Two-Dimensional Infrared Correlation Spectroscopy as a Probe of Sequential Events in the Thermal Unfolding of Cytochromesc†

Biochemistry ◽  
2001 ◽  
Vol 40 (28) ◽  
pp. 8256-8263 ◽  
Author(s):  
Angelo Filosa ◽  
Yan Wang ◽  
Ashraf A. Ismail ◽  
Ann M. English
Keyword(s):  
Correlation Spectroscopy ◽  
Thermal Unfolding ◽  
Two Dimensional

scholarly journals Two-dimensional IR correlation spectroscopy of mutants of the β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus identifies the mechanism of quaternary structure stabilization and unravels the sequence of thermal unfolding events

2004 ◽  
Vol 384 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Alessio AUSILI ◽  
Barbara DI LAURO ◽  
Beatrice COBUCCI-PONZANO ◽  
Enrico BERTOLI ◽  
Andrea SCIRÈ ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Ion Pairs ◽  
Sulfolobus Solfataricus ◽  
Correlation Spectroscopy ◽  
Glycoside Hydrolases ◽  
Thermal Unfolding ◽  
Two Dimensional ◽  
Wild Type ◽  
Hyperthermophilic Archaeon ◽  
2D Ir

β-Glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is a homotetramer with a higher number of ion pairs compared with mesophilic glycoside hydrolases. The ion pairs are arranged in large networks located mainly at the tetrameric interface of the molecule. In the present study, the structure and thermal stability of the wild-type β-glycosidase and of three mutants in residues R488 and H489 involved in the C-terminal ionic network were examined by FTIR (Fourier-transform IR) spectroscopy. The FTIR data revealed small differences in the secondary structure of the proteins and showed a lower thermostability of the mutant proteins with respect to the wild-type. Generalized 2D-IR (two-dimensional IR correlation spectroscopy) at different temperatures showed different sequences of thermal unfolding events in the mutants with respect to the wild-type, indicating that punctual mutations affect the unfolding and aggregation process of the protein. A detailed 2D-IR analysis of synchronous maps of the proteins allowed us to identify the temperatures at which the ionic network that stabilizes the quaternary structure of the native and mutant enzymes at the C-terminal breaks down. This evidence gives support to the current theories on the mechanism of ion-pair stabilization in proteins from hyperthermophilic organisms.

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