Computational engineering of bionanostructures Ram Samudrala University of Washington How can we analyse, design, & engineer peptides capable of specific binding properties and activities?

A comprehensive computational approach Sequence-based informatics - analyse sequence patterns responsible for binding specificity within experimentally characterised binders by creating specialised similarity matrices Structure-based informatics - analyse structural patterns within experimental characterised binders by performing de novo simulations both in the presence and absence of substrate Computational design - use de novo protocol to predict structures of the best candidate peptides or peptide assemblies, with validation by further experiment

Sequence-based informatics Create specialised similarity matrices by optimising the alignment scores such that strong, moderate, and weak binders for a given inorganic substrate cluster together – determines sequences patterns: Ersin Emre Oren (Sarikaya group)

Protein folding …-L-K-E-G-V-S-K-D-… …-CTA-AAA-GAA-GGT-GTT-AGC-AAG-GTT-… one amino acid Gene Protein sequence Unfolded protein Native biologically relevant state spontaneous self-organisation (~1 second) not unique mobile inactive expanded irregular

Protein folding …-L-K-E-G-V-S-K-D-… …-CTA-AAA-GAA-GGT-GTT-AGC-AAG-GTT-… one amino acid Gene Protein sequence Unfolded protein Native biologically relevant state spontaneous self-organisation (~1 second) unique shape precisely ordered stable/functional globular/compact helices and sheets not unique mobile inactive expanded irregular

Structure-based informatics: De novo prediction of protein structure astronomically large number of conformations 5 states/100 residues = = select hard to design functions that are not fooled by non-native conformations (“decoys”) sample conformational space such that native-like conformations are found

Semi-exhaustive segment-based folding EFDVILKAAGANKVAVIKAVRGATGLGLKEAKDLVESAPAALKEGVSKDDAEALKKALEEAGAEVEVK generate Make random moves to optimise what is observed in known structures …… minimise Find the most protein-like structures …… filter all-atom pairwise interactions, bad contacts compactness, secondary structure, consensus of generated conformations

CASP prediction for T Å C α RMSD for all 53 residues Ling-Hong Hung/Shing-Chung Ngan

CASP prediction for T Å C α RMSD for all 70 residues

CASP prediction for T Å Cα RMSD for 84 residues

CASP prediction for T Å Cα RMSD for 67 residues

CASP prediction for T Å Cα RMSD for all 69 residues

Structure-based informatics Make predictions of peptides without the presence of substrates using de novo protocol Make predictions of peptides in the presence of substrates using physics-based force-fields such as GROMACS Analyse for similarity of structures (local and global) as well as common contact patterns between atoms in amino acids – the structural similarities and patterns give us the structural patterns responsible for folding and inorganic substrate binding Perform higher-order simulations that involve many copies of a single or multiple peptides to generate sequences with specific stabilities and inorganic binding properties – larger assemblies for more controlled binding

Computational design Select the most promising candidate peptides generated from the sequence- and structure-based informatics for further simulation and design Simulations can be performed to ensure that active sites and/or topologies found in nature are grafted onto these peptides Experimental validation – synthesise peptides and check for binding activity Main goal here is to help with rational design of inorganic binding peptides and focus experimental efforts in a more optimal manner A good framework to obtain knowledge obtained experimentally with state of the protein structure prediction methodologies

oxidoreductase transferasehydrolaseligaselyase Grafting of biological active sites onto engineered peptides TIM barrel proteins 2246 with known structure

Acknowledgements Samudrala group: Aaron Chang Chuck Mader David Nickle Ekachai Jenwitheesuk Gong Cheng Jason McDermott Jeremy Horst Sarikaya group: Ersin Emre Oren National Institutes of Health National Science Foundation Searle Scholars Program (Kinship Foundation) Puget Sound Partners in Global Health UW Advanced Technology Initiative in Infectious Diseases Kai Wang Ling-Hong Hung Michal Guerquin Shing-Chung Ngan Stewart Moughon Tianyun Lu Zach Frazier