INTRODUCTION: The mouse Major Urinary Proteins constitute a family of pheromone binding proteins, while representing the major mouse allergen for humans. They belong to the Lipocalins superfamily sharing an eight-stranded, antiparallel β-barrel fold stabilized by a highly conserved disulfide bridge. Here, we have investigated the role of the disulfide bridge (C64-C157) and of the free C138 residue on the protein folding stability and ligand-binding property. METHODS: The three recombinant proteins bearing the substitutions C138A, C157A or C138-157A were studied by means of Circular Dichroism and intrinsic/extrinsic Fluorescence spectroscopies. RESULTS: Preliminary structural characterization confirmed that the mutants retain a native-like fold. The mutant C138A was able to bind the fluorescent probe N-phenyl-naphthylamine with the same affinity of the wt protein, while the mutants bearing the substitution of C157 revealed a weaker binding of the ligand. Thermal unfolding experiments attributed a significant loss of thermal stability only to the mutants C157A and C138-157A. Similarly, GndHCl unfolding studies revealed that the substitution of C157 residue determined an increased sensitivity to the denaturant. Complete reversibility of the denaturation process was obtained only with C138A which, after gradual sample cooling or progressive denaturant subtraction, recovered the native conformational properties. CONCLUSIONS: We ascribe the decrease of the binding affinity, of the thermal stability and of the resistance to GndHCl observed for C157A to the lack of the disulfide bridge. It is worth noting that the mutation C138A does not perturb the protein functionality, and in addition it suppresses the free and reactive thiol group that contributes to incorrect disulphide bond formation and irreversible unfolding. Further studies will take advantage of this stable and refolding protein to investigate its potential role both as carrier protein and human allergen.

Conserved cysteines mutagenesis in Major Urinary Protein: a new insight in folding stability and reversibility / Ferrari, Elena; Corsini, Romina; Rizzatello, Giulia; Spisni, Alberto. - ELETTRONICO. - (2015), pp. 204-204. ((Intervento presentato al convegno 58th National Meeting of the Italian Society of Biochemistry and Molecular Biology tenutosi a Urbino nel 14-16 Settembre 2015.

Conserved cysteines mutagenesis in Major Urinary Protein: a new insight in folding stability and reversibility

FERRARI, Elena;CORSINI, Romina;RIZZATELLO, GIULIA;SPISNI, Alberto
2015

Abstract

INTRODUCTION: The mouse Major Urinary Proteins constitute a family of pheromone binding proteins, while representing the major mouse allergen for humans. They belong to the Lipocalins superfamily sharing an eight-stranded, antiparallel β-barrel fold stabilized by a highly conserved disulfide bridge. Here, we have investigated the role of the disulfide bridge (C64-C157) and of the free C138 residue on the protein folding stability and ligand-binding property. METHODS: The three recombinant proteins bearing the substitutions C138A, C157A or C138-157A were studied by means of Circular Dichroism and intrinsic/extrinsic Fluorescence spectroscopies. RESULTS: Preliminary structural characterization confirmed that the mutants retain a native-like fold. The mutant C138A was able to bind the fluorescent probe N-phenyl-naphthylamine with the same affinity of the wt protein, while the mutants bearing the substitution of C157 revealed a weaker binding of the ligand. Thermal unfolding experiments attributed a significant loss of thermal stability only to the mutants C157A and C138-157A. Similarly, GndHCl unfolding studies revealed that the substitution of C157 residue determined an increased sensitivity to the denaturant. Complete reversibility of the denaturation process was obtained only with C138A which, after gradual sample cooling or progressive denaturant subtraction, recovered the native conformational properties. CONCLUSIONS: We ascribe the decrease of the binding affinity, of the thermal stability and of the resistance to GndHCl observed for C157A to the lack of the disulfide bridge. It is worth noting that the mutation C138A does not perturb the protein functionality, and in addition it suppresses the free and reactive thiol group that contributes to incorrect disulphide bond formation and irreversible unfolding. Further studies will take advantage of this stable and refolding protein to investigate its potential role both as carrier protein and human allergen.
Conserved cysteines mutagenesis in Major Urinary Protein: a new insight in folding stability and reversibility / Ferrari, Elena; Corsini, Romina; Rizzatello, Giulia; Spisni, Alberto. - ELETTRONICO. - (2015), pp. 204-204. ((Intervento presentato al convegno 58th National Meeting of the Italian Society of Biochemistry and Molecular Biology tenutosi a Urbino nel 14-16 Settembre 2015.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2801425
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