Design, synthesis and properties of synthetic chlorophyll proteins

Abstract

A chemoselective method is described for coupling chlorophyll derivatives with an aldehyde group to synthetic peptides or proteins modified with an aminoxyacetyl group at the :-amino group of a lysine residue. Three templateassembled antiparallel four-helix bundles were synthesized for the ligation of one or two chlorophylls. This was achieved by coupling unprotected peptides to cysteine residues of a cyclic decapeptide by thioether formation. The amphiphilic helices were designed to form a hydrophobic pocket for the chlorophyll derivatives. Chlorophyll derivatives Zn-methylpheophorbide b and Zn-methyl-pyropheophorbide d were used. The aldehyde group of these chlorophyll derivatives was ligated to the modified lysine group to form an oxime bond. The peptide±chlorophyll conjugates were characterized by electrospray mass spectrometry, analytical HPLC, and UV/visible spectroscopy. Two four-helix bundle chlorophyll conjugates were further characterized by sizeexclusion chromatography, circular dichroism, and resonance Raman spectroscopy.Keywords: synthetic proteins; chlorophylls; protein design.Proteins containing chlorophyll (Chl) and bacteriochlorophyll (BChl) are essential for the biological conversion of solar to chemical energy. They serve two general functions: (a) the absorption of light and energy transfer and (b) lightinduced ionization and stable charge separation. Interactions of the pigments with each other and with the protein, control their relative orientations and adjust their excited state and redox properties over a wide range. It is a challenge to design proteins with these functions for new applications. Several high-resolution structures of chlorophyll proteins are known that could guide such a design, including two water-soluble light-harvesting complexes, the FMO protein [1,2] and the peridinin-Chl a protein (PCP) [3], and several integral membrane proteins, namely the light-harvesting complexes LH2 [4] and LHCII [5], and reaction centers from purple bacteria [6,7] and photosystem I [8,9]. A resolution sufficient to recognize details of cofactor binding is only available for the FMO protein, PCP, LH2, and the bacterial reaction centers. The structures reveal rather complex binding pockets for these pigments, complicated particularly by the large hydrophobic phytyl tail of the chlorophylls whose principles of interaction with the molecular environment are currently only partly understood. The phytyl-tails are structurally well defined and interact with the protein, the carotenoids and the tails of neighboring chlorophylls. Taking the LH2 from Rhodobacter spheroides as an example, a single phenylalanine seems to be essential to lock such a hydrophobic cluster within the protein [10]. Even more complex properties are indicated by reconstitution studies of native or genetically modified LHCII [11±17]. As an example, a single tryptophan seems critical, in vitro, for the stepwise assembly of the protein with all 12 chlorophyll molecules. The crucial role of only a few amino acids (including a Trp) i...

A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

“…It has been proposed that the transmembrane helix domains of the ␣-polypeptides show a low level of sequence conservation when different proteobacteria are compared; the one exception is His31. The major determinant for the specific Bchl-B850 geometry may be found predominantly in the residues outside the transmembrane region (3, 7,32,36,43). It has been found that insertion of four residues (TTWA) towards the carboxy terminus of the transmembrane helix of the ␣-apoprotein leads to the absence of LH2 complexes (3).…”

Hydrogen Bonding in a Model Bacteriochlorophyll-binding Site Drives Assembly of Light Harvesting Complex

“…Our strategy to study the binding of BChl to membrane-embedded helices and the assembly to stable TMH/BChl pockets is based on the use of model BChl proteins, modified in particular near the BChl-binding sites. In previous works, ligation of the central magnesium atom of coherent Argon laser (Innova 100), to histidines of synthetic de novo peptides has been achieved by a chemical modeling approach (28,29). This approach, however, depends on the reconstitution with pigments in a non-native system and relies on aqueous systems, either by use of detergents or by rendering the helices water-soluble.…”

De novodesigned cyclic-peptide heme complexes

“…With the challenge of design making rapid progress, several groups have begun to look at the incorporation of functional cofactors in hopes of producing de novo catalytic proteins (8)(9)(10)(11)(12)(13)(14). Many enzymes require mono-and dinuclear metal ions (15), metal clusters (ferredoxin and nitrogenase) (16), heme (cytochromes and globins) (17), and organic cofactors (flavin-and quinone-based enzymes) (18).…”