title: Solution structure of oxidized rat microsomal cytochrome b5 in the presence of 2 M guanidinium chloride: monitoring the early steps in protein unfolding.
abstract: One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the oxidized state of the A-form of rat microsomal cytochrome b5 (cyt b5). The protein rapidly starts losing the heme at denaturant concentrations larger than approximately 2.0 M and a largely unfolded protein is eventually obtained. An estimate of the unfolding kinetics is obtained and, by use of a two-state model (folded left and right arrow unfolded), a value for DeltaG degrees. Below this concentration, small (</=0.15 ppm) but systematic chemical shift variations take place for the diamagnetic as well as the hyperfine-shifted signals, indicating that some structural changes occur. However, the protein core maintains its overall structure. The analysis of the two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY) maps has allowed the determination of the solution structure of the protein in the presence of 2 M GdmCl. By use of 1199 meaningful NOESY constraints (obtained from the assignment of 75% of the total protons) and 166 pseudocontact shifts, a family of 40 structures has been obtained through the program PSEUDYANA. The family was further refined through restrained energy minimization and the final root mean square deviation (RMSD) values with respect to the average structure are 0.67 +/- 0.10 A and 1.14 +/- 0.11 A for the backbone and heavy atoms, respectively. The quality of the present structure is equivalent to that of the one obtained recently for the native form [Arnesano et al. (1998) Biochemistry 37, 173-184], thus allowing a meaningful comparison between the two structures. Upon addition of 2 M GdmCl, significant local structural differences are induced to the protein backbone segments comprising residues 33-38 (helix alpha2) and 62-64 (end of helix alpha4-beginning of helix alpha5) while the overall folding scheme of the protein is still maintained. These protein regions form part of the "pocket" supporting the heme, whose plane is also rotated by approximately 10 degrees around an axis connecting the C2 and C8 carbon atoms. The initial steps of the unfolding process involve breaking of a few hydrogen bonds that stabilize local structural conformations. The hydrogen bond between Ser 64 and propionate 7, which stabilizes the heme binding to the protein frame, is broken in the presence of 2 M GdmCl. The same occurs for two hydrogen bonds between two beta-strands (beta2 and beta3), thus inducing the disruption of one of the antiparallel beta-sheets forming one side of the heme cavity. Our results are critically discussed in connection with the native-state protein local backbone mobility characteristics and point to the backbone carbons of Glu 37 and Ser 64 being the first "breaking points" of the protein frame once the global unfolding reaction is initiated at a somewhat higher concentration of denaturant.
authors.affiliation: Department of Chemistry, University of Florence, Florence, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Arnesano
authors.forename: L
authors.initials: L
authors.lastname: Banci
authors.forename: I
authors.initials: I
authors.lastname: Bertini
authors.forename: D
authors.initials: D
authors.lastname: Koulougliotis
mesh_terms: D000818:Animals; D003198:Computer Simulation; D015786:Cytochromes b5 / Q000737:chemistry*; D019791:Guanidine; D008861:Microsomes; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D011522:Protons; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D015786:Cytochromes b5; D019791:Guanidine