Jeremy H. Mills

Protein evolution with an expanded genetic code

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

Expanding the Product Profile of a Microbial Alkane Biosynthetic Pathway

Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.

Computational design of an α-gliadin peptidase

The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a kcat/KM of 568 M–1 s–1, representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.

Transition states. Trapping a transition state in a computationally designed protein bottle.

A transition state holds a pose The transition state of a chemical transformation is inherently fleeting because the structure is high in energy. Nonetheless, Pearson et al. trapped a classical example of a bond rotation transition state using a modified protein (see the Perspective by Romney and Miller). The biphenyl molecule passes through an energy maximum when its rings rotate through a parallel position. A pocket within the editing domain of threonyl–transfer RNA synthetase was modified to stabilize parallel biphenyl rings, allowing further characterization of this normally transient structure. Science, this issue p. 863; see also p. 829 A protein was designed to stabilize the coplanar geometry of the transition state for biphenyl ring rotation. [Also see Perspective by Romney and Miller] The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Herein we use packing interactions within the core of a protein to stabilize the planar TS for rotation around the central C-C bond of biphenyl so that it can be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis we identified a protein in which the side chain of p-biphenylalanine is kinetically trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.

A Genetically Encoded Direct Sensor of Antibody–Antigen Interactions

We have previously used the genetically encoded fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine[3] 1 and 2-amino-3-(5-(dimethylamino)naphthalene-1 sulfonamide) propanoic acid (dansylalanine)[4], as well as the bio-orthogonal chemical handles p-acetylphenylalanine[5], and alkyne and azide containing amino acids6 to introduce fluorescent reporters at specifically defined locations in proteins.[3,7] This allowed us to exploit the high sensitivities of fluorescent techniques, and the ability to selectively introduce a relatively small fluorescent probe at virtually any site in a protein. We hoped to extend this method by using the 7-hydroxycoumarin amino acid as a direct reporter of protein-protein interactions without the need for protein immobilization or a displacement assay. The fluorescent properties of hydroxycoumarins are well characterized, and have been shown to exhibit solvent dependent changes in fluorescence.[8,9] Because the pKAs of hydroxycoumarins are around 7.8, in many biologically relevant solvents they will exist in both neutral and ionized species[9] and can therefore also be used to probe pH in the environment of the protein. We and others have previously shown that antibodies that are chemically modified with small molecule fluorophores near the combining site fluorescently signal the interaction of the antibody and its ligand.[10,11] We sought to expand this method to the interaction of an antibody with a protein antigen using a one step genetic method rather than chemical modification to incorporate the fluorophore. We chose to study the interaction of the protein CD40 ligand (CD40L), with a neutralizing α-CD40L antibody, 5c8. CD40L is a tumor necrosis factor (TNF) homologue that is expressed on the surface of T-cells. It has been found to be involved in B-cell proliferation, and isotype switching,[12] as well as hyper-IgM syndrome.[13] Crystallographic characterization of the interaction of 5c8 and CD40L has been reported[14] (PDB id 1I9R). In order to monitor a protein-protein interaction by fluorescence, the fluorophore should be close enough to the interface that its environment is substantially modified on binding, but not so close that it significantly affects the binding interaction.[11] Analysis of the 5c8-CD40L co-crystal structure revealed a number of candidate sites for incorporation of 1 at the antibody-antigen interface which appear to fit the criteria above (Figure 1) including Ile98(L) which lies ~5.0 A from the nearest CD40L residue. Figure 1 Crystal structure of the 5c8 antibody in complex with CD40L antigen (PDB id 1I9R). The heavy and light chains are shown in grey and light blue respectively, and the protein antigen is shown in purple. The side chain of I98(L) is shown in sticks, and the ... To incorporate the coumarin containing amino acid, the codon for Ile98(L) was mutated to TAG. E. coli cells were co-transformed with plasmids encoding the mutant antibody as a humanized Fab in which the bicistronic heavy and light chains were under the control of a single araBAD promoter, and the previously engineered aminoacyl-tRNA synthetase (pEB-CouRS), and MjtRNACUATyr. The purified Fab was dialyzed into 150 mM sodium phosphate buffer at pH 7.4, and the fluorescent properties of the coumarin containing mutant were analyzed in the presence and absence of CD40L which was obtained by expression in Pichia pastoris. Replacement of the side chain of Ile 98 in the light chain of 5c8 with a 7-hydroxycoumarin moiety yielded a fluorescent antibody with an emission maximum at 450 nm as expected.[8] This residue is in proximity to, but does not directly contact the antigen in the co-crystal structure[14] suggesting that the fluorescent antibody would still bind CD40L. Interestingly, the emission signal of this mutant exhibited a 2–3 fold increase in intensity (depending on the excitation wavelength) in the presence of saturating concentrations of the antigen, but λmax did not change (Figure 3). To examine whether or not binding was affected, a titration of CD40L over a range of concentrations (50 nM – 7 µM) that spanned the dissociation constant (Kd) of 5c8 for CD40L was carried out. The fluorescent signal increased sigmoidally; a nonlinear fit of the binding curve (Figure S1) yielded a Kd of 120 nM. The Kds of wt 5c8 and I98(L) → 1 for CD40L were analyzed by Biacore, and found to be 7.0 nM and 28 nM, respectively. Although the Kd of the I98(L) → 1 mutant determined from Biacore analysis and fluorescence quenching differ (likely due to surface interactions which increase affinity in the former case) these data show that introduction of the hydroxycoumarin group leads to an ~4 fold decrease in CD40L binding affinity. In general such an effect is not expected to adversely affect the use of 1 as a direct sensor of antibody-antigen interactions, but will likely vary depending on the specific complex under investigation, and the site of modification (which can be varied by simple mutagenesis). Finally, the effect was shown to be antigen specific as the CD40L homologue TNF-α, (which binds 5c8 with 100 fold lower affinity than CD40L in an enzyme-linked immunosorbent assay) did not result in changes in fluorescence (Figure 4). Figure 3 Addition of CD40L to 5c8 I98(L)→ 1. Spectra shown are at CD40L concentrations of 0, 250 nM, 550 nM, 850 nM, and 1 µM. Excitation was at 316 nm. Fluorescence signal intensity at 450 nM increases with increasing concentrations of CD40L. Figure 4 5c8 I98(L)→1 alone (dashed line), in the presence of 1 µM TNF-α (dotted line), and in the presence of 1 µM TNF-α and 1µM CD40L (solid line). Excitation was at 316 nm. Only addition of CD40L results in an ... The fact that 7-hydroxycoumarins exist in both acid and base forms with different absorption maxima allows analysis of the local environment surrounding the fluorophore. Addition of saturating concentrations of antigen resulted in an increase in fluorescence of similar magnitude when the fluorophore was excited at 316 or 370 (2.1 and 2.3 fold respectively) suggesting no significant perturbation of the pKa of the phenolic proton of the 7-hydroxycoumarin occurs on addition of CD40L. Antibodies have found widespread application as bioanalytical reagents and as therapeutics.[15] Current methods for fluorescent labeling of proteins often rely on nucleophilic lysines or cysteines as handles for fluorophore attachment. Unfortunately, lysine conjugation is generally non-specific, resulting in high background fluorescence. Further, the presence of a number of disulfide bonds in the antibody scaffold (which are essential for correct folding) renders the application of cysteine conjugation chemistries difficult if not impossible in this system. Thus, by genetically encoding the fluorophore, we remove the necessity for chemical modification of the protein, and have shown that the fluorescent properties of 7-hydroxycoumarins can be exploited to monitor protein-protein interactions.

Computational design of a homotrimeric metalloprotein with a trisbipyridyl core

Significance This article reports the computational design of a threefold symmetric, self-assembling protein homotrimer containing a highly stable noncanonical amino acid-mediated metal complex within the protein interface. To achieve this result, recently developed protein–protein interface design methods were extended to include a metal-chelating noncanonical amino acid containing a bipyridine functional group in the design process. Bipyridine metal complexes can give rise to photochemical properties that would be impossible to achieve with naturally occurring amino acids alone, suggesting that the methods reported here could be used to generate novel photoactive proteins. Metal-chelating heteroaryl small molecules have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2′-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodology to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)3]2+ complexes at the interface between monomers. X-ray crystallographic analysis of the homotrimer showed that the design process had near-atomic-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophysical applications.

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

Trapping a transition state in a computationally designed protein bottle

A transition state holds a pose The transition state of a chemical transformation is inherently fleeting because the structure is high in energy. Nonetheless, Pearson et al. trapped a classical example of a bond rotation transition state using a modified protein (see the Perspective by Romney and Miller). The biphenyl molecule passes through an energy maximum when its rings rotate through a parallel position. A pocket within the editing domain of threonyl–transfer RNA synthetase was modified to stabilize parallel biphenyl rings, allowing further characterization of this normally transient structure. Science, this issue p. 863; see also p. 829 A protein was designed to stabilize the coplanar geometry of the transition state for biphenyl ring rotation. [Also see Perspective by Romney and Miller] The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Herein we use packing interactions within the core of a protein to stabilize the planar TS for rotation around the central C-C bond of biphenyl so that it can be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis we identified a protein in which the side chain of p-biphenylalanine is kinetically trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.

Computational Design of Multinuclear Metalloproteins Using Unnatural Amino Acids.

Multinuclear metal ion clusters, coordinated by proteins, catalyze various critical biological redox reactions, including water oxidation in photosynthesis, and nitrogen fixation. Designed metalloproteins featuring synthetic metal clusters would aid in the design of bio-inspired catalysts for various applications in synthetic biology. The design of metal ion-binding sites in a protein chain requires geometrically constrained and accurate placement of several (between three and six) polar and/or charged amino acid side chains for every metal ion, making the design problem very challenging to address. Here, we describe a general computational method to redesign oligomeric interfaces of symmetric proteins for the purpose of creating novel multinuclear metalloproteins with tunable geometries, electrochemical environments, and metal cofactor stability via first and second-shell interactions. The method requires a target symmetric organometallic cofactor whose coordinating ligands resemble the side chains of a natural or unnatural amino acid and a library of oligomeric protein structures featuring the same symmetry as the target cofactor. Geometric interface matches between target cofactor and scaffold are determined using a program that we call symmetric protein recursive ion-cofactor sampler (SyPRIS). First, the amino acid-bound organometallic cofactor model is built and symmetrically aligned to the axes of symmetry of each scaffold. Depending on the symmetry, rigid body and inverse rotameric degrees of freedom of the cofactor model are then simultaneously sampled to locate scaffold backbone constellations that are geometrically poised to incorporate the cofactor. Optionally, backbone remodeling of loops can be performed if no perfect matches are identified. Finally, the identities of spatially proximal neighbor residues of the cofactor are optimized using Rosetta Design. Selected designs can then be produced in the laboratory using genetically incorporated unnatural amino acid technology and tested experimentally for structure and catalytic activity.

Crystal Structure of a Kinetically Persistent Transition State in a Computationally Designed Protein Bottle

A transition state holds a pose The transition state of a chemical transformation is inherently fleeting because the structure is high in energy. Nonetheless, Pearson et al. trapped a classical example of a bond rotation transition state using a modified protein (see the Perspective by Romney and Miller). The biphenyl molecule passes through an energy maximum when its rings rotate through a parallel position. A pocket within the editing domain of threonyl–transfer RNA synthetase was modified to stabilize parallel biphenyl rings, allowing further characterization of this normally transient structure. Science, this issue p. 863; see also p. 829 A protein was designed to stabilize the coplanar geometry of the transition state for biphenyl ring rotation. [Also see Perspective by Romney and Miller] The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Here, we used packing interactions within the core of a protein to stabilize the planar TS conformation for rotation around the central carbon-carbon bond of biphenyl so that it could be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis, we identified a protein in which the side chain of p-biphenylalanine is trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.The fleeting lifetimes of the transition states (TSs) of chemical reactions make determination of their three-dimensional structures by diffraction methods a challenge. Herein we use packing interactions within the core of a protein to stabilize the planar TS for rotation around the central C-C bond of biphenyl so that it can be directly observed by x-ray crystallography. The computational protein design software Rosetta was used to design a pocket within threonyl-transfer RNA synthetase from the thermophile Pyrococcus abyssi that forms complementary van der Waals interactions with a planar biphenyl. This latter moiety was introduced biosynthetically as the side chain of the noncanonical amino acid p-biphenylalanine. Through iterative rounds of computational design and structural analysis we identified a protein in which the side chain of p-biphenylalanine is kinetically trapped in the energetically disfavored, coplanar conformation of the TS of the bond rotation reaction.