Our aim is to design new proteins that are capable of self-assembly with natural or modified pigments and redox cofactors. These assemblies should enable charge separation and/or energy transfer across the protein-bound arrays of cofactors. We focus primarily on iron-sulfur cluster, and chlorophyll proteins. In the next stage of the building process, we create an interface designed to couple these proteins with natural and/or artificial catalytic partners.
In collaboration with leading experts of protein computational design, we apply state-of-the-art computational tools such as the protCAD, and Rosetta software packages. In addition, we capitalize on the many medium- to high-resolution structures of redox- and photosynthetic protein complexes. These are used for a) taking multicofactor-binding domains out of their natural structural context, and adapting these to fold as small independent proteins, and b) identifying and implementing general features of natural cofactors binding sites in well established de novo designed protein scaffolds.
The products of the design stage are amino acid sequences. These are coded into DNA and over-expressed in E. coli bacteria. Since proteins are designed to spontaneously assemble with cofactors, preparing the holoprotein usually requires simply mixing the apoprotein with the desired cofactors. New designs are tested by a variety of analytical and spectroscopic methods, and the results are used for optimizing the previous designs in an iterative process.
Our iterative approach provides substantial insights into folding and assembly of protein-cofactor complexes, and the critical parameters affecting their function as energy- and electron-transfer relays. Most importantly, it can teach important lessons on how Nature achieves functional diversity by combining only a few basic modules into a variety of elaborate networks of long-distance inter- and intra-protein energy and electron-transfer reactions. These lessons may be used for designing and constructing custom-built networks of enzyme complexes to carry out chemical transformations of our choice, either in a non-biological context, or in a biological setting.
Iron-sulfur (FeS) clusters protein complexes are the most abundant natural electron relays. Some appear to be evolutionary remains of the era before earth’s atmosphere became oxygenic, hence FeS clusters are probably among the first inorganic complexes used for electron transfer (ET) by living organisms. The growing interest in biological and photobiological hydrogen production as a clean renewable alternative for fossil fuels is prompting greater interest in FeS proteins. Particularly, redox chains of four-iron four-sulfur clusters (Fe4S4), are receiving much attention as they facilitate long-range ET to and from the catalytic centers of hydrogenases. This functionality is critical to any practical application that requires interfacing hydrogenases either to electrodes or photoelectron sources. It is therefore very appealing to consider constructing artificial FeS proteins that can be coupled to natural catalytic centers via native-like protein-protein interfaces and provide ET relays between biocatalytic centers.
We employ a metal first strategy for the computational design of novel multi-center FeS cluster proteins (Fig.1). The method was implemented in the software package protCAD by our collaborator, Prof. Vikas Nanda, resulted in the successful computational design, preparation, and characterization of CCIS1 (Coiled-Coil Iron Sulfur protein 1), a novel Fe4S4 cluster protein with a single chain, α-helical coiled-coil fold (Grzyb et al. (2010) BBA-Bioenergetics 1797,406-413). This highly regular and completely non-natural fold for a Fe4S4 cluster protein is ideal for constructing multi-center redox proteins.
Currently, we are using CCIS1 as a template for new de novo designed multi-center FeS proteins that are tested and optimized first for efficient ET through the protein-embedded FeS redox chain, and then as electron transfer relays to or from natural biocatalytic redox centers.
Figure 1: The metal first strategy for designing CCIS1 - a four-helix bundle protein with a Fe4S4 cluster embeded within the coiled coil core
Natural selection has provided photosynthetic organisms with the photosystem as an elegant, highly efficient and dynamic apparatus for capturing incoming solar light. The key to the photosystem functionality is in organizing light absorbing pigments, primarily chlorophylls (Chls) and bacteriochlorophylls (BChls) in densely packed arrays within light-harvesting complexes (LHCs), at specific geometries. The particular organization provide high absorption cross-section without compromising efficient transfer of excitation energy from the LHCs to the reaction centers (RCs) where charge separation and the ensuing chemistry occur.
Intriguingly, Chls and BChls are also used as redox cofactors within the ET chains of RCs. Spatial organization and specific potein environment are the keys for adapting (B)Chls to their deiffernt functions. Furthermore, certain protein environments either prevent energy dissipation within the the Chl arrays therby optimizing their light-harvesting function, or actually induce strong excited state quenching. Photosynthetic organisms in a process called “non-photochemical quenching” (NPQ) indeed exploit the capability of natural LHCs to actively switch between these two extreme and opposite functions, in order to protect themselves against photodamage by high light intensities.
We are designing and preparing novel (B)Chl-protein complexes in order to provide minimal functional analogs of the natural photosynthetic RCs and LHCs. These are used as benchmarks for spectroscopic studies of the basic factors that control charge and energy transfer dynamics within photosynthetic pigment-protein complexes. Through the iterative process of designs, we are learning valuable lessons on binding and assembling multiple Chls and BChls within a protein scaffold. The new insights obtained from such studies are shedding light on mechanisms for switching between charge separation, light-harvesting, and photoprotection in natural photosystems. At the same time, they open new possibilities for constructing solar energy conversion devices.
We implement constraints and guidelines derived from our recent analysis of natural α-helical Chl- and BChl-binding proteins (Braun et al. (2010) Proteins: Struct. Func. Bioinfo. 79, (2), 463-476). Our starting templates are two classes of computationally designed water-soluble protein-pigment complexes. The first class includes four-helix bundles that were originally designed as heme-binding proteins (Fig. 2). We found that the HP7 protein of this class can bind up to three BChl derivatives per bundle (Cohen-Ofri et al. (2011) J. Am. Chem. Soc. 133 (24), 9526-9535). The second class includes water-soluble analogs of natural transmembranal Chl-binding motifs (Fig. 3).
Native Chls and BChls are water-insoluble with a very high tendency to self-aggregate in aqueous solutions, which usually prevents efficient incorporation into water-soluble proteins. This not withstanding, packing the aromatic macrocycle of the Chls within a hydrophobic binding pocket (such as the core of four-helix bundles) is a strong driving force for complex formation. We improve the water-solubility of Chls and BChls by hydrolyzing their phytil group. The resulting chlrorophyllide (Chlide) and bacteriochlorophyllide (BChlide) derivatives are better suited for incorporation into water-soluble proteins. Particularly, we find the zinc-substituted 132-OH-BChlide (ZnBChlide) and Chlide (ZnChlide) derivatives very useful as they are easily synthesized in large quantities and share very high chemical homology with the natural Mg containing pigments. The development of new water-soluble (B)Chl derivatives is carried out in close collaboration with the group of Prof. Avigdor Scherz in our department.
A.jpg) |
B
|
|
Figure 2. Titration of HP7, a de novo designed four-helix bundle protein, with ZnBChlide (A), a water-soluble BChl derivative. Absorption (B) and CD (C) spectra indicate a binding stoichiometry of up to 3 pigments per proetin, as well as excitonic interactions upon between the protein-bound ZnBChlide molecules.
Figure 3: Conversion of a transmembranal Chl-binding motif into a water-soluble protein. Sequence (A) and structure (B) alignment of four Chl-binding domains from PSI (PsaA in yellow, and PsaB in violet), and PSII (CP47 in cyan, and CP43 in magenta), reveal high similarity in sequence, fold, and Chl arrangement. This common motif binds five Chls, three of which, highlighted in yellow, have been conserved through evolution in all type I photosystems, and in PSII. The first design prototype, SCP1 (C), was based on PsaA after connecting the C-teminus of helix 3 (blue) to the N-terminus of helix 1 (green) with Loop 2. It maintained five Chl-binding sites (D); four histidines that do not bind Chls were replaced in order to simplify the structure. Then, membrane-facing hydrophobic residues were replaced by hydrophilic ones. These are marked in blue on the sequence (A) and by cyan spheres indicating their Cα atom positions on the model structure (C). We found that SCP1 is a water-soluble protein that dimerizes upon binding ZnBChlide molecules. Three out of the five histidines were found capable of binding ZnBChlide, thus, SCP1 binds up to six ZnBChlide molecules altogether. Intriguingly, the pigment-binding histidines in SCP1 correspond to the conserved histidines.
Hydrogen is a promising, clean alternative to fossil fuels. Unfortunately, there is currently no viable method for its production from renewable sources. Primary photosynthesis provides enough reducing power for driving the production of molecular hydrogen. In fact, certain unicellular algae and photosynthetic bacteria are capable of light-dependent hydrogen evolution. Unfortunately, the natural process is intermittent and short-lived (less than a couple of minutes). Although significant progress has been made in understanding the catalytic mechanisms of hydrogen evolution and sustaining hydrogen evolution for longer periods of time, it has not been matched by a similar advance in our ability to couple photosynthesis to hydrogen evolution. In order to address this challenge by using methods of computational protein design, we have initiated the Ligh2t project together with the labs of Prof. Thomas Happe, and Prof. David Baker. This project is funded by the Volkswagen foundation. Central to this methodology is the preparation of PSI core complexes, in which stromal ridge subunits have been stripped off, and their reconstitution with modified subunits. For this purpose, our lab we implement stripping and reconstitution protocols that were developed in the lab of Prof. John Golbeck.
The phycobilisomes (PBSs) are supra-molecular assemblies of multiple pigment and protein components that provide a peripheral light-harvesting system to the oxygenic photosystems of cyanobacteria. Their exceptional light-harvesting properties and unique modular architecture have prompted us to couple PBS components to artificial protein-pigment complexes or photochemical catalytic centers. This is a promising strategy for designing and constructing hybrid modular photosystems that may be utilized for solar energy conversion either in vitro as stand-alone molecular devices, or in vivo by heterologous expression in bacteria.
Our first prototype is a fusion between the natural low molecular weight hydrophobic allophycocyanin linker protein (Lc) of allophycoyanin (APC) of Thermosynechococcus vulcanus with a de novo designed porphyrin- and chlorophyll-binding protein (HP). It is a bifunctional water-soluble protein capable of binding porphyrins and chlorophylls at the HP domain, and self-assembling with APC at the Lc domain (Fig. 4). The increased solubility of the fusion protein circumvents the dificulties of APC-Lc in vitro reconstitution, which previously required partly denaturing conditions due to the hydrophobic nature of Lc. The new design makes it possible to explore energy transfer between the APC-Lc domain and pigments bound to the HP domain.
.jpg)
Figure 4. Self-assembly of APC with HP-Lc fusion protein yields typical APC-Lc absorption spectra (left). The HP domain is capable of bining various water soluble porphyrin, Chl, and BChl derivatives (right).
Natural oxygenic photosynthesis is not optimized for maximum solar energy conversion efficiency. Mismatches between the primary light-driven reactions and the ensuing biochemical reactions are significant sources of energy loss. Still, the natural photosynthetic apparatus offer unique advantages over artificial solar energy converters, particularly in providing elaborate and efficient catalytic modules for driving difficult redox reactions such as water oxidation.
To address this problem, we are developing a modular hybrid solar energy conversion system that is based on co-incorporating the natural oxygenic photosystems, PSI and PSII in sol-gel matrices. The photosystems are coupled by soluble redox-active molecules that shuttle electrons by diffusion through the porous sol-gel matrix. Thus, electrons abstracted from water by photo-oxidation at the oxidizing end of PSII are transferred all the way to the reducing end of PSI. This system excludes membranes as functional elements but maintains the use of redox carriers for coupling between the catalytic centers.
So far we were able to encapsulate active PSI and PSII into sol gel matrices (Fig. 5), and to demonstrate their effective coupling in solution by using dichlorophenol-indophenol (DCPIP) as redox mediator (Fig. 6).
A |
B |
C.jpg) |
Figure 5. Sol-gel enacpsulated PSI (A) and PSII (B) are capable of photooxidation, and photoreduction of DCPIP, respectively. Sol-gel encapsulated PSII retains its oxygen evolution activity (C).
|
In buffer solution |
In sol-gels |
Figure 6. When PSII and DCPIP are combined with PSI, DCPIP photoreduction by PSII upon actinic light illumination leads to accumulation of DCPIPH2 that is an external electron donor to PSI. Recycling of P700+. by DCPIPH2 oxidation sustains PSI activity. This was observed when PSI and PSII were either in solution (left), or co-encapsulated in sol-gels (right, orange trace), as well as when one photosystem was encapsulated in sol-gel and the other in solution (right, green and blue traces), but only in the presence of DCPIP.
The reducing end of PSI maintains three Fe4S4 clusters, FX, FA, and FB. While FX is bound by the integral membrane protein core of PSI, FA and FB are bound to a peripheral domain, named the “stromal ridge”, which associates non-covalently with the PSI core and protrudes into the solvent. The stromal ridge is comprised of three protein subunits, PsaC that binds FA and FB, and PsaD and PsaE that reinforce PsaC’s attachment to the core and make up, together with PsaC, a docking site for small soluble redox proteins that serve as the terminal electron acceptors, namely ferredoxin, and in some cases flavodoxin. Thus, the PSI core/stromal ridge system provides an excellent general model for the assembly of functional electron transfer (ET) complexes.
In order to understand, at the single-molecule level, the critical factors for making functional ET protein complexes, we are studying the assembly of the PSI core with its stromal ridge components by force spectroscopy. In collaboration with Prof. Ziv Reich we are constructing an experimental system whereby native or modified PSI particles are immobilized to a surface with their stromal side facing the bulk solution, and probed by an atomic force microscope (AFM). In this way, effects of many external factors on the PSI core/stromal ridge assembly can be evaluated with sensitivity far greater than that provided by methods relying on ensemble probing. These effects include the presence of other stromal ridge components, namely PsaD and PsaE, as well as modifications of specific amino acid residues. Uniquely, interaction forces can be probed at different redox states simply by changing the sample’s illumination conditions. This information may provide new insights about the role of the protein matrix in ET processes.
