Designed proteins to modulate cellular networks.
A major challenge of protein design is to create useful new proteins that interact specifically with biological targets in living cells. Such binding modules have many potential applications, including the targeted perturbation of protein networks. As a general approach to create such modules, we designed a library with approximately 10(9) different binding specificities based on a small 3-tetratricopeptide repeat (TPR) motif framework. We employed a novel strategy, based on split GFP reassembly, to screen the library for modules with the desired binding specificity. Using this approach, we identified modules that bind tightly and specifically to Dss1, a small human protein that interacts with the tumor suppressor protein BRCA2. We showed that these modules also bind the yeast homologue of Dss1, Sem1. Furthermore, we demonstrated that these modules inhibit Sem1 activity in yeast. This strategy will be generally applicable to make novel genetically encoded tools for systems/synthetic biology applications.
Cortajarena
Aitziber L
AL
Liu
Tina Y
TY
Hochstrasser
Mark
M
Regan
Lynne
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005095:Exoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D019151:Peptide Library; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins; C117424:SEM1 protein, S cerevisiae; C484606:SEM1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005095:Exoribonucleases; C098317:DSS1 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex