Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains.

Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be. Equilibrium studies show an increase in denaturant concentration required to unfold the domains with only a single unfolding transition; however, in some cases, this is not accompanied by the increase in m value, which would be expected if the protein is a truly cooperative, all-or-none system. We analyze the complex kinetics of spectrin domain pairs, both wild-type and carefully selected mutants. By comparing these pairs, we are able to demonstrate that equilibrium data alone are insufficient to describe the folding of multidomain proteins and to quantify the effects that one domain can have on its neighbor.