Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.
Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules.
Bismuto
Ettore
E
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Napoli, Italy. ettore.bismuto@unina2.it
Febbraio
Ferdinando
F
Limongelli
Simona
S
Briante
Raffaella
R
Nucci
Roberto
R
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005453:Fluorescence; D005470:Fluorometry; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase