Fundamental Limits on Wavelength, Efficiency and Yield of the Charge Separation Triad

In an attempt to optimize a high yield, high efficiency artificial photosynthetic protein we have discovered unique energy and spatial architecture limits which apply to all light-activated photosynthetic systems. We have generated an analytical solution for the time behavior of the core three cofactor charge separation element in photosynthesis, the photosynthetic cofactor triad, and explored the functional consequences of its makeup including its architecture, the reduction potentials of its components, and the absorption energy of the light absorbing primary-donor cofactor. Our primary findings are two: First, that a high efficiency, high yield triad will have an absorption frequency more than twice the reorganization energy of the first electron transfer, and second, that the relative distance of the acceptor and the donor from the primary-donor plays an important role in determining the yields, with the highest efficiency, highest yield architecture having the light absorbing cofactor closest to the acceptor. Surprisingly, despite the increased complexity found in natural solar energy conversion proteins, we find that the construction of this central triad in natural systems matches these predictions. Our analysis thus not only suggests explanations for some aspects of the makeup of natural photosynthetic systems, it also provides specific design criteria necessary to create high efficiency, high yield artificial protein-based triads

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes

Controlling complexity and water penetration in functional <i>de novo</i> protein design

Natural proteins are complex, and the engineering elements that support function and catalysis are obscure. Simplified synthetic protein scaffolds offer a means to avoid such complexity, learn the underlying principles behind the assembly of function and render the modular assembly of enzymatic function a tangible reality. A key feature of such protein design is the control and exclusion of water access to the protein core to provide the low-dielectric environment that enables enzymatic function. Recent successes in de novo protein design have illustrated how such control can be incorporated into the design process and have paved the way for the synthesis of nascent enzymatic activity in these systems

Simple structure, complex function

The four-helix bundle is a simple structural motif, widespread in nature, that is involved in numerous and fundamental processes. This portfolio is now expanded by the report of a four-helix bundle protein able to store copper for particulate methane monooxygenase, an enzyme that catalyzes methane oxidation

Elementary tetrahelical protein design for diverse oxidoreductase functions

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications

Design and engineering of O₂ transport protein

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. We introduce this method here with the first design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assemble a four helix bundle, position histidines to bis-his ligate hemes, and exploit helical rotation and glutamate burial on heme binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without heme oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines with the remarkable exception that O(2) binds tighter than CO