Solid phase proteomics: dramatic reinforcement of very weak protein-protein interactions.
Very weak protein-protein interactions may play a critical role in cell physiology but they are not easily detectable in "in vitro" experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein-protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein-protein complexes formed by strong protein-protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein-protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein-protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein-protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein-protein interactions.
Fuentes
Manuel
M
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Mateo
Cesar
C
Pessela
Benevides C C
BC
Batalla
Pilar
P
Fernandez-Lafuente
Roberto
R
Guisán
J M
JM
D000327:Adsorption; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D011506:Proteins