Analysis of stable and transient protein-protein interactions

Abstract

The assembly of proteins into defined complexes drives a plethora of cellular activities. These protein complexes often have a set of more stably interacting proteins as well as more unstable or transient interactions. Studying the in vivo components of these protein complexes is challenging as many of the techniques used for isolation result in the purification of only the most stable components and the transient interactions are lost. A technology called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) has been developed to identify these transiently interacting proteins as well as the stable interactions. Described here are the detailed methodological approaches used for a transient I-DIRT analysis of a multi-subunit complex, NuA3, that acetylates histone H3 and functions to activate gene transcription. Transcription is known to involve a concert of protein assemblies performing different activities on the chromatin/gene template, thus understanding the less stable or transient protein interactions with NuA3 will shed light onto the protein complexes that function synergistically, or antagonistically, to regulate gene transcription and chromatin remodeling.

Fig. 1

Transient I-DIRT technology for identifying stable and less stable protein–protein interactions. One culture containing an affinity-tagged protein is grown in isotopically light media and cross-linked with formaldehyde (FA). A second culture of wild-type cells is grown in isotopically heavy media (e.g., ¹³C₆-arginine) and also cross-linked with formaldehyde. Following independent harvesting and freezing, the cultures are mixed 1:1 and cryogenically lysed under liquid nitrogen temperature. The affinity-tagged protein is purified with associating proteins and resolved by SDS-PAGE. The gel lane is sliced into 2 mm sections and proteins are identified with mass spectrometry. Proteins interacting stably with the affinity-tagged protein complex have ~100% isotopically light tryptic peptides (i.e., these protein interactions have been preserved from the original isotopically light culture), while nonspecific proteins co-purifying with the affinity-tagged protein complex will have ~50% isotopically light tryptic peptides (i.e., contamination occurs during the purification procedure and these nonspecific protein associations have an equal probability to be isotopically light or heavy). Transiently interacting proteins will have an intermediate level of isotopically light tryptic peptides. Examples are shown of each interaction from a purification of the NuA3 histone acetyltrans-ferase. Following mass spectrometric analysis, the role of the specific protein interactions are explored with functional analyses.