Ravi R. Sonani

Role of N-terminal residues on folding and stability of C-phycoerythrin: simulation and urea-induced denaturation studies

The conformational state of biliproteins can be determined by optical properties of the covalently linked chromophores. Recently determined crystal structure of truncated form of α-subunit of cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue provides a new insight into the structure–function relationship of αC-PE. To compare their stabilities, we have measured urea-induced denaturation transitions of the full length αC-PE (FL-αC-PE) and truncated αC-PE (Tr-αC-PE) followed by observing changes in absorbance at 565 nm, fluorescence at 350 and 573 nm, and circular dichroism at 222 nm as a function of [urea], the molar concentration of urea. The transition curve of each protein was analyzed for ΔGD0, the value of Gibbs free energy change on denaturation (ΔGD) in the absence of urea; m, the slope (=∂∆GD/∂[urea]), and Cm, the midpoint of the denaturation curve, i.e. [urea] at which ΔGD = 0. A difference of about 10% in ΔGD0 observed between FL-αC-PE and Tr-αC-PE, suggests that the two proteins are almost equally stable, and the natural deletion of 31 residues from the N-terminal side of the full length protein does not alter its stability. Furthermore, normalization of probes shows that the urea-induced denaturation of both the proteins is a two-state process. Folding of both structural variants (Tr-αC-PE and FL-αC-PE) of P. tenue were also studied using molecular dynamics simulations at 300 K. The results show clearly that the stability of the proteins is evenly distributed over the whole structure indicating no significant role of N-terminal residues in the stability of both proteins.

Effects of PAR and UV Radiation on the Structural and Functional Integrity of Phycocyanin, Phycoerythrin and Allophycocyanin Isolated from the Marine Cyanobacterium Lyngbya sp. A09DM

An in vitro analysis of the effects of photosynthetically active and ultraviolet radiations was executed to assess the photostability of biologically relevant pigments phycocyanin (PC), phycoerythrin (PE) and allophycocyanin (APC) isolated from Lyngbya sp. A09DM. Ultraviolet (UV) irradiances significantly affected the integrity of PC, PE and APC; however, PAR showed least effect. UV radiation affected the bilin chromophores covalently attached to phycobiliproteins (PBPs). Almost complete elimination of the chromophore bands associated with α‐ and β‐subunit of PE and APC occurred after 4 h of UV‐B exposure. After 5 h of UV‐B exposure, the content of PC, PE and APC decreased by 51.65%, 96.8% and 96.53%, respectively. Contrary to PAR and UV‐A radiation, a severe decrease in fluorescence of all PBPs was observed under UV‐B irradiation. The fluorescence activity of extracted PBP was gradually inhibited immediately after 15–30 min of UV‐B exposure. In comparison to the PC, the fluorescence properties of PE and APC were severely lost under UV‐B radiation. Moreover, the present study indicates that UV‐B radiation can damage the structural and functional integrity of phycobiliproteins leading to the loss of their ecological and biological functions.

Cyanobacterial Sunscreen Scytonemin: Role in Photoprotection and Biomedical Research

Cyanobacteria are the most promising group of photosynthetic microorganisms capable of producing an array of natural products of industrial importance. Scytonemin is a small hydrophobic alkaloid pigment molecules present in the extracellular sheath of several cyanobacteria as a protective mechanism against short wavelength solar ultraviolet (UV) radiation. It has great efficacy to minimize the production of reactive oxygen species and formation of DNA lesions. The biosynthesis of scytonemin is regulated by different physico-chemical stressors. Scytonemin display multiple roles, functioning as a potent UV sunscreen and antioxidant molecules, and can be exploited in cosmetic and other industries for the development of new cosmeceuticals. Herein, we review the occurrence, biosynthesis, and potential application of scytonemin in photoprotection, pharmaceuticals, and biomedical research.

The high-energy radiation protectant extracellular sheath pigment scytonemin and its reduced counterpart in the cyanobacterium Scytonema sp. R77DM

A cyanobacterial extracellular sheath pigment from Scytonema sp. R77DM was partially characterized and investigated for its increased production under abiotic factors, and UV-screening function. HPLC with PDA detection, and ion trap liquid chromatography/mass spectrometry analysis revealed the presence of a pigment scytonemin and its reduced counterpart. Ultraviolet radiation showed more stimulative effects on scytonemin production. A significant synergistic enhancement of scytonemin synthesis was observed under combined stress of heat and UV radiation. Scytonemin also exhibited efficient UV-screening function by reducing the in vivo production of reactive oxygen species (ROS) and cyclobutane thymine dimer. UV-induced formation of ROS and thymine dimer was also reduced upon exposure of cyanobacterial cells to exogenous antioxidant, ascorbic acid; however, the effect was more significant when both scytonemin and ascorbic acid were applied in combination. Moreover, the results indicate the potential role of scytonemin pigment as natural photoprotectant against high energy solar insolation.

Recent advances in production, purification and applications of phycobiliproteins

An obligatory sunlight requirement for photosynthesis has exposed cyanobacteria to different quantity and quality of light. Cyanobacteria can exhibit efficient photosynthesis over broad region (450 to 650 nm) of solar spectrum with the help of brilliantly coloured pigment proteins called phycobiliproteins (PBPs). Besides light-harvesting, PBPs are found to involve in several life sustaining phenomena including photoprotection in cyanobacteria. The unique spectral features (like strong absorbance and fluorescence), proteineous nature and, some imperative properties like hepato-protective, anti-oxidants, anti-inflammatory and anti-aging activity of PBPs enable their use in food, cosmetics, pharmaceutical and biomedical industries. PBPs have been also noted to show beneficial effect in therapeutics of some disease like Alzheimer and cancer. Such large range of applications increases the demand of PBPs in commodity market. Therefore, the large-scale and coast effective production of PBPs is the real need of time. To fulfil this need, many researchers have been working to find the potential producer of PBPs for the production and purification of PBPs. Results of these efforts have caused the inventions of some novel techniques like mixotrophic and heterotrophic strategies for production and aqueous two phase separation for purification purpose. Overall, the present review summarises the recent findings and identifies gaps in the field of production, purification and applications of this biological and economically important proteins.

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

A stable and functional single peptide phycoerythrin (15.45 kDa) from Lyngbya sp. A09DM.

A functional and stable truncated-phycoerythrin (T-PE) was found as a result of spontaneous in vitro truncation. Truncation was noticed to occur during storage of purified native-phycoerythrin (N-PE) isolated from Lyngbya sp. A09DM. SDS and native-PAGE analysis revealed the truncation of N-PE, containing α (19.0 kDa)--and β (21.5 kDa)--subunits to the only single peptide of ∼15.45 kDa (T-PE). The peptide mass fingerprinting (PMF) and MS/MS analysis indicated that T-PE is the part of α-subunit of N-PE. UV-visible absorption peak of N-PE was found to split into two peaks (540 and 565 nm) after truncation, suggesting the alterations in its folded state. The emission spectra of both N-PE and T-PE show the emission band centered at 581 nm (upon excitation at 559 nm) suggested the maintenance of fluorescence even after significant truncation. Urea-induced denaturation and Gibbs-free energy (ΔGD°) calculations suggested that the folding and structural stability of T-PE was almost similar to that of N-PE. Presented bunch of evidences revealed the truncation in N-PE without perturbing its folding, structural stability and functionality (fluorescence), and thereby suggested its applicability in fluorescence based biomedical techniques where smaller fluorescence molecules are more preferable.

Antioxidant Potential of Phycobiliproteins: Role in Anti-Aging Research

Aging research has made significant progress over recent years, particularly after the formulation of ‘Oxidative stress theory of aging’. According to this theory, aging and its associated abnormalities may be prevented, at least to some extent, by application of certain antioxidants. Cyanobacterial phycobiliproteins (PBPs), the major light harvesting pigment proteins are widely characterized for their in vivo and in vitro antioxidant activity. Since, reactive oxygen species (ROS) are considered as important factors to cause aging, PBPs can be used as an effective free radical scavengers and be a potent candidate to develop the anti-aging drug. The use of PBPs in preventing the oxidative stress mediated abnormalities or aging is rationally debated. The present review enlightens the recent advances in the field of antioxidant function of PBPs and major challenges in the application of these pigment proteins in anti-aging research. Also included is the possible mechanism behind the anti-aging capacity of these ecologically as well as economically important biomolecules.

Phormidium phycoerythrin forms hexamers in crystals: a crystallographic study.

The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus-2 synchrotron. The crystals diffracted to about 2.1 Å resolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108.4 Å, c = 116.6 Å, α = 78.94, β = 82.50, γ = 60.34°. The molecular-replacement solution confirmed the presence of 12 αβ monomers in the P1 cell. The Phormidium PE elutes as an (αβ)3 trimer of αβ monomers from a molecular-sieve column and exists as [(αβ)3]2 hexamers in the crystal lattice. Unlike red algal PE proteins, the hexamers of Phormidium PE do not form higher-order structures in the crystals. The existence of only one characteristic visual absorption band at 564 nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly.

Probing pH sensitivity of αC-phycoerythrin and its natural truncant: A comparative study

Cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue exists in two natural forms named as full length (FL-αC-PE) and truncated (Tr-αC-PE). FL-αC-PE and Tr-αC-PE are produced when cyanobacterium is grown in the optimal medium and nutrient deficient medium, respectively. Despite of N-terminal deletion, both proteins show similar spectroscopic properties. In this study, different optical properties of these two natural variants of C-PE were measured in the pH range 1.0-12.0 (1.0 ≤ pH ≤ 12.0). It was observed that: (i) their absorption, fluorescence and CD spectra remain unchanged within the range adjacent to neutral pH, 5.5-8.75, (ii) at pH values higher than 8.75 and lower than 5.5 their absorption, fluorescence and CD spectral signatures are changed significantly, and (iii) emission spectra of the covalently linked tetrapyrrole chromophores and Trp residue are perturbed at extreme pH values in the range 8.75<pH<5.5. Refolding experiments further suggest that pH-induced denaturation of both forms of C-PE is reversible in the pH range 2.5-11.0, but irreversible beyond this range on both sides of pH extremes. The pH-induced denaturation of both the full length and truncated αC-PEs follows a two-state mechanism.