Systems Biology

Protein–protein interactions facilitate cellular functions through the creation of networks and multi-protein complexes. Mapping the interactions within and between protein networks and elucidating the composition of protein complexes provides critical insight into biological processes. Interactions among soluble cytoplasmic proteins have been extensively investigated through the application of immunoaffinity capture as well as conventional nuclear two-hybrid testing. The integrated membrane yeast two-hybrid provides a method to investigate protein–protein interactions between integral membrane proteins in their native membrane environment. This procedure makes use of the ability of the amino-terminal fragment of ubiquitin (Nub) and the carboxyl-terminal fragment of ubiquitin (Cub) to refold reconstituting functional ubiquitin, which can be recognized by a ubiquitin peptidase. Appending a fusion protein composed of Cub fused to LexA and VP16 (CLV) to a candidate "bait" protein and Nub to candidate "prey" proteins allows a test of their interaction. If the two proteins interact closely, the CLV fragment is cleaved and enters the nucleus to activate the expression of reporter genes, signaling the interaction. When the bait and prey proteins are tagged with CLV and NubG, respectively, at their genomic loci, they are only copies of the bait and prey in the cell and are expressed under the regulation of their native promoters. This avoids overexpression artifacts that can occur if the tagged proteins are expressed from plasmids while the untagged chromosomally encoded copies of the bait and prey continue to be expressed.

Agrobacterium-mediated transient expression has greatly contributed to research in molecular plant biology but has low efficiency and inconsistency in Arabidopsis thaliana (Arabidopsis). Here, we describe a simple, efficient and fast protocol to make transient gene expression in NahG Arabidopsis plants using Agrobacterium tumefaciens. This protocol has been successfully used to assess protein sub-cellular localization and accumulation, enzyme activity, and protein-protein interaction. In addition, this assay overcomes the use of Nicotiana benthamiana plants as a surrogate system for transient gene expression assays. Finally, the use of this protocol does not require complex inoculation methods or specific growth conditions, and can be used with different Agrobacterium strains with similar results.

Molecular dynamics (MD) simulations have become one of the most important tools in understanding the behavior of bio-molecules on nanosecond to microsecond time scales. In this protocol, we provide a general approach and standard setup protocol for MD simulations by using the Gromacs MD suite.

Protein-protein interactions are at the core of a plethora of developmental, physiological and biochemical processes. Consequently, insights into the origin and evolutionary dynamics of protein-protein interactions may provide information on the constraints and dynamics of specific biomolecular circuits and their impact on the organismal phenotype.

This protocol describes how ancestral protein-protein interaction patterns can be inferred using a set of known protein interactions from phylogenetically informative species. Although this protocol focuses on protein-protein interaction data, character-state reconstructions can in general be performed with other kinds of binary data in the same way.