Brian Kuhlman

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 Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines.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.

A genetically encoded photoactivatable Rac controls the motility of living cells

The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV–Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins.

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.

Computer-based redesign of a protein folding pathway

A fundamental test of our current understanding of protein folding is to rationally redesign protein folding pathways. We use a computer-based design strategy to switch the folding pathway of protein G, which normally involves formation of the second, but not the first, β-turn at the rate limiting step in folding. Backbone conformations and amino acid sequences that maximize the interaction density in the first β-hairpin were identified, and two variants containing 11 amino acid replacements were found to be ∼4 kcal mol−1 more stable than wild type protein G. Kinetic studies show that the redesigned proteins fold ∼100× faster than wild type protein and that the first β-turn is formed and the second disrupted at the rate limiting step in folding.

Labelling and optical erasure of synaptic memory traces in the motor cortex

Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.

An improved protein decoy set for testing energy functions for protein structure prediction.

We have improved the original Rosetta centroid/backbone decoy set by increasing the number of proteins and frequency of near native models and by building on sidechains and minimizing clashes. The new set consists of 1,400 model structures for 78 different and diverse protein targets and provides a challenging set for the testing and evaluation of scoring functions. We evaluated the extent to which a variety of all‐atom energy functions could identify the native and close‐to‐native structures in the new decoy sets. Of various implicit solvent models, we found that a solvent‐accessible surface area–based solvation provided the best enrichment and discrimination of close‐to‐native decoys. The combination of this solvation treatment with Lennard Jones terms and the original Rosetta energy provided better enrichment and discrimination than any of the individual terms. The results also highlight the differences in accuracy of NMR and X‐ray crystal structures: a large energy gap was observed between native and non‐native conformations for X‐ray structures but not for NMR structures. Proteins 2003.

E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer

During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.

Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface

Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain–light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

A "solvated rotamer" approach to modeling water-mediated hydrogen bonds at protein-protein interfaces

Water‐mediated hydrogen bonds play critical roles at protein–protein and protein–nucleic acid interfaces, and the interactions formed by discrete water molecules cannot be captured using continuum solvent models. We describe a simple model for the energetics of water‐mediated hydrogen bonds, and show that, together with knowledge of the positions of buried water molecules observed in X‐ray crystal structures, the model improves the prediction of free‐energy changes upon mutation at protein–protein interfaces, and the recovery of native amino acid sequences in protein interface design calculations. We then describe a “solvated rotamer” approach to efficiently predict the positions of water molecules, at protein–protein interfaces and in monomeric proteins, that is compatible with widely used rotamer‐based side‐chain packing and protein design algorithms. Finally, we examine the extent to which the predicted water molecules can be used to improve prediction of amino acid identities and protein–protein interface stability, and discuss avenues for overcoming current limitations of the approach. Proteins 2005.

RosettaDesign server for protein design

The RosettaDesign server identifies low energy amino acid sequences for target protein structures (). The client provides the backbone coordinates of the target structure and specifies which residues to design. The server returns to the client the sequences, coordinates and energies of the designed proteins. The simulations are performed using the design module of the Rosetta program (RosettaDesign). RosettaDesign uses Monte Carlo optimization with simulated annealing to search for amino acids that pack well on the target structure and satisfy hydrogen bonding potential. RosettaDesign has been experimentally validated and has been used previously to stabilize naturally occurring proteins and design a novel protein structure.