Modular enzyme design: regulation by mutually exclusive protein folding.
A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topological constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodynamic tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.
Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St, Syracuse, NY 13210, USA.
Jeung-Hoi
JH
Ha
James S
JS
Butler
Diana M
DM
Mitrea
Stewart N
SN
Loh
D000818:Animals; D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D020134:Catalytic Domain; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D008024:Ligands; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease