both the recruitment and positioning
of important enzymatic complexes at a gene promoter and represent
a technical advancement in the discovery of new cellular binding targets
of transcriptional activators