Measurement of In Vivo Protein Binding Affinities in a Signaling Network with Mass Spectrometry.
Protein interaction networks play a key role in signal processing. Despite the progress in identifying the interactions, the quantification of their strengths lags behind. Here we present an approach to quantify the in vivo binding of proteins to their binding partners in signaling-transcriptional networks, by the pairwise genetic isolation of each interaction and by varying the concentration of the interacting components over time. The absolute quantification of the protein concentrations was performed with targeted mass spectrometry. The strengths of the interactions, as defined by the apparent dissociation constants, ranged from subnanomolar to micromolar values in the yeast galactose signaling network. The weak homodimerization of the Gal4 activator amplifies the signal elicited by glucose. Furthermore, combining the binding constants in a feedback loop correctly predicted cellular memory, a characteristic network behavior. Thus, this genetic-proteomic binding assay can be used to faithfully quantify how strongly proteins interact with proteins, DNA and metabolites.
Biozentrum, University of Basel , Klingelbergstrasse 50/70, 4056 Basel, Switzerland.
Mumun
M
Gencoglu
Biozentrum, University of Basel , Klingelbergstrasse 50/70, 4056 Basel, Switzerland.
Alexander
A
Schmidt
Biozentrum, University of Basel , Klingelbergstrasse 50/70, 4056 Basel, Switzerland.
Attila
0000-0002-9462-0934
A
Becskei
D005686:Galactokinase / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C055002:GAL80 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C524219:GAL1 protein, S cerevisiae; D005686:Galactokinase
GAL1; GAL80; Saccharomyces cerevisiae; bistability; equilibrium dissociation constant; protein half-life