File S1 - The Key to the Extraordinary Thermal Stability of P. furiosus Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure

Supporting information and figures. Figure S1, Panel a : Control solutions lacking ferrozine. Tube-1 contains a ferric chloride solution. Tube-2 contains ferric chloride and beta-mercaptoethanol. Tube-3 contains ferric chloride, beta-mercaptoethanol and guanidium hydrochloride. Panel b : Sample solutions containing ferrozine. Tube-1 contains ferrozine added to ferric chloride. Tube-2 contains ferrozine added to ferric chloride pre-mixed with beta-mercaptoethanol. Tube-3 contains ferrozine added to ferric chloride pre-mixed with beta-mercaptoethanol and guanidium hydrochloride. Tube-4 contains ferrozine added to ferrous sulphate. Figure S2, Panel a : Elution of free ferrozine (∼17 ml and ∼20 ml) on a Superdex Peptide (GE) column in the absence of any iron or protein. Panel b : Fe2+-bound ferrozine (two eluting species at 12.5 ml and 14.0 ml) separated from free ferrozine (∼17 ml) on the same column. Panel c : Fe2+-bound ferrozine (∼12.5 ml and ∼14.0 ml) separated from free ferrozine (∼17 ml and 20 ml) and PfRd protein (∼10 ml) on the same column. Panel d: Elution of PfRd protein (∼10 ml) on a Superdex Peptide (GE) column in the absence of any ferrozine. Figure S3, Panel a : MALDI-TOF MS spectrum of Apo-2 PfRd (including the N-terminal 6xHis tag), showing that the protein has a mass of 7292 Da. The theoretically expected mass is ∼7294 Da. Panel b : MALDI-TOF MS spectrum of N-terminally 6xHis tagged Apo-2 PfRd alkylated by iodoacetic acid (IAA) after treatment with beta mercaptoethanol. The masses of 7534, and 7409 Da represent species carrying four, and two, IAA aductions, respectively, indicating that PfRd's four cysteine residues are free and available to be alkylated in the presence of beta-mercaptoethanol. Panel c : MALDI-TOF MS spectrum of N-terminally 6xHis tagged Apo-2 PfRd alkylated by iodoacetic acid (IAA) without any treatment with beta mercaptoethanol. The masses of ∼7523.95, ∼7462.27, ∼7406 and ∼7345 Da represent species carrying four, three, two, and one IAA aductions, respectively, with the mass peak with the highest intensity representing the population with all four of PfRs's cysteine residues modified. The molecule's cysteine residues are thus free and available to be alkylated (and not disulfide bonded). Figure S4, Organization of the aromatic cluster in holo-PfRd, showing different aromatic interactions amongst the molecules six aromatic residues, namely W3 (green), Y10 (orange), Y12 (magenta), F29 (blue), W36 (red) and F48 (black). Figure S5, Panels a and b : Changes in the CD MRE signal at 222 nm of aliphatic (Panel a) and aromatic (Panel b) substitution mutants as a function of increasing temperature, in the absence of denaturant. Panels c and d : Changes in the CD MRE signal at 222 nm of aliphatic (Panel c) and aromatic (Panel d) substitution mutants as a function of increasing temperature, in the presence of 6 M Gdm. HCl. Panels e and f : Time course of changes in the CD MRE signal at 222 nm of aliphatic (Panel e) and aromatic (Panel f) substitution mutants at 95 degrees Centigrade, in the presence of 6 M Gdm.HCl. (PDF)