DnaJ proteins are ubiquitous co-chaperones of the DnaK/Hsp70 molecular chaperone family, which are involved in the folding of client proteins and in the prevention of aggregate formation 
. Although DnaJs display a high degree of diversity in terms of their architectural domains and subcellular localizations, all are characterized by the presence of a J-domain, which is responsible for binding to DnaK/Hsp70 and stimulating its ATPase activity 
. In addition, DnaJs bind to client proteins through hydrophobic interactions and present them to DnaK/Hsp70 
, but some can also function as chaperones through their own activity 
DnaJ proteins contain four conserved domains/regions () 
. The highly conserved α-helical J-domain defines DnaJ proteins and is at the N-terminus in Types I and II. Secondly, a disordered Gly/Phe rich region (G/F region) is present which is responsible for flexibility and may function as a linker between the J-domain and the C-terminal region of DnaJ proteins 
. Beyond that, there is no conservation among Type I and II, although some Type II proteins share some C-terminal homology with Type I proteins, but this is not a necessary criteria.
Structural domain arrangements of wild-type Sis1 and mutants.
The sequence and position of the domains defines DnaJ Types I and II 
. In Type I, such as in yeast Ydj1, the central portion between the G/F domain and the C-terminus contains a zinc-finger-like region (ZFLR) whereas in Type II, such as in yeast Sis1, this is replaced by a G/M-rich (Glycine/Methionine-rich) domain () 
. The region known as the C-terminal Domain (CTD) is formed by two structurally similar subdomains (named CTDI and CTDII) composed of β-sheet structure (see ). These two domains dimerize through the C-terminus region and fold into a bent horseshoe dimer 
. Both the CTD and ZFLR are responsible for the chaperone activity of Hsp40s 
Interestingly, Type I and II DnaJs are not equivalent. For example, Ydj1 and Sis1 display divergent activities on yeast Hsp70 and chaperone function 
. Fan et al. 
present interaction data among chaperone substrate models for Ydj1 and Sis1 and two engineered chimeras in which the central part of Sis1 (residues 122–257) was exchanged with the central part of Ydj1 (residues 101–255) and vice versa, generating the chimeras SYS and YSY, respectively. These chimeras switch the specificity for binding substrates and in stimulating firefly luciferase refolding activity by DnaK/Hsp70 
. These results suggest that DnaJ substrate-binding sites are needed for substrate selection in DnaJs, and that the central portion of this protein is important for chaperone activity specification 
. In addition, human Type I and II DnaJs have divergent structures and substrate binding selectivity, possibly regulating the activity of DnaK/Hsp70 by multiple binding site interactions 
. Similarly, structural solution studies suggest that the switch of the central part of Sis1 and Ydj1 also induced an exchange in the structures of the chimeras (SYS and YSY) 
. Interestingly, deletion of the central part of Sis1 did not result in a large change in its overall shape, whereas deletion of the central part of Ydj1 largely altered its overall shape to a conformation similar to that seen for Ydj1 
. All of these observations directly suggest that the central region of DnaJs encodes their structure.
In this study, we applied unfolding strategies for studying the stability of Sis1, two deletion mutants of its central region (Sis1_Δ124–174
) and a chimera formed by the central region of Ydj1 and the N- and C-terminal regions of Sis1 named SYS (Sis11–121
) () 
. Based on thermal unfolding followed by differential scanning calorimetry (DSC), chemical unfolding followed by circular dichroism (CD), analytical ultracentrifugation (AUC), and size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) studies, we suggest that Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. The importance of the C-terminus to Sis1 stabilization and dimerization is addressed below. In addition, we observed by thermodynamic data and nuclear magnetic resonance (NMR) experiments that the G/M region of Sis1 is intrinsically disordered.