Chiti F, Mangione P, Andreola A, Giorgetti S, Stefani M, Dobson C M, Bellotti V, Taddei N
Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
J Mol Biol. 2001 Mar 16;307(1):379-91. doi: 10.1006/jmbi.2000.4478.
beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis. The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds. Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules. Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state. The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus. Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation.
β2微球蛋白是一种小的、与主要组织相容性复合体I类相关的蛋白质，由于长期血液透析，它会发生聚集并作为淀粉样沉积物在人体组织中积累。通过在pH 7.4的复性缓冲液中稀释盐酸胍变性的蛋白质，并借助多种光谱探针监测折叠过程，从而在体外研究了这种淀粉样蛋白的折叠过程，这些光谱探针能够在蛋白质天然结构形成时对其进行检测。这些技术包括荧光光谱、远紫外和近紫外圆二色性、8-苯胺基-1-萘磺酸结合以及双跳跃测定。所有光谱探针均表明，在停流测量的死时间（<5毫秒）内会形成大量结构。折叠反应通过一个快速相和一个缓慢相完成，在水中其速率常数分别约为5.1和0.0030 s(-1)。展开-折叠双跳跃实验以及肽基脯氨酰异构酶的使用表明，β2微球蛋白折叠的缓慢相并非根本由X-Pro肽键的顺/反异构化决定。其他展开-折叠双跳跃实验也表明，折叠的快速相和缓慢相与不同蛋白质分子群体的独立折叠无关。相反，我们提供了一种折叠顺序机制的证据，即变性的β2微球蛋白折叠成一组部分折叠的构象（I(1)），随后这些构象折叠成结构更紧密的物种（I(2)），最终达到天然状态。部分折叠的物种I(2)似乎与先前研究的β2微球蛋白的淀粉样形成形式非常相似，例如该蛋白在轻度酸性pH值下以及在N端缺失六个残基的变体所采用的形式。由于体内淀粉样形成源于β2微球蛋白在有利于折叠过程的条件下的部分变性，这里检测到的长寿命、部分结构化的物种在某些生理条件下可能大量存在，因此可能在淀粉样形成过程中起重要作用。
