title: Can template-based protein models guide the design of sequence fitness for enhanced thermal stability of single domain antibodies?
abstract: We investigate the practical use of comparative (template-based) protein models in replica-exchange simulations of single-domain antibody (sdAb) chains to evaluate if the models can correctly predict in rank order the thermal susceptibility to unfold relative to experimental melting temperatures. The baseline model system is the recently determined crystallographic structure of a llama sdAb (denoted as A3), which exhibits an unusually high thermal stability. An evaluation of the simulation results for the A3 comparative model and crystal structure shows that, despite the overall low Cα root-mean-square deviation between the two structures, the model contains misfolded regions that yields a thermal profile of unraveling at a lower temperature. Yet comparison of the simulations of four different comparative models for sdAb A3, C8, A3C8 and E9, where A3C8 is a design of swapping the sequence of the complementarity determining regions of C8 onto the A3 framework, discriminated among the sequences to detect the highest and lowest experimental melting transition temperatures. Further structural analysis of A3 for selected alanine substitutions by a combined computational and experimental study found unexpectedly that the comparative model performed admirably in recognizing substitution 'hot spots' when using a support-vector machine algorithm.
authors.affiliation: Department of Cell Biology and Biochemistry, Molecular and Translational Sciences Division, USAMRIID, Frederick, MD, USA molson@compbiophys.org.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Olson
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Zabetakis
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC, USA.
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Legler
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC, USA.
authors.forename: Kendrick B
authors.initials: KB
authors.lastname: Turner
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC, USA.
authors.forename: George P
authors.initials: GP
authors.lastname: Anderson
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC, USA.
authors.forename: Ellen R
authors.initials: ER
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics*; D013696:Temperature
chemical_list: D061905:Single-Domain Antibodies
keywords: Langevin dynamics; comparative modeling; mutational studies; protein engineering; thermal stability