Abstract:
Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of βγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein\'s tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.
摘要:
在希腊关键基序内形成酪氨酸角的芳香残基对于折叠至关重要,稳定性,和βγ-晶状体蛋白的顺序,因此晶状体透明度。为了描述CRYGS突变体p.F10_Y11delinsLN的N末端结构域酪氨酸角中的双氨基酸取代如何导致青少年常染色体显性皮质层状白内障,将人γS-晶状体蛋白c-DNA克隆到pET-20b()中,并通过定点诱变产生p.F10_Y11delinsLN突变体,过度表达,并使用离子交换和尺寸排阻色谱法纯化。结构,稳定性,使用荧光光谱法和圆二色性测定了溶液在热和化学胁迫下的聚集特性。在良性条件下,p.F10_Y11delinsLN突变不会影响蛋白质骨架,但会稍微改变其色氨酸微环境。突变体对热和GuHCl诱导的胁迫不太稳定,在热应力下进行两态转变,中点为60.4°C(野生型73.1°C),并表现出三态转变,中点为1.25和2.59MGuHCl(野生型:两态转变,Cm=2.72MGuHCl)。在60°C加热后,突变体自聚集,被α-晶状体蛋白和还原剂抑制。因此,人γS晶状体蛋白中的F10_Y11delinsLN突变损害了蛋白质的色氨酸微环境,削弱其在热和化学应力下的稳定性,导致自聚集,晶状体混浊,和白内障。