Striking stabilization of Arc repressor by an engineered disulfide bond.
A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.
Robinson
C R
CR
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Sauer
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RT
D017100:Bacteriophage P22 / Q000235:genetics*; D004220:Disulfides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000379:methods; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics; D014764:Viral Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D004220:Disulfides; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D019791:Guanidine