Thermodynamic analysis of an antagonistic folding-unfolding equilibrium between two protein domains.
A simple model is formulated for analyzing the coupled folding-unfolding equilibrium present in a unique class of molecular switch proteins. We previously fused two single-domain proteins, barnase and ubiquitin, such that the free energy stored in the folded structure of one subunit is used to drive unfolding of the other. Here, we present a thermodynamic test of that mechanism. The antagonistic interaction is represented by a coupling free energy term DeltaGX. DeltaGX is the penalty imposed on folding of one domain by the native structure of the other. If DeltaGX=0, then neither domain senses the other and they fold and unfold independently. If DeltaGX>0, then destabilizing one domain will stabilize the other, and vice versa. In the limit where DeltaGX is greater than the intrinsic stability of either protein, then only one domain can be folded at any given time. We estimate DeltaGX by measuring stability parameters for a series of mutants that destabilize either the barnase or ubiquitin domains. Fitting the data to the model leads to a DeltaGX value of approximately 4 kcal mol(-1). DeltaGX is proposed to depend on both the length of the linker peptides used to join the two proteins, and on the inherent structural plasticity of each domain. We predict that shortening the linkers from their current lengths of two and three amino acid residues will increase structural and thermodynamic coupling.
Cutler
Thomas A
TA
Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Loh
Stewart N
SN
D001426:Bacterial Proteins; D003198:Computer Simulation; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D025801:Ubiquitin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D025801:Ubiquitin; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D019791:Guanidine