title: Prediction of folding stability and degradability of the de novo designed protein MB-1 in cow rumen.
abstract: The authors have recently reported on the design of a protein (MB-1) enriched in methionine, threonine, lysine, and leucine. The protein is intended to be produced by rumen bacteria, in a way that would provide high producing lactating cows with limiting amino acids. In this report, MB-1 stability in the rumen is assessed, i.e., where the protein might be found after cell lysis or after being secreted by rumen bacteria. Current in vitro methods used to predict proteolytic degradability in the rumen were used for MB-1, as well as other natural proteins for comparison. MB-1 was found to be more susceptible to degradation than cytochrome c and ribonuclease A. Data indicate that MB-1 will be rapidly degraded if exposed to the rumen environment without protection. The contribution of folding stability to proteolytic stability was also examined. Rumen liquor components were selected to formulate a solution compatible with constraints of thermal denaturation studies. Denaturation curves show that the natural proteins were folded at rumen temperature. The MB-1 denaturation curves indicated that MB-1 does not unfold in a cooperative transition when heated from 20 to 70 degrees C. This suggests that MB-1 structure may be progressively modified as temperature increases, and that a continuum of conformations are available to MB-1. At 39 degrees C, a significant (50%) portion of MB-1 molecules had their tertiary structure unfolded, contributing to proteolytic degradability. Despite the unusual constraints used in MB-1 design (i.e., a maximized content in selected essential amino acids), results show that MB-1 has structural properties similar to previously reported de novo designed proteins.
authors.affiliation: Chemistry Department, University of Prince Edward Island, Charlottetown, Canada.
authors.forename: J D
authors.initials: JD
authors.lastname: MacCallum
authors.forename: M A
authors.initials: MA
authors.lastname: Hefford
authors.forename: S
authors.initials: S
authors.lastname: Omar
authors.forename: M
authors.initials: M
authors.lastname: Beauregard
mesh_terms: D000601:Amino Acids, Essential / Q000008:administration & dosage / Q000378:metabolism; D000821:Animal Feed; D000818:Animals; D002417:Cattle; D004044:Dietary Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012417:Rumen / Q000378:metabolism* / Q000382:microbiology; D012996:Solutions
chemical_list: D000601:Amino Acids, Essential; D004044:Dietary Proteins; D005227:Fatty Acids; C107457:MB-1synthetic dietary protein; D011994:Recombinant Proteins; D012996:Solutions