title: Different folding pathways taken by highly homologous proteins, goat alpha-lactalbumin and canine milk lysozyme.
abstract: Is the folding pathway conserved in homologous proteins? To address this question, we compared the folding pathways of goat alpha-lactalbumin and canine milk lysozyme using equilibrium and kinetic circular dichroism spectroscopy. Both Ca(2+)-binding proteins have 41% sequence identity and essentially identical backbone structures. The Phi-value analysis, based on the effect of Ca(2+) on the folding kinetics, showed that the Ca(2+)-binding site was well organized in the transition state in alpha-lactalbumin, although it was not yet organized in lysozyme. Equilibrium unfolding and hydrogen-exchange 2D NMR analysis of the molten globule intermediate also showed that different regions were stabilized in the two proteins. In alpha-lactalbumin, the Ca(2+)-binding site and the C-helix were weakly organized, whereas the A- and B-helices, both distant from the Ca(2+)-binding site, were well organized in lysozyme. The results thus provide an example of highly homologous proteins taking different folding pathways. To understand the molecular origin of this difference, we investigated the native three-dimensional structures of the proteins in terms of non-local contact clusters, a parameter based on the residue-residue contact map and known to be well correlated with the folding rate of non-two-state proteins. There were remarkable differences between the proteins in the distribution of the non-local contact clusters, and these differences provided a reasonable explanation of the observed difference in the folding initiation sites. In conclusion, the protein folding pathway is determined not only by the backbone topology but also by the specific side-chain interactions of contacting residues.
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.forename: Katsuaki
authors.initials: K
authors.lastname: Tomoyori
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Mukaiyama
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004285:Dogs; D006041:Goats; D066298:In Vitro Techniques; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008892:Milk / Q000201:enzymology*; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D011994:Recombinant Proteins; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium