Paula Braun

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.

Structural Role of (Bacterio)chlorophyll Ligated in the Energetically Unfavorable β-Position

Chlorophyll is attached to apoprotein in diastereotopically distinct ways, by β- and α-ligation. Both the β- and α-ligated chlorophylls of photosystem I are shown to have ample contacts to apoprotein within their proteinaceous binding sites, in particular, at C-13 of the isocyclic ring. The H-bonding patterns for the C-131 oxo groups, however, are clearly distinct for the β-ligated and α-ligated chlorophylls. The β-ligated chlorophylls frequently employ their C-131 oxo in H-bonds to neighboring helices and subunits. In contrast, the C-131 oxo of α-ligated chlorophylls are significantly less involved in H-bonding interactions, particularly to neighboring helices. Remarkably, in the peripheral antenna, light harvesting complex (LH2) from Rhodobacter sphaeroides, a single mutation in the α-subunit, introduced to eliminate H-bonding to the β-bacteriochlorophyll-B850, which is ligated in the “β-position,” results in significant thermal destabilization of the LH2 in the membrane. In addition, in comparison with wild type LH2, the expression level of the LH2 lacking this H-bond is significantly reduced. These findings show that H-bonding to the C-131 keto group ofβ-ligated (bacterio)-chlorophyll is a key structural motif and significantly contributes to the stability of bacteriochlorophyll proteins in the native membrane. Our analysis of photosystem I and II suggests that this hitherto unrecognized motif involving H-bonding to β-ligated chlorophylls may be equally critical for the stable assembly of the inner core antenna of these multicomponent chlorophyll proteins.

Design and Assembly of Functional Light-Harvesting Complexes

Two complementary model systems are described, which are used to study the assembly of functional light-harvesting (LH) complexes. One system is based on rational design of cofactor-binding motifs and their capacity to assembly model LH2 complexes via expression in native-like membranes. The second takes advantage of the highly reversible self-assembly of the LH1 complex in artificial membranes and provides a convenient tool for design of model complexes with modified cofactors. In essence, re-design of the cofactor binding pockets in LH2 enables exploration of the underlying principles that enable particular amino acid combinations to sustain stable and functional assembly of LH-active arrays. Cofactor-binding motifs predicted in silico are tested in the context of the LH2 complex. In this way, H-bonding at the bacteriochlorophyll (BChl)/protein interface and the presence of aromatic residues were identified as critical for assembly of BChl and carotenoid (Crt). Moreover, the volumes of particular residues in the vicinity of BChl were shown to be critical for fine-tuning the spectroscopic properties. The LH1 reconstitution system, on the other hand, provides new information on the cofactor-related determinants of formation and functioning of this LH complex. Using the excitation trap approach, the coupling between BChl and excitation delocalization over the LH1 ring could be evaluated, while, by the replacement of Crts, their contribution to the assembly was assessed and for the first time a Crt-binding intermediate of LH1 assembly was identified. A new challenge is to make the two model approaches more interchangeable, thus allowing us to compare the same factors in different LH complexes, and eventually to identify on a molecular level what renders these apparently similar complexes so different.

Fine tuning of the spectral properties of LH2 by single amino acid residues

The peripheral light-harvesting complex, LH2, of Rhodobacter sphaeroides consists of an assembly of membrane-spanning α and β polypeptides which assemble the photoactive bacteriochlorophyll and carotenoid molecules. In this study we systematically investigated bacteriochlorophyll-protein interactions and their effect on functional bacteriochlorophyll assembly by site-directed mutations of the LH2 α-subunit. The amino acid residues, isoleucine at position −1 and serine at position −4 were replaced by 12 and 13 other residues, respectively. All residues replacing isoleucine at position −1 supported the functional assembly of LH2. The replacement of isoleucine by glycine, glutamine or asparagine, however, produced LH2 complex with significantly altered spectral properties in comparison to LH2 WT. As indicated by resonance Raman spectroscopy extensive rearrangement of the bacteriochlorophyll-B850 macrocycle(s) took place in LH2 in which isoleucine −1 was replaced by glycine. The replacement results in disruption of the H-bond between the C3 acetyl groups and the aromatic residues +13/+14 without affecting the H-bond involving the C131 keto group. In contrast, nearly all amino acid replacements of serine at position −4 resulted in shifting of the bacteriochlorophyll-B850 red most absorption maximum. Interestingly, the extent of shifting closely correlated with the volume of the residue at position −4. These results illustrate that fine tuning of the spectral properties of the bacteriochlorophyll-B850 molecules depend on their packing with single amino acid residues at distinct positions.