Kinetically Robust Monomeric Protein from a Hyperthermophile

Abstract

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl-and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40°C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50°C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (∆G(H 2 O)) at 50°C was 43.6 kJ mol -1 . The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90°C h -1 was higher than at a scan rate of 5°C h -1 . The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k u and ln k r values depended linearly on the denaturant concentration between 10 and 50°C. The ∆G(H 2 O) value obtained from the rate constant in water using the two-state model at 50°C, 44.5 kJ mol -1 , was coincident with that from the equilibrium study, 43.6 kJ mol -1 , suggesting the two-state folding of TkRNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.Microorganisms can be classified into psychrophiles, mesophiles, thermophiles, or hyperthermophiles on the basis of the differences in their optimal growth temperatures. The stabilities of proteins from these microorganisms exhibit different properties. Proteins from hyperthermophiles generally reveal greater stability than those from any other microorganisms. Therefore, proteins from hyperthermophiles are expected to provide unique information about protein folding, stability, and function (1-7).The stability of proteins in solution is evaluated by the Gibbs energy changes (∆G) upon denaturation, when they are reversible under experimental conditions. The value of ∆G is obtained from equilibrium and kinetic experiments. Studies concerning the stability of proteins from hyperthermophiles have focused primarily on the equilibrium aspects (8-13). The results suggested that extremely high stability of these proteins can be achieved by increasing the number of ionic interactions and the extent of hydrophobic surface burial. Several reports have recently appeared in which the equilibrium...

Thermostability of Irreversible Unfolding α-Amylases Analyzed by Unfolding Kinetics

“…Similar to numerous thermophilic proteins, which exhibit very slow unfolding processes, we obtained extremely small unfolding rates for the thermostable BLA originating from a mesophilic source. In contrast to reversible unfolding proteins, where either kinetic or thermodynamic stability can be the key feature in thermostabilization (40), only kinetic barriers determine thermostability for the ␣-amylases with a fast irreversible process. Based on this, mutant studies often revealed enhanced stabilities that are determined kinetically rather than thermodynamically.…”

“…In particular a comparison between mesophilic and thermophilic proteins was the focus of these studies. Besides some exceptions (40,42), most studies revealed significant slower unfolding rates for thermophilic proteins as compared with their mesophilic counterparts. At a reference temperature of 100°C for rubredoxin a difference in unfolding rates of 2-3 orders of magnitude was obtained (35).…”

Hydrophobic Effect on the Stability and Folding of a Hyperthermophilic Protein

“…In our previous study, Tk-RNase HII was found to possess high kinetic stability and the T m value was found to depend on the scan rate of heating. 18 Therefore, the heat-induced unfolding experiments on Tk-RNase HII cannot be evaluated quantitatively due to kinetic stability. However, these results demonstrate that every mutant protein is destabilized against heatinduced unfolding compared with the WT protein.…”

“…[15][16][17] Tk-RNase HII is highly stable, and its stabilization mechanism is characterized by its remarkably slow unfolding. [18][19][20] It has also been reported that the *Corresponding author. E-mail address: ktakano@mls.eng.osaka-u.ac.jp.…”

Structural and dynamical features contributing to thermostability in α-amylases

“…Following transition state theory, not only DG but also the barrier height of the transition state determines the folding and unfolding rates and therefore controls protein stability with respect to the corresponding time regime. Recently it was demonstrated in some cases that a higher thermostability was accompanied by significantly smaller unfolding rates (K u ) as compared with their mesophilic homologues [8,[56][57][58]. In contrast, at least for proteins with reversible unfolding, only small differences in the (re-)folding rates (K f ) were observed [56].…”