Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.
Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.
Doucet
Alain
A
Groupe de Recherche en Energie et Information Biomoléculaire, Département de Chimie-Biologie, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec G9A 5H7, Canada.
Williams
Martin
M
Gagnon
Mylene C
MC
Sasseville
Maxime
M
Beauregard
Marc
M
D002942:Circular Dichroism; D004044:Dietary Proteins; D004220:Disulfides / Q000737:chemistry*; D009154:Mutation; D010316:Particle Size; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins
D004044:Dietary Proteins; D004220:Disulfides; C107457:MB-1synthetic dietary protein; D011506:Proteins