We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.The study of the folding, association, and disassembly of large multimeric proteins is complicated by their size, the general irreversibility of the reactions involved, and the frequent occurrence of off-pathway intermediates. However, the significant advances already made hold promise for a detailed understanding of these processes (1). Spherical virus capsids are large, multimeric proteins (2-5) and constitute attractive models for the study of the association, stability, and disassembly of very large protein complexes (for reviews see Refs. 4 and 6 -13). In addition, viral capsids are highly dynamic entities and have evolved unique structural solutions in response to the diverse, sometimes conflicting functions they must perform during the virus life cycle (4, 7, 11, 14 -16). Thus, they provide good opportunities to understand finely tuned structure-function relationships in proteins and to develop new antiviral approaches based on the inhibition of assembly or uncoating (15,17, 18).The icosahedral T ϭ 1 capsids of parvoviruses (19 -24) are formed by 60 protein subunits contributed by three nonidentical polypeptide chains that show, however, identical -fold and core sequence. VP2 is the major capsid protein and can selfassemble into empty (DNA-free) capsids (viral-like particles or VLPs).1 VP1, a minor component of natural capsids, includes...