Dee Derek, Pencer Jeremy, Nieh Mu-Ping, Krueger Susan, Katsaras John, Yada Rickey Y
Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada.
Biochemistry. 2006 Nov 28;45(47):13982-92. doi: 10.1021/bi061270i.
A zymogen-derived protein, pepsin, appears to be incapable of folding to the native state without the presence of the prosegment. To better understand the nature of the irreversible denaturation of pepsin, the present study reports on the characterization of the stability and low-resolution tertiary and secondary structures of native, alkaline unfolded and acid refolded porcine pepsin. Through a combination of small-angle neutron scattering (SANS), CD, and DSC, acid refolded pepsin (Rp) was shown to have secondary and tertiary structures intermediate between the alkaline denatured and native forms but was found to be thermodynamically stable relative to the native state. It was also observed that the acid refolded state of pepsin was dependent on the protein concentration during refolding because CD and SANS data revealed that both the secondary and tertiary structures of concentrated-refolded pepsin (>10 mg/mL) (CRp) were native-like, in contrast to the intermediate nature of Rp, refolded under dilute concentration (<10 mg/mL). Despite a native-like conformation, CRp was more stable and had substantially reduced activity compared to that of the native state, suggesting that the protein was misfolded. It is proposed that the stable but misfolded, acid-refolded states are evidence that pepsin in its native conformation was metastable. Furthermore, the disruption of the active site cleft in the denatured states could be discerned by modeling of the SANS data.
一种由酶原衍生的蛋白质——胃蛋白酶，在没有前肽存在的情况下似乎无法折叠成天然状态。为了更好地理解胃蛋白酶不可逆变性的本质，本研究报告了天然、碱性变性和酸性复性猪胃蛋白酶的稳定性以及低分辨率三级和二级结构的特征。通过小角中子散射（SANS）、圆二色性（CD）和差示扫描量热法（DSC）相结合的方法，结果表明酸性复性胃蛋白酶（Rp）具有介于碱性变性形式和天然形式之间的二级和三级结构，但相对于天然状态在热力学上是稳定的。还观察到胃蛋白酶的酸性复性状态取决于复性过程中的蛋白质浓度，因为CD和SANS数据显示，与在稀浓度（<10 mg/mL）下复性的Rp的中间性质相反，浓缩复性胃蛋白酶（>10 mg/mL）（CRp）的二级和三级结构都类似天然结构。尽管CRp具有类似天然的构象，但与天然状态相比，它更稳定且活性大幅降低，这表明该蛋白质发生了错误折叠。有人提出，稳定但错误折叠的酸性复性状态证明天然构象的胃蛋白酶是亚稳态的。此外，通过对SANS数据进行建模，可以识别变性状态下活性位点裂隙的破坏情况。
