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We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 A) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 A along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.

We computationally designed a de novo protein-protein interaction between wild-type ubiquitin and a redesigned scaffold. Our strategy was to incorporate zinc at the designed interface to promote affinity and orientation specificity. A large set of monomeric scaffold surfaces were computationally engineered with three-residue zinc coordination sites, and the ubiquitin residue H68 was docked to the open coordination site to complete a tetrahedral zinc site. This single coordination bond was intended as a hotspot and polar interaction for ubiquitin binding, and surrounding residues on the scaffold were optimized primarily as hydrophobic residues using a rotamer-based sequence design protocol in Rosetta. From thousands of independent design simulations, four sequences were selected for experimental characterization. The best performing design, called Spelter, binds tightly to zinc (Kd

Structure-based protein design tests our understanding of the minimal determinants of protein structure and function. Previous studies have demonstrated that placing zinc binding amino acids (His, Glu, Asp or Cys) near each other in a folded protein in an arrangement predicted to be tetrahedral is often sufficient to achieve binding to zinc. However, few designs have been characterized with high-resolution structures. Here, we use X-ray crystallography, binding studies and mutation analysis to evaluate three alternative strategies for designing zinc binding sites with the molecular modeling program Rosetta. While several of the designs were observed to bind zinc, crystal structures of two designs reveal binding configurations that differ from the design model. In both cases, the modeling did not accurately capture the presence or absence of second-shell hydrogen bonds critical in determining binding-site structure. Efforts to more explicitly design second-shell hydrogen bonds were largely unsuccessful as evidenced by mutation analysis and low expression of proteins engineered with extensive primary and secondary networks. Our results suggest that improved methods for designing interaction networks will be needed for creating metal binding sites with high accuracy.

Summary Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 degrees C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the protein for arginine and to elucidate structural changes associated with arginine binding. The protein binds arginine with a dissociation constant of 20 muM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor.

Computational design of proteins with altered ligand specificity is an emerging method for the creation of new biosensing systems. In this work, we investigated the outcome of site-directed mutagenesis on the Escherichia coli ribose binding protein (RBP), which is frequently used as a design scaffold for computational searches. A ribose biosensor was first constructed whereby an environmentally sensitive fluorescent probe was covalently attached to RBP at position S265C. This protein conjugate displayed a 54% decrease in emission intensity upon the addition of saturating ribose concentrations and exhibited an apparent dissociation constant (K(d) ) of 3.4 microM. Site-directed mutants within the RBP binding pocket were created and examined for ribose binding ability and overall structural stability. Because as many as 12 mutations are needed to alter ligand specificity in RBP, we measured the effect of single and multiple alanine mutations on stability and signal transduction potential of the ribose biosensor. Single alanine mutations had significant impact on both stability and signaling. Mutations of N190A and F214A each produced melting temperatures8 degrees C below those observed for the wild-type protein. Residue Q235, located in the hinge region of RBP, appeared to be a hot spot for global protein stability as well. Additional single alanine mutations demonstrated as much as 200-fold increase in apparent K(d) but retained overall protein stability. The data collected from this study may be incorporated into design algorithms to help create more stable biosensors and optimize signal transduction properties for a variety of important analytes.

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscopic observations with specific secondary structural elements. The data determined the presence of a buried beta-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding of bacterial binding protein family members and their potential biotechnological applications.

Fluorescent protein biosensors, which exhibit a significant change in fluorescence based on the physical interaction between protein and ligand, may prove to be effective tools to measure a variety of analytes. In particular, real-time monitoring of glucose levels has potential applications in bioprocess monitoring and in minimizing health complications caused by diabetes. In this work, site-directed mutagenesis of the Escherichia coli glucose/galactose binding protein (GGBP) was used to engineer double-cysteine mutations that allowed selective covalent attachment of thiol-reactive dyes. Because GGBP undergoes a large conformational change on the addition of glucose, rational placement of these sites allowed glucose-dependent spatial realignment of the two fluorophores, which was monitored as a change in fluorescence intensity and extinction coefficients. Using targeted mutagenesis of the GGBP binding pocket, glucose biosensors were created to measure concentrations spanning five orders of magnitude (0.04–12,000 μM). The glucose biosensor retained its function in complex solutions that contained realistic concentrations of protein and potential interfering agents found in blood serum. In addition to the development of a fluorescent protein sensor for glucose, this work helps to expand the spectroscopic tools used for the detection of conformational movements within a single polypeptide chain.

Reengineering protein surfaces to exhibit high net charge, referred to as "supercharging", can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from -11 to -61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and -30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.

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.