File S1 - The Key to the Extraordinary Thermal Stability of <i>P. furiosus</i> 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)辅助信息及附图。图S1(a)：不含菲洛嗪（ferrozine）的对照溶液。管1为氯化铁溶液；管2为氯化铁与β-巯基乙醇（beta-mercaptoethanol）的混合溶液；管3为氯化铁、β-巯基乙醇与盐酸胍（guanidium hydrochloride）的混合溶液。(b) 含菲洛嗪的样品溶液：管1为向氯化铁溶液中加入菲洛嗪；管2为向预先混合了β-巯基乙醇的氯化铁溶液中加入菲洛嗪；管3为向预先混合了β-巯基乙醇与盐酸胍的氯化铁溶液中加入菲洛嗪；管4为向硫酸亚铁溶液中加入菲洛嗪。图S2(a)：无任何铁离子或蛋白质存在时，游离菲洛嗪（洗脱峰约17 ml与约20 ml）在Superdex Peptide（GE）凝胶过滤柱上的洗脱曲线。(b) 相同色谱柱上，Fe²⁺结合型菲洛嗪（两个洗脱组分分别在12.5 ml与14.0 ml处）与游离菲洛嗪（约17 ml处）的分离效果。(c) 相同色谱柱上，Fe²⁺结合型菲洛嗪（约12.5 ml与约14.0 ml）、游离菲洛嗪（约17 ml与20 ml）与PfRd蛋白质（约10 ml）的分离效果。(d) 无任何菲洛嗪存在时，PfRd蛋白质（约10 ml）在Superdex Peptide（GE）凝胶过滤柱上的洗脱曲线。图S3(a)：带N端6×His标签的脱辅基PfRd（Apo-2 PfRd）的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）谱图，测得该蛋白质分子量为7292 Da，理论预期分子量约为7294 Da。(b) 经β-巯基乙醇处理后，用碘乙酸（IAA）烷基化的带N端6×His标签的脱辅基PfRd的MALDI-TOF MS谱图：分子量7534 Da与7409 Da的组分分别携带4个与2个IAA修饰基团，表明PfRd的4个半胱氨酸残基处于游离状态，可在β-巯基乙醇存在下发生烷基化反应。(c) 未经β-巯基乙醇处理，直接用碘乙酸（IAA）烷基化的带N端6×His标签的脱辅基PfRd的MALDI-TOF MS谱图：分子量约7523.95 Da、7462.27 Da、7406 Da与7345 Da的组分分别携带4个、3个、2个与1个IAA修饰基团，其中信号强度最高的峰对应4个半胱氨酸残基均被修饰的组分，由此证明该蛋白质的半胱氨酸残基处于游离状态，可发生烷基化反应（未形成二硫键）。图S4：全辅基PfRd（holo-PfRd）中芳香族残基簇的组织结构，展示该蛋白质6个芳香族残基间的不同芳香相互作用，分别为W3（绿色）、Y10（橙色）、Y12（品红色）、F29（蓝色）、W36（红色）与F48（黑色）。图S5(a)与(b)：无变性剂存在时，脂肪族取代突变体(a)与芳香族取代突变体(b)的圆二色谱（CD）平均残基摩尔椭圆率（MRE）信号在222 nm波长处随温度升高的变化趋势。(c)与(d)：6 M盐酸胍（Gdm·HCl）存在时，脂肪族取代突变体(c)与芳香族取代突变体(d)的CD MRE信号在222 nm波长处随温度升高的变化趋势。(e)与(f)：6 M盐酸胍（Gdm·HCl）存在、95℃条件下，脂肪族取代突变体(e)与芳香族取代突变体(f)的CD MRE信号随时间的变化过程。(PDF)