Folding and stability of endoglucanase III, a single-domain cellulase from Trichoderma reesei.
The reversible folding of an endoglucanase (EGIII) from the filamentous fungus Trichoderma reesei was investigated by activity, tryptophan fluorescence, and peptide CD measurements. Equilibrium stability was determined by urea denaturation at various pH and temperature values. Unfolding and refolding rates were measured over a range of urea concentrations. The data from the equilibrium and kinetic studies fit a simple two-state model, except at lower urea concentrations, where the folding kinetics indicate a transient intermediate. Unfolding is very slow, with a half-life of about 2 h in 8 M urea at pH 5.5 and 25 degrees C. Comparison of the urea dependence of the folding kinetics and equilibrium indicates the protein undergoes 93% of its total change in solvent exposure on going from the unfolded state to the transition state. Thus, the transition state is quite compact. The presence of dithiothreitol destabilized the protein by 7 kcal/mol, indicating the presence of an unusually strong disulfide linkage between the two cysteines in the molecule. Protein stability is dramatically reduced at alkaline pH values; this can be attributed to a titratable shift (pKa = 7.8) in the slope of the urea dependence of unfolding.
Arunachalam
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U
Genencor International, Inc., Palo Alto, California 94304, USA.
Kellis
J T
JT
D001426:Bacterial Proteins; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014242:Trichoderma / Q000201:enzymology*; D014364:Tryptophan / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D004220:Disulfides; D014364:Tryptophan; D014508:Urea; D002480:Cellulase; C061269:cel-3 protein, Fibrobacter succinogenes; D004229:Dithiothreitol