Biophysical and structural characterization of the small heat shock protein HspA from Thermosynechococcus vulcanus in 2 M urea.

Small heat shock proteins (sHSPs) belong to the superfamily of molecular chaperones. They prevent aggregation of partially unfolded or misfolded client proteins, providing protection to organisms under stress conditions. Here, we report the biophysical and structural characterization of a small heat shock protein (HspA) from a thermophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 M urea. HspA has been shown to be important for the protection of Photosystem II and the Phycobilisome antenna complex at elevated temperatures. Heterologously expressed HspA requires the presence of 1-2 M urea to maintain its solubility at concentrations required for most characterization methods. Spectroscopic studies reveal the presence of the ß-sheet structure and intactness of the tertiary fold in HspA. In vitro assays show that the HspA maintains chaperone-like activity in protecting soluble proteins from thermal aggregation. Chromatography and electron microscopy show that the HspA exists as a mixture of oligomeric forms in the presence of 2 M urea. HspA was successfully crystallized only in the presence of 2 M urea. The crystal structure of HspA shows urea-induced loss of about 30% of the secondary structure without major alteration in the tertiary structure of the protein. The electron density maps reveal changes in the hydrogen bonding network which we attribute to the presence of urea. The crystal structure of HspA demonstrates a mixture of both direct interactions between urea and protein functionalities and interactions between urea and the surrounding solvent that indirectly affect the protein, which are in accordance with previously published studies.

Structural heterogeneity leads to functional homogeneity in A. marina phycocyanin.

The major light harvesting antenna in all cyanobacterial species is the phycobilisome (PBS). The smallest PBS identified to date is that of Acaryochloris marina (A. marina), composed of a single four-hexamer rod. We have determined the crystal structure of phycocyanin (AmPC), the major component of the A. marina PBS (AmPBS) to 2.1 Å. The basic unit of the AmPC is a heterodimer of two related subunits (α and ß), and we show that the asymmetric unit contains a superposition of two α and two ß isoforms, the products of the simultaneous expression of different genes. This is the first time to our knowledge that isolated proteins crystallized with such identifiable heterogeneity. We believe that the presence of the different isoforms allows the AmPBS to have a significant bathochromic shift in its fluorescence emission spectrum, allowing, in the total absence of allophycocyanin, a better overlap with absorption of the chlorophyll d-containing reaction centers. We show that this bathochromic shift exists in intact AmPBS as well as in its disassembled components, thus suggesting that AmPC can efficiently serve as the AmPBS terminal emitter.

The Structural Basis for the Extraordinary Energy-Transfer Capabilities of the Phycobilisome.

Light absorption is the initial step in the photosynthetic process. In all species, most of the light is absorbed by dedicated pigment-protein complexes called light harvesting complexes or antenna complexes. In the case of cyanobacteria and red-algae, photosynthetic organisms found in a wide variety of ecological niches, the major antenna is called the Phycobilisome (PBS). The PBS has many unique characteristics that sets it apart from the antenna complexes of other organisms (bacteria, algae and plants). These differences include the type of light absorbing chromophores, the protein environment of the chromophores, the method of assembly and association and the intercellular location with respect to the photosynthetic reaction centers (RCs). Since the final goal of all antenna complexes is the same - controlled absorption and transfer of the energy of the sun to the RCs, the unique structural and chemical differences of the PBS also require unique energy transfer mechanisms and pathways. In this review we will describe in detail the structural facets that lead to a mature PBS, followed by an attempt to understand the energy transfer properties of the PBS as they have been measured experimentally.

Structure of the bovine COPI δ subunit µ homology domain at 2.15 Å resolution.

The heptameric COPI coat (coatomer) plays an essential role in vesicular transport in the early secretory system of eukaryotic cells. While the structures of some of the subunits have been determined, that of the δ-COP subunit has not been reported to date. The δ-COP subunit is part of a subcomplex with structural similarity to tetrameric clathrin adaptors (APs), where δ-COP is the structural homologue of the AP µ subunit. Here, the crystal structure of the µ homology domain (MHD) of δ-COP (δ-MHD) obtained by phasing using a combined SAD-MR method is presented at 2.15 Å resolution. The crystallographic asymmetric unit contains two monomers that exhibit short sections of disorder, which may allude to flexible regions of the protein. The δ-MHD is composed of two subdomains connected by unstructured linkers. Comparison between this structure and those of known MHD domains from the APs shows significant differences in the positions of specific loops and ß-sheets, as well as a more general change in the relative positions of the protein subdomains. The identified difference may be the major source of cargo-binding specificity. Finally, the crystal structure is used to analyze the potential effect of the I422T mutation in δ-COP previously reported to cause a neurodegenerative phenotype in mice.