P. Douglas Renfrew

Rosetta3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules

We have recently completed a full re-architecturing of the ROSETTA molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid prototyping of novel protocols by providing easy-to-use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as ROSETTA3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This chapter describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform.

Serverification of Molecular Modeling Applications: The Rosetta Online Server That Includes Everyone (ROSIE)

The Rosetta molecular modeling software package provides experimentally tested and rapidly evolving tools for the 3D structure prediction and high-resolution design of proteins, nucleic acids, and a growing number of non-natural polymers. Despite its free availability to academic users and improving documentation, use of Rosetta has largely remained confined to developers and their immediate collaborators due to the code’s difficulty of use, the requirement for large computational resources, and the unavailability of servers for most of the Rosetta applications. Here, we present a unified web framework for Rosetta applications called ROSIE (Rosetta Online Server that Includes Everyone). ROSIE provides (a) a common user interface for Rosetta protocols, (b) a stable application programming interface for developers to add additional protocols, (c) a flexible back-end to allow leveraging of computer cluster resources shared by RosettaCommons member institutions, and (d) centralized administration by the RosettaCommons to ensure continuous maintenance. This paper describes the ROSIE server infrastructure, a step-by-step ‘serverification’ protocol for use by Rosetta developers, and the deployment of the first nine ROSIE applications by six separate developer teams: Docking, RNA de novo, ERRASER, Antibody, Sequence Tolerance, Supercharge, Beta peptide design, NCBB design, and VIP redesign. As illustrated by the number and diversity of these applications, ROSIE offers a general and speedy paradigm for serverification of Rosetta applications that incurs negligible cost to developers and lowers barriers to Rosetta use for the broader biological community. ROSIE is available at http://rosie.rosettacommons.org.

A Preliminary Survey of the Peptoid Folding Landscape

We present an analysis of the conformational preferences of N-substituted glycine peptoid oligomers. We survey the backbone conformations observed in experimentally determined peptoid structures and provide a comparison with high-level quantum mechanics calculations of short peptoid oligomers. The dominant sources of structural variation derive from: side-chain dependent cis/trans isomerization of backbone amide bonds, side chain stereochemistry, and flexibility in the psi dihedral angle. We find good agreement between the clustering of experimentally determined peptoid torsion angles and local torsional minima predicted by theory for a disarcosine model. The calculations describe a well-defined conformational map featuring distinct energy minima. The general features of the peptoid backbone conformational landscape are consistent across a range of N-alkyl glycine side chains. Alteration of side chain types, however, creates subtle but potentially significant variations in local folding propensities. We identify a limited number of low energy local conformations, which may be preferentially favored by incorporation of particular monomer units. Greater variation in backbone dihedral angles are accessible in peptoids featuring trans amide bond geometries. These results confirm that computational approaches can play a valuable role in guiding the design of complex peptoid architectures and may lead to strategies for introducing constraints that select among a limited number of low energy local conformations.

Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design

Noncanonical amino acids (NCAAs) can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs) to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source), auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/).

The Rosetta All-Atom Energy Function for Macromolecular Modeling and Design

Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosettas success is the energy function: a model parametrized from small-molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, called the Rosetta Energy Function 2015 (REF15). Applying these concepts, we explain how to use Rosetta energies to identify and analyze the features of biomolecular models. Finally, we discuss the latest advances in the energy function that extend its capabilities from soluble proteins to also include membrane proteins, peptides containing noncanonical amino acids, small molecules, carbohydrates, nucleic acids, and other macromolecules.

Adding Diverse Noncanonical Backbones to Rosetta: Enabling Peptidomimetic Design

Peptidomimetics are classes of molecules that mimic structural and functional attributes of polypeptides. Peptidomimetic oligomers can frequently be synthesized using efficient solid phase synthesis procedures similar to peptide synthesis. Conformationally ordered peptidomimetic oligomers are finding broad applications for molecular recognition and for inhibiting protein-protein interactions. One critical limitation is the limited set of design tools for identifying oligomer sequences that can adopt desired conformations. Here, we present expansions to the ROSETTA platform that enable structure prediction and design of five non-peptidic oligomer scaffolds (noncanonical backbones), oligooxopiperazines, oligo-peptoids, -peptides, hydrogen bond surrogate helices and oligosaccharides. This work is complementary to prior additions to model noncanonical protein side chains in ROSETTA. The main purpose of our manuscript is to give a detailed description to current and future developers of how each of these noncanonical backbones was implemented. Furthermore, we provide a general outline for implementation of new backbone types not discussed here. To illustrate the utility of this approach, we describe the first tests of the ROSETTA molecular mechanics energy function in the context of oligooxopiperazines, using quantum mechanical calculations as comparison points, scanning through backbone and side chain torsion angles for a model peptidomimetic. Finally, as an example of a novel design application, we describe the automated design of an oligooxopiperazine that inhibits the p53-MDM2 protein-protein interaction. For the general biological and bioengineering community, several noncanonical backbones have been incorporated into web applications that allow users to freely and rapidly test the presented protocols (http://rosie.rosettacommons.org). This work helps address the peptidomimetic communitys need for an automated and expandable modeling tool for noncanonical backbones.

Engineered coiled-coil protein microfibers.

The fabrication of de novo proteins able to self-assemble on the nano- to meso-length scales is critical in the development of protein-based biomaterials in nanotechnology and medicine. Here we report the design and characterization of a protein engineered coiled-coil that not only assembles into microfibers, but also can bind hydrophobic small molecules. Under ambient conditions, the protein forms fibers with nanoscale structure possessing large aspect ratios formed by bundles of α-helical homopentameric assemblies, which further assemble into mesoscale fibers in the presence of curcumin through aggregation. Surprisingly, these biosynthesized fibers are able to form in conditions of remarkably low concentrations. Unlike previously designed coiled-coil fibers, these engineered protein microfibers can bind the small molecule curcumin throughout the assembly, serving as a depot for encapsulation and delivery of other chemical agents within protein-based 3D microenvironments.

Using quantum mechanics to improve estimates of amino acid side chain rotamer energies.

Amino acid side chains adopt a discrete set of favorable conformations typically referred to as rotamers. The relative energies of rotamers partially determine which side chain conformations are more often observed in protein structures and accurate estimates of these energies are important for predicting protein structure and designing new proteins. Protein modelers typically calculate side chain rotamer energies by using molecular mechanics (MM) potentials or by converting rotamer probabilities from the protein database (PDB) into relative free energies. One limitation of the knowledge‐based energies is that rotamer preferences observed in the PDB can reflect internal side chain energies as well as longer‐range interactions with the rest of the protein. Here, we test an alternative approach for calculating rotamer energies. We use three different quantum mechanics (QM) methods (second order Møller‐Plesset (MP2), density functional theory (DFT) energy calculation using the B3LYP functional, and Hartree‐Fock) to calculate the energy of amino acid rotamers in a dipeptide model system, and then use these pre‐calculated values in side chain placement simulations. Energies were calculated for over 36,000 different conformations of leucine, isoleucine, and valine dipeptides with backbone torsion angles from the helical and strand regions of the Ramachandran plot. In a subset of cases these energies differ significantly from those calculated with standard molecular mechanics potentials or those derived from PDB statistics. We find that in these cases the energies from the QM methods result in more accurate placement of amino acid side chains in structure prediction tests. Proteins 2008.

A rotamer library to enable modeling and design of peptoid foldamers.

Peptoids are a family of synthetic oligomers composed of N-substituted glycine units. Along with other “foldamer” systems, peptoid oligomer sequences can be predictably designed to form a variety of stable secondary structures. It is not yet evident if foldamer design can be extended to reliably create tertiary structure features that mimic more complex biomolecular folds and functions. Computational modeling and prediction of peptoid conformations will likely play a critical role in enabling complex biomimetic designs. We introduce a computational approach to provide accurate conformational and energetic parameters for peptoid side chains needed for successful modeling and design. We find that peptoids can be described by a “rotamer” treatment, similar to that established for proteins, in which the peptoid side chains display rotational isomerism to populate discrete regions of the conformational landscape. Because of the insufficient number of solved peptoid structures, we have calculated the relative energies of side-chain conformational states to provide a backbone-dependent (BBD) rotamer library for a set of 54 different peptoid side chains. We evaluated two rotamer library development methods that employ quantum mechanics (QM) and/or molecular mechanics (MM) energy calculations to identify side-chain rotamers. We show by comparison to experimental peptoid structures that both methods provide an accurate prediction of peptoid side chain placements in folded peptoid oligomers and at protein interfaces. We have incorporated our peptoid rotamer libraries into ROSETTA, a molecular design package previously validated in the context of protein design and structure prediction.

Improved Stability and Half-Life of Fluorinated Phosphotriesterase Using Rosetta

Recently we demonstrated that incorporating p‐fluorophenylalanine (pFF) into phosphotriesterase dramatically improved folding, thereby leading to enhanced stability and function at elevated temperatures. To further improve the stability of the fluorinated enzyme, Rosetta was used to identify multiple potential stabilizing mutations. One such variant, pFF‐F104A, exhibited enhanced activity at elevated temperature and maintained activity over many days in solution at room temperature.