Solution structure of oxidized rat microsomal cytochrome (b5) in the presence of 2 m guanidinium chloride: Monitoring the early steps in protein unfolding

One- and two-dimensional proton NMR spectroscopy has been employed in order to study the denaturation effect of guanidinium chloride (GdmCl) on the ox idized state of the A-form of rat microsomal cytochrome b(5) (cyt b(5)), Th e protein rapidly starts losing the heme at denaturant concentrations large r than similar to 2.0 M and a largely unfolded protein is eventually obtain ed. An estimate of the unfolding kinetics is obtained and, by use of a two- state model (folded <--> unfolded), a value for Delta G degrees. Below this concentration, small (less than or equal to 0.15 ppm) but systematic chemi cal shift variations take place for the diamagnetic as well as the hyperfin e-shifted signals, indicating that some structural changes occur. However, the protein core maintains its overall structure. The analysis of the two-d imensional nuclear Overhauser effect spectroscopy (2D NOESY) maps has allow ed the determination of the solution structure of the protein in the presen ce 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 t he final root mean square deviation (RMSD) values with respect to the avera ge structure are 0.67 +/- 0.10 Angstrom and 1.14 +/- 0.11 Angstrom for the backbone and heavy atoms, respectively. The quality of the present structur e is equivalent to that of the one obtained recently for the native form [A rnesano et al. (1998) Biochemistry 37, 173-184], thus allowing a meaningful comparison between the two structures. Upon addition of 2 M GdmCl, signifi cant local structural differences are induced to the protein backbone segme nts comprising residues 33-38 (helix alpha 2) and 62-64 tend of helix alpha 4-beginning of helix alpha 5) while the overall folding scheme of the prot ein is still maintained. These protein regions form part of the "pocket" su pporting the heme, whose plane is also rotated by approximately 10 degrees around an axis connecting the C-2 and C-8 carbon atoms. The initial steps o f the unfolding process involve breaking of a few hydrogen bonds that stabi lize local structural conformations. The hydrogen bond between Ser 64 and p ropionate 7, which stabilizes the heme binding to the protein frame, is bro ken in the presence of 2 M GdmCl. The same occurs for two hydrogen bonds be tween two beta-strands (beta 2 and beta 3), thus inducing the disruption of one of the antiparallel beta-sheets forming one side of the heme cavity. O ur results are critically discussed in connection with the native-state pro tein local backbone mobility characteristics and point to the backbone carb ons of Glu 37 and Ser 64 being the first "breaking points" of the protein f rame once the global unfolding reaction is initiated at a somewhat higher c oncentration of denaturant.