Detection of two partially structured species in the folding process of the amyloidogenic protein beta 2-microglobulin

beta2-Microglobulin is a small, major histocompatibility complex class I-as sociated protein that undergoes aggregation and accumulates as amyloid depo sits in human tissues as a consequence of long-term haemodialysis. The fold ing process of this amyloidogenic protein has been studied in vitro by dilu ting the guanidine hydrochloride-denatured protein in refolding buffer at p H 7.4 and monitoring the folding process by means of a number of spectrosco pic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and n ear-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant a mount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase follo wed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in wa ter, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of foldin g of <beta>2-microglobulin is not fundamentally determined by cis/trans iso merisation of X-Pro peptide bonds. Other folding-unfolding double jump expe riments also suggest that the fast and slow phases of folding are not relat ed to independent folding of different populations of protein molecules. Ra ther, we provide evidence for a sequential mechanism of folding where denat ured beta2-microglobulin collapses to an ensemble of partially folded confo rmations (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 beta2-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. Si nce amyloid formation in vivo originates from partial denaturation of beta2 -microglobulin under conditions favouring the folding process, the long-liv ed, partially structured species detected here might be significantly popul ated under some physiological conditions and hence might play an important role in the process of amyloid formation. (C) 2001 Academic Press.