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.
Keywords: Langevin dynamics; comparative modeling; mutational studies; protein engineering; thermal stability.
Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.