title: Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain.
abstract: Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100°C, a ΔG(D-N) of 15.5 kcal mol(-1) and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 Å and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Porebski
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Adrian A
authors.initials: AA
authors.lastname: Nickson
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia.
authors.forename: David E
authors.initials: DE
authors.lastname: Hoke
authors.affiliation: Centre for Brain Research and Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Morag R
authors.initials: MR
authors.lastname: Hunter
authors.affiliation: Faculty of Information Technology, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Liguang
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Sheena
authors.initials: S
authors.lastname: McGowan
authors.affiliation: Faculty of Information Technology, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Geoffrey I
authors.initials: GI
authors.lastname: Webb
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia ashley.buckle@monash.edu.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Buckle
mesh_terms: D018360:Crystallography, X-Ray; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D005353:Fibronectins; C501364:fibronectin type III like peptide, human
keywords: FN3; consensus design; fibronectin type III; molecular dynamics; stability