Structural basis of the destabilization produced by an amino-terminal tag in the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus.
We have previously shown that the major ion-pairs network of the tetrameric beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the beta-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85-95 degrees C and decreased the Tm of the protein of 6 degrees C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 degrees C whilst that of the wild type enzyme is stable up to 98 degrees C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the beta-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.
Ausili
A
A
Institute of Biochemistry, Università Politecnica delle Marche, Via Ranieri, 60131 Ancona, Italy.
Cobucci-Ponzano
B
B
Di Lauro
B
B
D'Avino
R
R
Scirè
A
A
Rossi
M
M
Tanfani
F
F
Moracci
M
M
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013329:Structure-Activity Relationship; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase