AlphaFold accurately predicts distinct conformations based on the oligomeric state of a de novo designed protein.

Using the molecular modeling program Rosetta, we designed a de novo protein, called SEWN0.1, which binds the heterotrimeric G protein Gαq. The design is helical, well-folded, and primarily monomeric in solution at a concentration of 10 µM. However, when we solved the crystal structure of SEWN0.1 at 1.9 Å, we observed a dimer in a conformation incompatible with binding Gαq . Unintentionally, we had designed a protein that adopts alternate conformations depending on its oligomeric state. Recently, there has been tremendous progress in the field of protein structure prediction as new methods in artificial intelligence have been used to predict structures with high accuracy. We were curious if the structure prediction method AlphaFold could predict the structure of SEWN0.1 and if the prediction depended on oligomeric state. When AlphaFold was used to predict the structure of monomeric SEWN0.1, it produced a model that resembles the Rosetta design model and is compatible with binding Gαq , but when used to predict the structure of a dimer, it predicted a conformation that closely resembles the SEWN0.1 crystal structure. AlphaFold's ability to predict multiple conformations for a single protein sequence should be useful for engineering protein switches.

Toward complete rational control over protein structure and function through computational design.

The grand challenge of protein design is a general method for producing a polypeptide with arbitrary functionality, conformation, and biochemical properties. To that end, a wide variety of methods have been developed for the improvement of native proteins, the design of ideal proteins de novo, and the redesign of suboptimal proteins with better-performing substructures. These methods employ informatic comparisons of function-structure-sequence relationships as well as knowledge-based evaluation of protein properties to narrow the immense protein sequence search space down to an enumerable and often manually evaluable set of structures that meet specified criteria. While arbitrary manipulation of protein-protein interfaces and molecular catalysis remains an unsolved problem, and no protein shape or behavior manipulation algorithm is universally applicable, the promising results thus far are a strong indicator that a general approach to the arbitrary manipulation of polypeptides is within reach.

Author Correction: Optogenetic control of cofilin and αTAT in living cells using Z-lock.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Computational Protocol for Regulating Protein Binding Reactions with a Light-Sensitive Protein Dimer.

Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. On activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 on stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.

Optogenetic control of cofilin and αTAT in living cells using Z-lock.

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Protocols for Requirement-Driven Protein Design in the Rosetta Modeling Program.

We have developed a set of protocols in the molecular modeling program Rosetta for performing requirement-driven protein design. First, the user specifies a set of structural features that need to be present in the designed protein. These requirements can be general (e.g., "create a protein with five helices"), or they can be very specific and require the correct placement of a set of amino acids to bind a ligand. Next, a large set of protein models are generated that satisfy the design requirements. The models are built using a method that we recently introduced into Rosetta, called SEWING, that rapidly assembles novel protein backbones by combining pieces of naturally occurring proteins. In the last step of the process, rotamer-based sequence optimization and backbone refinement are performed with Rosetta, and a variety of quality metrics are used to pick sequences for experimental characterization. Here we describe the input files and user options needed to run SEWING and perform requirement-driven design and provide detailed instructions for two specific applications of the process: the design of new structural elements at a protein-protein interface and the design of ligand binding sites.

Improving computational efficiency and tractability of protein design using a piecemeal approach. A strategy for parallel and distributed protein design.

MOTIVATION: Accuracy in protein design requires a fine-grained rotamer search, multiple backbone conformations, and a detailed energy function, creating a burden in runtime and memory requirements. A design task may be split into manageable pieces in both three-dimensional space and in the rotamer search space to produce small, fast jobs that are easily distributed. However, these jobs must overlap, presenting a problem in resolving conflicting solutions in the overlap regions. RESULTS: Piecemeal design, in which the design space is split into overlapping regions and rotamer search spaces, accelerates the design process whether jobs are run in series or in parallel. Large jobs that cannot fit in memory were made possible by splitting. Accepting the consensus amino acid selection in conflict regions led to non-optimal choices. Instead, conflicts were resolved using a second pass, in which the split regions were re-combined and designed as one, producing results that were closer to optimal with a minimal increase in runtime over the consensus strategy. Splitting the search space at the rotamer level instead of at the amino acid level further improved the efficiency by reducing the search space in the second pass. AVAILABILITY AND IMPLEMENTATION: Programs for splitting protein design expressions are available at www.bioinfo.rpi.edu/tools/piecemeal.html CONTACT: bystrc@rpi.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Origin of the dengue virus outbreak in Martin County, Florida, USA 2013.

After a 75-year absence from Florida, substantial local transmission of dengue virus (DENV) occurred in Key West, Monroe County, Florida in 2009 and continued in 2010. The outbreak culminated in 85 reported cases. In 2011 and 2012, only isolated cases of local DENV transmission were reported in Florida, none were reported in Key West. In 2013, a new outbreak occurred, but this time in Martin County about 275 miles North of Key West with 22 reported cases. As the Key West and Martin County outbreaks involved DENV serotype 1 (DENV-1), we wanted to investigate whether the same strain or a different strain of DENV was responsible for the outbreaks. In this study, we report the sequence and phylogenetic analysis of the E generegion from a patient diagnosed with dengue in Martin County. Our results indicate that the 2013 Martin County DENV-1 strain is distinct from the 2009-2010 Key West DENV-1 and that it is most closely related to viruses from a recent expansion of South American DENV-1 strains into the Caribbean. We conclude that the 2013 Martin County outbreak was the result of a new introduction of DENV-1 in Florida.