title: Design and folding of a multidomain protein.
abstract: To test whether the folding process of a large protein can be understood on the basis of the folding behavior of the domains that constitute it, we coupled two well-studied small -helical proteins, the B-domain of protein A (60 amino acids) and Rd-apocytochrome b562 (Rd-apocyt b562, 106 amino acids), by fusing the C-terminal helix of the B-domain of protein A with the N-terminal helix of Rd-apocyt b562 without changing their hydrophobic core residues. The success of the design was confirmed by determining the structure of the engineered protein with multidimensional NMR methods. Kinetic studies showed that the logarithms of the folding/unfolding rate constants of the engineered protein are linearly dependent on concentrations of guanidinium chloride in the measurable range from 1.7 to 4 M. Their slopes (m-values) are close to those of Rd-apocyt b562. In addition, the 1H-15N HSQC spectrum taken at 1.5 M guanidinium chloride reveals that only the Rd-apocyt b562 domain in the designed protein remained folded. These results suggest that the two domains have weak energetic coupling. Interestingly, the redesigned protein folds faster than Rd-apocyt b562, suggesting that the fused helix stabilizes the rate-limiting transition state.
authors.affiliation: Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhou
authors.forename: Hanqiao
authors.initials: H
authors.lastname: Feng
authors.forename: Hongyi
authors.initials: H
authors.lastname: Zhou
authors.forename: Yaoqi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Yawen
authors.initials: Y
authors.lastname: Bai
mesh_terms: D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003573:Cytochrome b Group; D013205:Staphylococcal Protein A