Dror Noy

The bacterial flagellar switch complex is getting more complex

The mechanism of function of the bacterial flagellar switch, which determines the direction of flagellar rotation and is essential for chemotaxis, has remained an enigma for many years. Here we show that the switch complex associates with the membrane‐bound respiratory protein fumarate reductase (FRD). We provide evidence that FRD binds to preparations of isolated switch complexes, forms a 1:1 complex with the switch protein FliG, and that this interaction is required for both flagellar assembly and switching the direction of flagellar rotation. We further show that fumarate, known to be a clockwise/switch factor, affects the direction of flagellar rotation through FRD. These results not only uncover a new component important for switching and flagellar assembly, but they also reveal that FRD, an enzyme known to be primarily expressed and functional under anaerobic conditions in Escherichia coli, nonetheless, has important, unexpected functions under aerobic conditions.

Fluorescence and excited state absorption in modified pigments of bacterial photosynthesis a comparative study of metal-substituted bacteriochlorophylls a

Abstract A series of transmetalated bacteriochlorophylls, where the central magnesium has been replaced by Pd, Ni, Zn, Cd, Cu, have been investigated by linear and non-linear laser spectroscopic methods. A strong dependence of the fluorescence on the central metal was obtained. Differences in fluorescence lifetimes and quantum yields are caused mainly by different efficiencies of intersystem crossing. Strong excited state absorptions were found, in particular, the Pd-compound has a strong transient absorption in the 550–670 nm range. The results are of interest with respect to a possible oxygen-free sensitizing action of Pd-BPhe a for photodynamic tumor therapy.

Zinc-bacteriochlorophyllide dimers in de novo designed four-helix bundle proteins. A model system for natural light energy harvesting and dissipation.

Photosynthetic organisms utilize interacting pairs of chlorophylls and bacteriochlorophylls as excitation energy donors and acceptors in light harvesting complexes, as photosensitizers of charge separation in reaction centers, and maybe as photoprotective quenching centers that dissipate excess excitation energy under high light intensities. To better understand how the pigments local environment and spatial organization within the protein tune its ground- and excited-state properties to perform different functions, we prepared and characterized the simplest possible system of interacting bacteriochlorophylls within a protein scaffold. Using HP7, a high-affinity heme-binding protein of the HP class of de novo designed four-helix bundles, we incorporated 13(2)-OH-zinc-bacteriochlorophyllide-a (ZnBChlide), a water-soluble bacteriochlorophyll derivative, into specific binding sites within the four-helix bundle protein core. We capitalized on the rich and informative optical spectrum of ZnBChlide to rigorously characterize its complexes with HP7 and two variants, in which a single heme-binding site is eliminated by replacing histidine residues at positions 7 or 42 by phenylalanine. Surprisingly, we found the ZnBChlide binding capacity of HP7 and its variants to be higher than for heme: up to three ZnBChlide pigments bind per HP7, or two per each single histidine variant. The formation of dimers within HP7 results in dramatic quenching of ZnBChlide fluorescence, reducing its quantum yield by about 80%, and the singlet excited-state lifetime by 2 orders of magnitudes compared to the monomer. Thus, HP7 and its variants are the first examples of a simple protein environment that can isolate a self-quenching pair of photosynthetic pigments in pure form. Unlike its complicated natural analogues, this system can be constructed from the ground up, starting with the simplest functional element, increasing the complexity as needed.

Empirical and computational design of iron-sulfur cluster proteins.

Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Fine Tuning of Chlorophyll Spectra by Protein-Induced Ring Deformation.

The ability to tune the light-absorption properties of chlorophylls by their protein environment is the key to the robustness and high efficiency of photosynthetic light-harvesting proteins. Unfortunately, the intricacy of the natural complexes makes it very difficult to identify and isolate specific protein-pigment interactions that underlie the spectral-tuning mechanisms. Herein we identify and demonstrate the tuning mechanism of chlorophyll spectra in type II water-soluble chlorophyll binding proteins from Brassicaceae (WSCPs). By comparing the molecular structures of two natural WSCPs we correlate a shift in the chlorophyll red absorption band with deformation of its tetrapyrrole macrocycle that is induced by changing the position of a nearby tryptophan residue. We show by a set of reciprocal point mutations that this change accounts for up to 2/3 of the observed spectral shift between the two natural variants.

Bimodal intramolecular excitation energy transfer in a multichromophore photosynthetic model system: hybrid fusion proteins comprising natural phycobilin- and artificial chlorophyll-binding domains.

The phycobilisomes of cyanobacteria and red-algae are highly efficient peripheral light-harvesting complexes that capture and transfer light energy in a cascade of excitation energy transfer steps through multiple phycobilin chromophores to the chlorophylls of core photosystems. In this work, we focus on the last step of this process by constructing simple functional analogs of natural phycobilisome-photosystem complexes that are based on bichromophoric protein complexes comprising a phycobilin- and a chlorophyll- or porphyrin-binding domain. The former is based on ApcE(1-240), the N-terminal chromophore-binding domain of the phycobilisomes L(CM) core-membrane linker, and the latter on HP7, a de novo designed four-helix bundle protein that was originally planned as a high-affinity heme-binding protein, analogous to b-type cytochromes. We fused a modified HP7 protein sequence to ApcEΔ, a water-soluble fragment of ApcE(1-240) obtained by excising a putative hydrophobic loop sequence of residues 77-153. HP7 was fused either to the N- or the C-terminus of ApcEΔ or inserted between residues 76 and 78, thereby replacing the native hydrophobic loop domain. We describe the assembly, spectral characteristics, and intramolecular excitation energy transfer of two unique systems: in the first, the short-wavelength absorbing zinc-mesoporphyrin is bound to the HP7 domain and serves as an excitation-energy donor to the long-wavelength absorbing phycocyanobilin bound to the ApcE domain; in the second, the short-wavelength absorbing phycoerythrobilin is bound to the ApcE domain and serves as an excitation energy donor to the long-wavelength absorbing zinc-bacteriochlorophyllide bound to the HP7 domain. All the systems that were constructed and tested exhibited significant intramolecular fluorescence resonance energy transfer with yields ranging from 21% to 50%. This confirms that our modular, covalent approach for studying EET between the cyclic and open chain tetrapyrroles is reasonable, and may be extended to larger structures mimicking light-harvesting in cyanobacteria. The design, construction, and characterization process demonstrated many of the advances in constructing such model systems, particularly in our ability to control the fold and aggregation state of protein-based systems. At the same time, it underlines the potential of exploiting the versatility and flexibility of protein-based systems in assembling multiple pigments into effective light-harvesting arrays and tuning the spectral properties of multichromophore systems.

Design principles for chlorophyll‐binding sites in helical proteins

The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, and hemes are ubiquitous cofactors of biological catalysis that are involved in a multitude of reactions. One systematic approach for understanding how Nature achieves functional diversity with only this handful of cofactors is by designing de novo simple and robust protein scaffolds with heme and/or (bacterio)chlorophyll [(B)Chls]‐binding sites. This strategy is currently mostly implemented for heme‐binding proteins. To gain more insight into the factors that determine heme‐/(B)Chl‐binding selectivity, we explored the geometric parameters of (B)Chl‐binding sites in a nonredundant subset of natural (B)Chl protein structures. Comparing our analysis to the study of a nonredundant database of heme‐binding helical histidines by Negron et al. (Proteins 2009;74:400–416), we found a preference for the m‐rotamer in (B)Chl‐binding helical histidines, in contrast to the preferred t‐rotamer in heme‐binding helical histidines. This may be used for the design of specific heme‐ or (B)Chl‐binding sites in water‐soluble helical bundles, because the rotamer type defines the positioning of the bound cofactor with respect to the helix interface and thus the protein‐binding site. Consensus sequences for (B)Chl binding were identified by combining a computational and database‐derived approach and shown to be significantly different from the consensus sequences recommended by Negron et al. (Proteins 2009;74:400–416) for heme‐binding helical proteins. The insights gained in this work on helix‐ (B)Chls‐binding pockets provide useful guidelines for the construction of reasonable (B)Chl‐binding protein templates that can be optimized by computational tools. Proteins 2011.

A minimal phycobilisome: Fusion and chromophorylation of the truncated core-membrane linker and phycocyanin

Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.

Electron transport between photosystem II and photosystem I encapsulated in sol-gel glasses.

Photosystem I (PSI) and photosystem II (PSII) are the primary solar-energy-converting enzymes of oxygenic photosynthetic organisms. PSI is a robust, potent, and highly efficient photosensitizer capable of providing electrons at a high reduction potential ( 0.55 V vs. normal hydrogen electrode, NHE) from its reducing end. These properties have prompted many attempts to integrate PSI into biohybrid systems for solar energy conversion and storage. In a biological setting, water photooxidation by PSII provides a source of electrons for the reductive processes driven by PSI, but in contrast to PSI, integration of PSII into nonbiological systems is very difficult. Both photosystems were successfully “wired” to conductive electrode surfaces, but their integration into a photocatalytic bioelectrochemical device has not been reported to date. Herein we demonstrate a simple scheme for mediated coupling of PSII and PSI in solution; this coupling enables electron flow from water photooxidized by PSII all the way to the reducing end of PSI. Furthermore, we show that the same scheme can be reconstituted either when both photosystems are coencapsulated in sol–gel glasses, or when one photosystem is encapsulated and the other is in solution. The sol– gel trapping technique is a proven method for encapsulating a wide variety of biological materials, from intact whole cells to functional individual enzymes. 7] In addition, sol–gel systems are porous and optically transparent, which makes them ideal scaffolds for photoinduced electron transfer systems such as the photosynthetic machinery. The main difference between the sol–gel and solution samples is that the photosystem complexes are immobilized within the sol–gel cavities instead of diffusing freely in solution. This property suggests interesting possibilities of segregating PSI and PSII in distinct microenvironments while maintaining electron flow between the photosystems. PSII is not naturally directly coupled to PSI (Figure 1, top). Instead, electrons flow from PSII to a pool of membrane-soluble plastoquinones that diffuse between the acceptor and donor sides of PSII, and cytochrome b6f (b6f), respectively. Only a fraction of the electrons extracted from water at the oxygen-evolving complex of PSII end up at the acceptor side of PSI. The rest are cycled between PSII and b6f whereby directed diffusion of quinones, the release of protons on the water oxidizing face, and their consumption on the reducing face of the membrane actively pumps protons across the membrane, and generates proton-motive force. By using the amphipathic quinone analogue 2,6dichlorophenolindophenol (DCPIP) as an electron carrier, we were able to bypass b6f and set up an alternative pathway of electron flow from PSII to PSI (Figure 1, bottom). Although it is well established that DCPIP can be reduced by PSII, and the reduced form DCPIPH2 is an electron donor to PSI, DCPIP-mediated electron flow from PSII to PSI was not reported to date, to the best of our knowledge. This Figure 1. Electron and proton flow in native photosynthetic membranes (top), and mediated electron flow between sol–gel encapsulated PSI and PSII (bottom). Pink arrows indicate proton flow, solid blue and dashed black arrows indicate electron transfer by tunneling and diffusion of soluble carriers, respectively. The oxidized and reduced states of the redox carriers are shown by orange and blue colors, respectively. Fd = ferredoxin, PC = plastocyanin, b6f = cytochrome b6f, PQ = plastoquinone, OEC = oxygen-evolving complex, FNR= ferredoxin NADP reductase.

Structural principles for computational and de novo design of 4Fe-4S metalloproteins.

Iron-sulfur centers in metalloproteins can access multiple oxidation states over a broad range of potentials, allowing them to participate in a variety of electron transfer reactions and serving as catalysts for high-energy redox processes. The nitrogenase FeMoCO cluster converts di-nitrogen to ammonia in an eight-electron transfer step. The 2(Fe4S4) containing bacterial ferredoxin is an evolutionarily ancient metalloprotein fold and is thought to be a primordial progenitor of extant oxidoreductases. Controlling chemical transformations mediated by iron-sulfur centers such as nitrogen fixation, hydrogen production as well as electron transfer reactions involved in photosynthesis are of tremendous importance for sustainable chemistry and energy production initiatives. As such, there is significant interest in the design of iron-sulfur proteins as minimal models to gain fundamental understanding of complex natural systems and as lead-molecules for industrial and energy applications. Herein, we discuss salient structural characteristics of natural iron-sulfur proteins and how they guide principles for design. Model structures of past designs are analyzed in the context of these principles and potential directions for enhanced designs are presented, and new areas of iron-sulfur protein design are proposed. This article is part of a Special issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, protein networks, edited by Ronald L. Koder and J.L Ross Anderson.