The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology

TNfn3, the third fibronectin type III domain of human tenascin, is an immun oglobulin-like protein that is a good model for experimental and theoretica l analyses of Greek key folding. The third fibronectin type III domain of h uman tenascin folds and unfolds in a hue-state fashion over a range of temp erature and pH values, and in the presence of stabilising salts. Here, we p resent a high resolution protein engineering analysis of the single rate de termining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the pr otein exhibit high Phi-values, indicating that they are partially structure d in the transition state. First, a common-core ring of four positions in t he central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed ar e the C' region and the E-F loop. The Phi-values gradually decrease away fr om these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglo bulin-like proteins in which the common-core "ring" forms the nucleus for f olding, whilst the C' and E-F regions are constrained by topology to pack e arly. Folding characteristics of a group of structurally related proteins a ppear to support this model. (C) 2000 Academic Press.