title: Design of high essential amino acid proteins: two design strategies for improving protease resistance of the nutritious MB-1 protein.
abstract: Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations we face in this endeavour is achieving stable proteins despite a highly biased amino acid content. We report here the synthesis and characterisation of two mutants derived from our MB-1 designer protein. The first mutant contains a disulphide bridge designed to cross-link remote segments of the polypeptide chain. The second one is a Tyr62-Trp mutant, where position 62 is buried in the core of the protein. Both mutants were found to be largely helical as per design, and based on thermal denaturation experiments, were substantially more stable than the MB-1 parent molecule. Enhancement of conformational stability in MB-1Trp translated into an impressive improvement of its ability to resist proteolytic degradation. Furthermore, digestion experiments intended to model degradation of proteins in a cow's rumen revealed that MB-1Trp's resistance to degradation compared to that of cytochrome c. Design strategies used for these mutants are discussed with regards to their applicability in creating efficient nutritional proteins.
authors.affiliation: Groupe de recherche en énergie et information biomoléculaire, Département de chimie-biologie, Université du Québec à Trois-Rivières, CP 500, Trois-Rivières, Que., Canada G9A 5H7.
authors.forename: M
authors.initials: M
authors.lastname: Williams
authors.forename: M C
authors.initials: MC
authors.lastname: Gagnon
authors.forename: A
authors.initials: A
authors.lastname: Doucet
authors.forename: M
authors.initials: M
authors.lastname: Beauregard
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000601:Amino Acids, Essential / Q000378:metabolism; D000821:Animal Feed; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004044:Dietary Proteins / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D011402:Pronase / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011506:Proteins
chemical_list: D000601:Amino Acids, Essential; D004044:Dietary Proteins; D004220:Disulfides; C107457:MB-1synthetic dietary protein; D011506:Proteins; D010450:Endopeptidases; D011402:Pronase; D003545:Cysteine