ABSTRACT
Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination--a well-studied model system for proton transfer from carbon--we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10(8)-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.
Subject(s)
Biocatalysis , Directed Molecular Evolution , Enzymes/chemistry , Enzymes/metabolism , Protein Engineering , Carbon/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzymes/genetics , Kinetics , Models, Molecular , Protons , Triazoles/chemistry , Triazoles/metabolism , Triose-Phosphate Isomerase/metabolismABSTRACT
A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.
Subject(s)
Computational Biology/methods , Protein Engineering/methods , Algorithms , Catalysis , Catalytic Domain , Crystallography, X-Ray/methods , Ligands , Models, Chemical , Molecular Conformation , Molecular Dynamics Simulation , Point Mutation , Protons , SoftwareABSTRACT
The construction and characteristics of the stable and well-structured alpha(4)W protein are described. The 117-residue, single-chain protein has a molecular weight of 13.1 kDa and is designed to fold into a four-helix bundle. Experimental characterization of the expressed and purified protein shows a 69.8 +/- 0.8% helical content over a 5.5-10.0 pH range. The protein is thermostable with a T(M) > 355 K and has a free energy of unfolding as measured by chemical denaturation of -4.7 kcal mol(-1) at 25 degrees C and neutral pH. One-dimensional (1D) proton and 2D (15)N-HSQC spectra show narrow, well-dispersed spectral lines consistent with a uniquely structured alpha-helical protein. Analytical ultracentrifugation and NMR data show that the protein is monomeric over a broad protein concentration range. The 324 nm emission maximum of the unique Trp-106 is consistent with a sequestered position of the aromatic residue. Additionally, differential pulse voltammetry characterization indicates an elevated peak potential for Trp-106 when the protein is folded (pH range 7.0-8.5) relative to partly unfolded (pH range 11.4-13.2). The oxidation of Trp-106 is coupled to proton release as shown by a 53 +/- 3 mV/pH unit dependence of the peak potential over the 7.0-8.5 pH range.
Subject(s)
Oxidation-Reduction , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Circular Dichroism , Gene Expression , Magnetic Resonance Spectroscopy , Protein Binding , Protein Conformation , Protein Folding , Protein Renaturation , Temperature , Thioredoxins/chemistry , Tryptophan/analysis , UltracentrifugationABSTRACT
The incorporation of small-molecule transition state structures into protein design calculations poses special challenges because of the need to represent the added translational, rotational, and conformational freedoms within an already difficult optimization problem. Successful approaches to computational enzyme design have focused on catalytic side-chain contacts to guide placement of small molecules in active sites. We describe a process for modeling small molecules in enzyme design calculations that extends previously described methods, allowing favorable small-molecule positions and conformations to be explored simultaneously with sequence optimization. Because all current computational enzyme design methods rely heavily on sampling of possible active site geometries from discrete conformational states, we tested the effects of discretization parameters on calculation results. Rotational and translational step sizes as well as side-chain library types were varied in a series of computational tests designed to identify native-like binding contacts in three natural systems. We find that conformational parameters, especially the type of rotamer library used, significantly affect the ability of design calculations to recover native binding-site geometries. We describe the construction and use of a crystallographic conformer library and find that it more reliably captures active-site geometries than traditional rotamer libraries in the systems tested.
Subject(s)
Combinatorial Chemistry Techniques/methods , Drug Design , Enzymes/chemistry , Catalytic Domain , Chorismate Mutase/chemistry , Computer Simulation , Databases, Protein , Escherichia coli/enzymology , Ligands , Models, Molecular , Molecular Structure , Saccharomyces cerevisiae/enzymology , Streptavidin/chemistry , Thermodynamics , Triose-Phosphate Isomerase/chemistryABSTRACT
Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.
Subject(s)
Amino Acids/chemistry , Enzymes/chemistry , Free Radicals/chemistry , Models, Chemical , Protein Engineering/methods , Azurin/chemistry , Oxidation-Reduction , Photosynthetic Reaction Center Complex Proteins/chemistry , Protein Conformation , Protein Subunits , Thermodynamics , Tryptophan/chemistry , Tyrosine/chemistryABSTRACT
The de novo design, synthesis, and characterization of a four-alpha-helix bundle scaffold containing heme ligated by 4-beta-(pyridyl)-l-alanine (Pal) is presented. The protein scaffold is highly helical, stable, and conformationally specific in the apo-state. Incorporation of heme using the designed bis-Pal axial ligation is shown using UV-visible and EPR spectroscopies. The observed heme midpoint reduction potential, +58 mV versus SHE, is 287 mV (6.8 kcal/mol) higher than the analogous bis-histidine-ligated heme protein.