Partially Unfolded States of  2 -Microglobulin and Amyloid Formation in Vitro ² Victoria J. McParland, ‡,§ Neil M. Kad, ‡,§ Arnout P. Kalverda, ‡ Anthony Brown, # Patricia Kirwin-Jones, # Michael G. Hunter, # Margaret Sunde, ⊥ and Sheena E. Radford* ,‡ School of Biochemistry and Molecular Biology, UniVersity of Leeds, Leeds LS2 9JT, U.K., British Biotech Pharmaceuticals Ltd., Watlington Road, Oxford OX4 5LY, U.K., and Department of Biochemistry, UniVersity of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K. ReceiVed February 4, 2000; ReVised Manuscript ReceiVed May 2, 2000 ABSTRACT: Dialysis-related amyloidosis (DRA) involves the aggregation of  2 -microglobulin ( 2 m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric  2 m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are ∼10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric  2 m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK a of 4.7 destabilizes native  2 m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional 1 H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for  2 m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies. A number of proteins have been shown to aggregate into amyloid fibrils in vivo, leading to a pathological disorder known as amyloidosis (1). This term is generically applied to diseases that involve the conversion of normally soluble proteins or peptides into insoluble fibrillar arrays, although the clinical manifestations of each disease are specific to the identity of the aggregating protein. One such disorder, known as dialysis-related amyloidosis (DRA), involves the aggrega- tion of full-length, wild-type, human  2 -microglobulin ( 2 m) into amyloid fibrils (2, 3). As its name implies, DRA arises in patients with chronic renal failure and results in the deposition of  2 m amyloid fibrils systemically (4), most typically in the musculo-skeletal system. The disease is a common and serious complication of long-term hemodialysis, with deposits of  2 m amyloid developing in joints within 18 months of the commencement of dialysis (5). Serious complications develop in more than 90% of patients under- going dialysis for a period of 10 or more years (6). Clinical symptoms of the disease include carpal tunnel syndrome, destructive arthropathy, and pathological bone fractures.  2 m is the light chain of the type I major histocompatibility complex. The protein is small (99 residues in length) and nonpolymorphic, and its 7-stranded -sandwich structure is typical of proteins in the immunoglobulin superfamily (Figure 1). The two -sheets are linked by a single disulfide bond (7). In vivo,  2 m is continuously shed from the surface of cells displaying MHC class I molecules. It is then carried in the plasma to the kidneys, where it is degraded and excreted. As a consequence of renal failure, the  2 m levels accumulate in the plasma [its concentration increases 25- 35-fold (8)]. Together with a possible multitude of other factors, including the age of the patient and the duration of dialysis, pathogenic amyloid fibrils are formed (6, 9). Analyses of  2 m fibrils extracted ex vivo have identified full- length, as well as truncated, versions of the wild-type protein (2-4, 10),  2 m modified with advanced-glycation end products, glycosaminoglycans (11), and auxiliary proteins such as apoE and serum amyloid P component (12-14). In addition, amyloid deposits have been observed in patients prior to the initiation of dialysis, suggesting that the process ² We acknowledge with thanks financial support from The University of Leeds, The Wellcome Trust, and the BBSRC. V.J.M. and N.M.K. are supported by the BBSRC, A.P.K. is funded by The Wellcome Trust, and M.S. is a Royal Society University Research Fellow. V.J.M., N.M.K., A.P.K., and S.E.R. are members of the Astbury Centre for Structural Molecular Biology, which is part of the North of England Structural Biology Centre and is funded by the BBSRC. * To whom correspondence should be addressed. Phone: +44 113 233 3170. Fax: +44 113 233 3167. E-mail: s.e.radford@leeds.ac.uk. ‡ University of Leeds. # British Biotech Pharmaceuticals Ltd. ⊥ University of Cambridge. § These authors contributed equally to this work. 1 Abbreviations: 2m, human 2-microglobulin; SAP, serum amyloid component-P; thio-T, thioflavin-T; ANS, 1-anilinonaphthalene-8-sul- fonic acid; DRA, dialysis-related amyloidosis; EM, electron microscopy; TTR, transthyretin. 8735 Biochemistry 2000, 39, 8735-8746 10.1021/bi000276j CCC: $19.00 © 2000 American Chemical Society Published on Web 07/01/2000