Interlandi G Wetzel SK Settanni G Plückthun A Caflisch A 2008 Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between beta-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus. application/pdf https://sonar.ch/global/documents/4313 eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2007.09.042 info:eu-repo/semantics/altIdentifier/pmid/18048057 info:eu-repo/semantics/openAccess Journal of molecular biology. - 2008 Amino Acid Sequence Ankyrin Repeat Ankyrins Arginine Binding Sites Circular Dichroism Combinatorial Chemistry Techniques Computer Simulation Consensus Sequence Escherichia coli Glutamic Acid Histidine Hot Temperature Hydrogen Bonding Hydrogen-Ion Concentration Hydrophobic and Hydrophilic Interactions Light Models, Chemical Molecular Sequence Data Point Mutation Protein Binding Protein Conformation Protein Denaturation Protein Engineering Protein Structure, Secondary Protein Structure, Tertiary Reproducibility of Results Scattering, Radiation Solubility Temperature Thermodynamics Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments. http://purl.org/coar/resource_type/c_6501