Residual Structures in the Acid-Unfolded States of Vλ6 Proteins Affect Amyloid Fibrillation Tomonori Mishima 1 , Takatoshi Ohkuri 1 , Akira Monji 2 , Takaaki Kanemaru 3 , Yoshito Abe 1 and Tadashi Ueda 1 ⁎ 1 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan 2 Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan 3 Morphology Core Unit, Kyushu University Hospital, Fukuoka 812-8582, Japan Received 17 March 2009; received in revised form 27 June 2009; accepted 27 July 2009 Available online 6 August 2009 Many proteins form amyloid-like fibrils in vitro under partially or highly unfolding conditions. Recently, we showed that the residual structure in highly unfolded state is closely related to amyloid fibril formation in hen lysozyme. Thus, to better understand the role of the residual structure on amyloid fibril formation, we focused on AL amyloidosis, which results from the extracellular deposition of monoclonal immunoglobulin light-chain variable domains (V L s) as insoluble fibrils. We examined the relationship between the residual structure and amyloid fibril formation on three λ6 recombinant V L (rVλ6) proteins, wild type, Jto, and Wil. Although rVλ6 proteins are highly unfolded in pH 2, 15 N NMR transverse relaxation experiments revealed nonrandom structures in regions, which include some hydrophobic residues and a single disulfide bond, indicating the existence of residual structure in rVλ6 proteins. However, the residual structure of Wil was markedly disrupted compared with those of the other proteins, despite there being no significant differences in amino acid sequences. Fibrillation experiments revealed that Wil had a longer lag time for fibril formation than the others. When the single disulfide bond was reduced and alkylated, the residual structure was largely disrupted and fibril formation was delayed in all three rVλ6 proteins. It was suggested that the residual structure in highly unfolded state has a crucial role in amyloid fibril formation in many proteins, even pathogenic ones. © 2009 Elsevier Ltd. All rights reserved. Edited by S. Radford Keywords: amyloid fibril; NMR relaxation; residual structure; hydrophobic interaction; disulfide bond Introduction A number of human diseases such as Alzheimer's, Parkinson's, and Creutzfeldt–Jakob originate from the accumulation of misfolded and aggregated proteins. 1–3 Although toxic proteins related to these diseases do not have special characteristics in terms of their three-dimensional structures or amino acid sequences, 4 all are known to form fibrillar aggregates, termed amyloid fibrils. It has also been shown that many proteins unrelated to diseases are able to form amyloid-like fibrils under appropriate conditions, 5–10 suggesting that amyloid fibril formation is a generic property of polypeptide chains. 11 It is widely believed that globular proteins need to unfold, at least partially, to aggregate into amyloid fibrils. 11–13 Moreover, amyloid fibril formation is observed in natively unfolded proteins, such as amyloid-β peptide in Alzheimer's disease, 14,15 and even in small polypep- tides of two to six residues. 16–18 These findings suggest that the protein conformation in the unfolded state is involved in amyloid fibril formation. Various NMR techniques such as 15 N relaxation, 19– 21 paramagnetic relaxation enhancement, 22 and resid- ual dipolar couplings 23,24 have been used to study the unfolded states of proteins. NMR relaxation analysis revealed that hen egg-white lysozyme (HEL) in the reduced form at pH 2 has a residual structure characterized by six hydrophobic clusters, 19,25 despite the lack of any secondary structure. The residual structure of HEL in the reduced form is characterized by six hydrophobic clusters containing tryptophan residues, that is, cluster 2 (Trp28), cluster 3 (Trp62 and Trp63), cluster 5 (Trp108 and Trp111), and cluster 6 *Corresponding author. E-mail address: ueda@phar.kyushu-u.ac.jp. Abbreviations used: EM, electron microscopy; HEL, hen egg-white lysozyme; HSQC, heteronuclear single quantum coherence; ThT, thioflavin T; V L , light-chain variable domain; Vλ6, lambda 6 variable domain. doi:10.1016/j.jmb.2009.07.078 J. Mol. Biol. (2009) 392, 1033–1043 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.