Both the C- and the N-terminal domains, when in isolation, fold through collapsed, partly folded, transiently populated intermediates before reaching the fully folded state (as evidenced by rollover in the folding limbs of the chevron plots).Parker et al. one domain (Apic et al., 2001;Ekman et al., 2005;Gerstein, 1998;Liu and Rost, 2004;Teichmann et al., 1999). In the context of this review the term domain is defined as a structural, functional, and evolutionary component of proteins, which can often be expressed as a single unit (Murzin et al., 1995). This definition distinguishes multidomain proteins from proteins such as lysozyme, which have two structural domains, but the individual domains are not stable alone and are not found in other protein contexts [and thus are defined together as a single domain in both the SCOP and Pfam databases (http:scop.mrc-lmb.cam.ac.uk and http:pfam.sanger.ac.uk)]. Furthermore, this definition does not include the linear repeat proteins made up of small repeating units which cannot be expressed singly (seeKloss et al., 2008;Main et al., 2005, for recent reviews). Approximately 40%65% of prokaryotic proteins contain multiple domains and the proportion is even higher in eukaryotic proteins (65%80%). Thus,in vivo, most domains fold in the context Rabbit polyclonal to DDX6 of a much larger protein, with neighboring domains. The domains in a multidomain protein often have significant interfaces and can either be attached by short, structured linkers or by longer flexible PROTO-1 linkers. The linkers themselves may be important for the proteins function (Gokhale and Khosla, 2000). There have been few systematic studies of the folding of domains in the context of PROTO-1 larger, multidomain systems. Some of the earlier studies have been discussed in depth in a detailed review byJaenicke (1999), and a more comprehensive review of the folding and evolution of multidomain proteins was published more recently byHan et al. (2007). This work does not aim to be a comprehensive account of all the work that has been done. Instead we aim to illustrate what has been learned PROTO-1 about how multidomain protein systems should be analyzed. A few seminal and careful studies have highlighted some general experimental problems in the analysis of multidomain proteins, which may affect the conclusions drawn. In principle, in a two domain protein system there are three regimes which can occur: (1) Each domain is entirely independent of the other. In this case the stability of each domain will be unaffected by its neighbor and the folding and unfolding rate constants will be the same whether the domain is alone or in the tandem pair. (2) The two domains interact in the native state only. In this case the native state of both domains will be stabilized by the interaction in the fully folded protein. If this is the only effect, then the unfolding rate constants of each domain will be slowed but the folding rate constants will be unaffected. (3) Folding of one domain catalyzes the folding of the second domain. In this case the folding rate constant of the second domain will be affected. Note that, additionally, nonnative interactions PROTO-1 between the two domains can have the effect of slowing folding significantly. == DISTINGUISHING SPECIFIC FROM nonspecific EFFECTS == (i) Deciding on domain boundaries. This is nontrivial. It has been established for some time that cutting a domain too short can lead to a loss in stability of the domain (Hamill et al., 1998;Pfuhl et al., 1997). This arises from the difficulty of establishing precise domain boundaries. Domain boundaries may be determined using sequence alignments, by analysis of proteolytic fragments, by determining regions that crystallize, or by examining crystal or nuclear magnetic resonance (NMR) structures; however, none of these methods is perfect. The best solution is probably by alignment of sequences, although even this may not be successful. In Ig-like domains, for instance, the terminal A and G strands show most variation and are often not used in sequence alignment algorithms, so domain boundaries are hard to define. Structural alignments have to be used in this case. The 16th alpha helical repeat domain of chicken brain -spectrin (R16) has been the subject of a number of studies into the effect of website boundaries on stability, and illustrates the difficulty of determining where domains start and end. There are different opinions as to which residues in R16 should be counted as making up the entire repeat. Pfam (Bateman et al., 2004) defines the repeating unit as 105 residues (with a single one-residue linker between repeats) and gives the boundaries of R16 as residues 17691873. MacDonald and co-workers define a 106 repeat as 17721876 (MacDonald et al., 1994;MacDonald.