Catalysis of protein folding by an immobilized small-molecule dithiol.
The isomerization of non-native disulfide bonds often limits the rate of protein folding. Small-molecule dithiols can catalyze this process. Here, a symmetric trithiol, tris(2-mercaptoacetamidoethyl)amine, is designed on the basis of criteria known to be important for efficient catalysis of oxidative protein folding. The trithiol is synthesized and attached to two distinct solid supports via one of its three sulfhydryl groups. The resulting immobilized dithiol has an apparent disulfide E degrees ' = -208 mV, which is close to that of protein disulfide isomerase (E degrees ' = -180 mV). Incubation of the dithiol immobilized on a TentaGel resin with a protein containing non-native disulfide bonds produced only a 2-fold increase in native protein. This dithiol appeared to be inaccessible to protein. In contrast, incubation of the dithiol immobilized on styrene-glycidyl methacrylate microspheres with the non-native protein produced a 17-fold increase in native protein. This increase was 1.5-fold greater than that of a monothiol immobilized on the microspheres. Thus, the choice of both the solid support and thiol can affect catalysis of protein folding. The use of dithiol-decorated microspheres is an effective new strategy for preparative protein folding in vitro.
Woycechowsky
Kenneth J
KJ
Department of Biochemistry and Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Hook
Brad A
BA
Raines
Ronald T
RT
D002384:Catalysis; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000737:chemistry*; D004793:Enzyme Reactivators / Q000737:chemistry*; D008863:Microspheres; D011485:Protein Binding; D017510:Protein Folding; D012117:Resins, Synthetic / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D014050:Toluene / Q000031:analogs & derivatives* / Q000737:chemistry*
D020099:Coated Materials, Biocompatible; D004793:Enzyme Reactivators; D012117:Resins, Synthetic; D014050:Toluene; D012259:Ribonuclease, Pancreatic; C004848:dithiol