THE EFFECT OF BOUNDARY SELECTION ON THE STABILITY AND FOLDING OF THE 3RD FIBRONECTIN TYPE-III DOMAIN FROM HUMAN TENASCIN

Correct selection of domain boundaries is critical for structural anal ysis of single domains from multimodular proteins. Folding and stabili ty studies of the third fibronectin type III domain from human tenasci n (TNfn3(1-90)) have shown that it is moderately stable (Delta G(D-N)( H2O) similar to 5 kcal mol(-1)) and folds with two-state kinetics. In an attempt to stabilize the protein, five domains were constructed wit h different combinations of extensions to the N- and C-termini. Therma l denaturation studies show that a specific two amino acid (Gly-Leu) e xtension to the C-terminus is primarily responsible for a significant increase in stability. The Delta Delta G(D-N)(H2O) of the Gly-Leu exte nsion (TNfn3(1-92)) is 2.7 +/- 0.3 kcal mol(-1). Refolding kinetics do not differ significantly, but unfolding is slowed 40-fold. Mutation o f leucine 92 to alanine does not affect stability, indicating that the stability of the extension does not come from the packing of the leuc ine side chain. Hydrogen exchange data suggest that the extension adds new hydrogen bonds and strengthens existing hydrogen bonds in the C-t erminal interaction with the A-B and E-F loops. Removal of a very smal l number of hydrogen bonds substantially increases the unfolding rate, a phenomenon which may be important in stress-relaxation of FNIII-con taining muscle proteins such as titin. These experiments demonstrate t he importance of a small number of additional long-range interactions in the overall formation of a compact independently folding beta-sheet module.