title: Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy.
abstract: A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.
authors: Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors: Pietro
authors: P
authors: Alfarano
authors: Gautham
authors: G
authors: Varadamsetty
authors: Christina
authors: C
authors: Ewald
authors: Fabio
authors: F
authors: Parmeggiani
authors: Riccardo
authors: R
authors: Pellarin
authors: Oliver
authors: O
authors: Zerbe
authors: Andreas
authors: A
authors: Plückthun
authors: Amedeo
authors: A
authors: Caflisch
mesh_terms: D000818:Animals; D051186:Armadillo Domain Proteins / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: D051186:Armadillo Domain Proteins; D029701:Saccharomyces cerevisiae Proteins