The International Journal of Biochemistry & Cell Biology 35 (2003) 1452–1460 A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein Elvira R. Rafikova a , Boris I. Kurganov b , Alexander M. Arutyunyan a , Stanislav V. Kust a , Vladimir A. Drachev a , Evgeny N. Dobrov a,∗ a A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 11999, Russia b Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia Received 19 December 2002 Abstract To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 ◦ C, the TMV CP passed very rapidly (in less than 15 s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Tobacco mosaic virus coat protein; Aggregation; Denaturation; Kinetics; Ionic strength 1. Introduction Mature, relatively ordered protein aggregates called amyloid fibrils, or plaques have been implicated in pathogenesis of prion infections and amyloid diseases for over several recent years, which constitute the whole history of their molecular biological studies. However, newer evidence suggests that some nonfib- Abbreviations: CP, coat protein; PB, K + /K + phosphate buffer; PVX, potato virus X; TMV, tobacco mosaic virus; ToMV, tomato mosaic virus ∗ Corresponding author. Tel.: +7-95-939-5008; fax: +7-95-939-3181. E-mail address: dobrov@belozersky.msu.ru (E.N. Dobrov). rillar intermediate products, rather than mature amy- loid fibrils, play the major role in their pathogenesis (Conway et al., 2000; Hartley et al., 1999; Lambert et al., 1998). Interestingly, under certain conditions, amyloid fibrils can originate from misfolding of nor- mal proteins and peptides, not associated with any pathology (Chiti et al., 1999; Fandrich, Fletcher, & Dobson, 2001; Guijarro, Sunde, Jones, Campbell, & Dobson, 1998). In April 2002, a study from Prof. C.M. Dobson’s laboratory was published demon- strating that prefibrillar (amorphous and granular) aggregates of fragments of “normal” proteins, but not the mature fibrils, produced cytotoxic effects in cell cultures (Bucciantini et al., 2002). These results im- plicate amorphous “nonspecific” protein aggregates 1357-2725/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1357-2725(03)00106-7