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

Abstract

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 Ala can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor.

The novel anti-cancer feature of Brazzein through activating of hTLR5 by integration of biological evaluation: molecular docking and molecular dynamics simulation

“…Given the high thermal stability and low chemical stability of Brazzein reported in the previous studies, the presence of disulfide bonds profoundly influenced the structural integrity of the protein 5 . In summary, the antithetical properties of p.A19K mutants on the protein activity revealed that the presence of charged residue at this position may exert a profound effect on the interaction between protein and corresponding receptor.…”

“…Further, Brazzein protein is isolated from the fruit of Pentadiplandra brazzeana Baillon , composing of the three strands of antiparallel β-sheet, as well as a short α-helix connected by loops. It consists of 54 residues and has a molecular weight of about 6.5 kDa, in the structure of which eight Cys residues generate four disulfide bonds between the beta-sheets 5 , 6 . The protein is predicted to exhibit antibacterial and antifungal properties due to specific structural characteristics.…”

“…Regarding the Brazzein, the loops are mostly on the surface, at which the p.A19K mutation (point mutation) is located. The mutant is a positive charge which can more interact with its environment 5 , 6 .…”

Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris

“…Although some models of receptor activation by brazzein have been proposed, the exact mechanism is not known. Interestingly, substitutions of some amino acid residues have been shown to significantly increase the sweetness of brazzein [7,10,11].…”

Positive Charges on the Surface of Thaumatin Are Crucial for the Multi-Point Interaction with the Sweet Receptor

“…Since sweet-tasting proteins are useful as low-calorie sweeteners as well as a substitute for sucrose, a number of investigations have been performed to clarify why sweet-tasting proteins elicit a sweet taste, and how sweet-tasting proteins interact with sweet receptor (Kohmura et al, 1992 ; Kim and Weickmann, 1994 ; Slootstra et al, 1995 ; Somoza et al, 1995 ; Assadi-Porter et al, 2000 , 2010a , b ; Kaneko and Kitabatake, 2001 ; Temussi, 2002 , 2006 , 2011 ; Jin et al, 2003 ; Jiang et al, 2004 ; Tancredi et al, 2004 ; Masuda et al, 2005a , b , 2013 , 2016 ; Esposito et al, 2006 ; Walters and Hellekant, 2006 ; Koizumi et al, 2007 , 2015 ; Kurimoto et al, 2007 ; Li et al, 2008 ; Nakajima et al, 2008 , 2011 ; Ohta et al, 2008 , 2011a , b ; Ide et al, 2009 ; Walters et al, 2009 ; Xue et al, 2009 ; Dittli et al, 2011 ; Templeton et al, 2011 ; Liu et al, 2012 , 2016 ; Cornilescu et al, 2013 ; Lee et al, 2013 ; Rega et al, 2015 ; Ghanavatian et al, 2016 ; Leone et al, 2016 ; Lim et al, 2016 ; Singarapu et al, 2016 ).…”