Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus.


Abstract

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate. Study holds ProTherm entries: 20117, 20118, 20119, 20120, 20121, 20122, 20123, 20124 Extra Details: Heating scan is 0.17 K/min. The data are for teh transition from native to denatured state. thermal unfolding, TIM-barrel protein, unfolding intermediate state, calorimetry, circular dichroism.

Submission Details

ID: 6AQ6oJkU3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Pyrpassopoulos S;Vlassi M;Tsortos A;Papanikolau Y;Petratos K;Vorgias CE;Nounesis G,Proteins (2006) Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus. PMID:16685709
Additional Information

Sequence Assay Result Units