Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.
Proteins. 2011 Jan;79(1):244-60. doi: 10.1002/prot.22878.
Cross-strand disulfides bridge two cysteines in a registered pair of antiparallel β-strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross-strand disulfides. Seventy-six cross-strand disulfides were found of which 75 and 1 occurred at non-hydrogen-bonded (NHB) and hydrogen-bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T(m). All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG⁰ = -3.3 to -6.7 kcal/mol). The data demonstrate that introduction of cross-strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity.
二硫键将两个半胱氨酸桥连在注册的反平行β-折叠的一对中。使用包含 2311 个二硫键的 5025 个多肽的非冗余数据集研究了链间二硫键。发现了 76 个链间二硫键，其中 75 个和 1 个分别位于非氢键（NHB）和氢键（HB）注册对。构象分析和建模研究表明，HB 对中二硫键的形成必然需要至少一个半胱氨酸残基具有非常罕见的正值 χ¹ 值。HB 位置的二硫键也与主链有更多不利的空间排斥。在四个模型蛋白：亮氨酸结合蛋白（LBP）、亮氨酸、异亮氨酸、缬氨酸结合蛋白（LIVBP）、麦芽糖结合蛋白（MBP）和 Top7 中，在 NHB 和 HB 对中引入了 13 对二硫键。LIVBP T247C V331C 所有突变体在纯化或用氧化剂处理时均显示二硫键形成。所有突变体的氧化和还原状态的蛋白质稳定性均进行了测量。与野生型相比，LBP 和 MBP 突变体在化学变性方面不稳定，尽管唯一暴露的 NHB LBP 突变体的 Tm 增加了 3.1°C。所有 Top7 突变体均通过硫氰酸胍化学变性进行了稳定性表征。两个暴露的和三个埋藏的 NHB 突变体中的两个都得到了明显的稳定。所有四个 HB Top7 突变体均不稳定（ΔΔG⁰=-3.3 至-6.7 kcal/mol）。数据表明，在暴露的 NHB 对中引入链间二硫键是提高蛋白质稳定性的一种稳健方法。所有四个暴露的 Top7 二硫键突变体均显示出轻微的氧化还原活性。
