title: Stabilization of the third fibronectin type III domain of human tenascin-C through minimal mutation and rational design.
abstract: Non-antibody scaffolds are increasingly used to generate novel binding proteins for both research and therapeutic applications. Our group has developed the tenth fibronectin type III domain of human tenascin-C (TNfn3) as one such scaffold. As a scaffold, TNfn3 must tolerate extensive mutation to introduce novel binding sites. However, TNfn3's marginal stability (T(m) ∼ 59°C, ΔG(unfolding) = 5.7 kcal/mol) stands as a potential obstacle to this process. To address this issue, we sought to engineer highly stable TNfn3 variants. We used two parallel strategies. Using insights gained from structural analysis of other FN3 family members, we (1) rationally designed stabilizing point mutations or (2) introduced novel stabilizing disulfide bonds. Both strategies yielded highly stable TNfn3 variants with T(m) values as high as 83°C and ΔG(unfolding) values as high as 9.4 kcal/mol. Notably, only three or four mutations were required to achieve this level of stability with either approach. These results validate our rational design strategies and illustrate that substantial stability increases can be achieved with minimal mutation. One TNfn3 variant reported here has now been successfully used as a scaffold to develop two promising therapeutic molecules. We anticipate that other variants described will exhibit similar utility.
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, MD 20878, USA.
authors.forename: R N
authors.initials: RN
authors.lastname: Gilbreth
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, MD 20878, USA.
authors.forename: B M
authors.initials: BM
authors.lastname: Chacko
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, MD 20878, USA.
authors.forename: L
authors.initials: L
authors.lastname: Grinberg
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, MD 20878, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Swers
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, MD 20878, USA bacam@medimmune.com.
authors.forename: M
authors.initials: M
authors.lastname: Baca
mesh_terms: D000595:Amino Acid Sequence; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000235:genetics*; D016415:Sequence Alignment; D019063:Tenascin / Q000737:chemistry* / Q000235:genetics*
chemical_list: D005353:Fibronectins; D019063:Tenascin
keywords: alternative scaffold; disulfide engineering; fibronectin type III; protein stability engineering; structure-guided design