title: The effect of boundary selection on the stability and folding of the third fibronectin type III domain from human tenascin.
abstract: Correct selection of domain boundaries is critical for structural analysis of single domains from multimodular proteins. Folding and stability studies of the third fibronectin type III domain from human tenascin (TNfn31-90) have shown that it is moderately stable (Delta G(D-N)(H2O) approximately 5 kcal mol-1) and folds with two-state kinetics. In an attempt to stabilize the protein, five domains were constructed with different combinations of extensions to the N- and C-termini. Thermal denaturation studies show that a specific two amino acid (Gly-Leu) extension at the C-terminus is primarily responsible for a significant increase in stability. The Delta Delta G(D-N)(H2O) of the Gly-Leu extension (TNfn3(1-92)) is 2.7 +/- 0.3 kcal mol-1. Refolding kinetics do not differ significantly, but unfolding is slowed 40-fold. Mutation of leucine 92 to alanine does not affect stability, indicating that the stability of the extension does not come from the packing of the leucine side chain. Hydrogen exchange data suggest that the extension adds new hydrogen bonds and strengthens existing hydrogen bonds in the C-terminal interaction with the A-B and E-F loops. Removal of a very small number of hydrogen bonds substantially increases the unfolding rate, a phenomenon which may be important in stress-relaxation of FNIII-containing muscle proteins such as titin. These experiments demonstrate the importance of a small number of additional long-range interactions in the overall formation of a compact independently folding beta-sheet module.
authors.affiliation: MRC Centre for Protein Engineering, Department of Chemistry, University of Cambridge, U.K.
authors.forename: S J
authors.initials: SJ
authors.lastname: Hamill
authors.forename: A E
authors.initials: AE
authors.lastname: Meekhof
authors.forename: J
authors.initials: J
authors.lastname: Clarke
mesh_terms: D000409:Alanine / Q000235:genetics; D002942:Circular Dichroism; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006859:Hydrogen; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019063:Tenascin / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D005353:Fibronectins; D019063:Tenascin; D006859:Hydrogen; D007930:Leucine; D000409:Alanine