Two-dimensional IR correlation spectroscopy of mutants of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus identifies the mechanism of quaternary structure stabilization and unravels the sequence of thermal unfolding events.
Beta-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 beta-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.
Ausili
Alessio
A
Institute of Biochemistry, Faculty of Sciences, Università Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy.
Di Lauro
Barbara
B
Cobucci-Ponzano
Beatrice
B
Bertoli
Enrico
E
Scirè
Andrea
A
Rossi
Mosè
M
Tanfani
Fabio
F
Moracci
Marco
M
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D006639:Histidine; D001120:Arginine; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase; D000409:Alanine