Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine

• Published in: Biochimie 2016-12, Vol.131, p.20-28
• Main Authors: Ghanavatian, Parisa, Khalifeh, Khosrow, Jafarian, Vahab
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Alanine - genetics; Alanine - metabolism; Amino Acid Substitution; Brazzein; Circular Dichroism; Crystallography, X-Ray; Docking; Equilibrium; Interaction; Models, Molecular; Mutation; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Protein Binding; Protein Denaturation; Protein Domains; Protein Folding; Protein Structure, Secondary; Site-directed mutagenesis; Sweetening Agents - chemistry; Sweetening Agents - metabolism; Sweetness; Thermodynamics
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2016.09.006

Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor. •Position 19 of Brazzein was identified as a key point for enhancing the protein sweetness.•A19K mutant as more sweetener mutants can forms more stabilized complex with sweet taste receptor.•A19D mutant forms more destabilized complex with sweet taste receptor and has lower sweetening power.•Structural Stability of A19G mutant is greater that of other variants.