Concerted enhanced-sampling simulations to elucidate the helix-fibril transition pathway of intrinsically disordered α-Synuclein.

Fibril formation of α-synuclein is linked with Parkinson's disease. The intrinsically disordered nature of α-syn provides extensive conformational plasticity and becomes difficult to characterize its transition pathway from native monomeric to disease-associated fibril form. We implemented different simulation methods such as steered dynamics-umbrella sampling, and replica-exchange and conventional MD simulations to access various conformational states of α-syn. Nineteen distinct intermediate structures were identified by free energy landscape and cluster analysis. They were then sorted based on secondary structure and solvent exposure of fibril-core residues to illustrate the fibril dissociation pathway. The analysis showed that following the initial dissociation of the polypeptide chain from the fibril, α-syn might form either compact-conformations by long-range interactions or extended-conformations stabilized by local interactions. This leads α-syn to adapt two different pathways. The secondary structure, solvation, contact distance, interaction energies and backbone dihedrals of thirty-two selected residues were analyzed for all the 19 intermediates. The results suggested that formation of ß-turns, reorganization of salt bridges, and dihedral changes in the hydrophobic regions are the major driving forces for helix-fibril transition. Structural features of the intermediates also correlated with the earlier experimental and computational studies. The study provides critical information on the fibrillation pathway of α-syn.

A conserved Plasmodium structural integrity maintenance protein (SIMP) is associated with sporozoite membrane and is essential for maintaining shape and infectivity.

Plasmodium sporozoites are extracellular forms introduced during mosquito bite that selectively invade mammalian hepatocytes. Sporozoites are delimited by a cell membrane that is linked to the underlying acto-myosin molecular motor. While membrane proteins with roles in motility and invasion have been well studied, very little is known about proteins that maintain the sporozoite shape. We demonstrate that in Plasmodium berghei (Pb) a conserved hypothetical gene, PBANKA_1422900 specifies sporozoite structural integrity maintenance protein (SIMP) required for maintaining the sporozoite shape and motility. Sporozoites lacking SIMP exhibited loss of regular shape, extensive membrane blebbing at multiple foci, and membrane detachment. The mutant sporozoites failed to infect hepatocytes, though the altered shape did not affect the organization of cytoskeleton or inner membrane complex (IMC). Interestingly, the components of IMC failed to extend under the membrane blebs likely suggesting that SIMP may assist in anchoring the membrane to IMC. Endogenous C-terminal HA tagging localized SIMP to membrane and revealed the C-terminus of the protein to be extracellular. Since SIMP is highly conserved among Plasmodium species, these findings have important implications for utilizing it as a novel sporozoite-specific vaccine candidate.

Spectroscopic studies on the stability and nucleation-independent fibrillation of partially-unfolded proteins in crowded environment.

Fibril formation of globular proteins is driven by attaining an appropriate partially-unfolded conformation. Excluded volume effect exerted by the presence of other macromolecules in the solution, as found in the cellular interior, might affect the conformational state of proteins and alter their fibril formation process. The change in structure, stability and rate of fibril formation of aggregation-prone partially-unfolded states of lysozyme (Lyz) and α-lactalbumin (ALA) in the presence of different sizes of polyethylene glycol (PEG) is examined using spectroscopic methods. Thermal denaturation and far-UV CD studies suggest that Lyz is stabilized by PEGs and the stability increases with increasing concentration of PEGs. However, the stability of ALA depends on the size and concentration of PEG. The change in enthalpy of unfolding indicates the existence of soft-interactions between the proteins and PEG along with excluded volume effect. Fibrillation rate of Lyz is not significantly altered in the presence of lower concentrations of PEGs suggesting that the crowding effect dominates the viscosity-induced retardation of protein association whereas at higher concentrations the rates are reduced. In case of ALA, the rate of fibrillation is drastically reduced; however, there is a marginal increase with the increasing concentration of PEG. The results suggest that the fibril formation is influenced by change in initial conformation of the partially-unfolded states of the proteins and their stability in the presence of the crowding agent. Further, the size and concentration of the crowding agent, and the soft-interaction between the proteins and PEG also affects the fibrillation.

Polyols, increasing global stability of cytochrome c, destabilize the thermal unfolding intermediate.

Studies on the intermediate states of proteins provide essential information on folding pathway and energy landscape of proteins. Osmolytes, known to alter the stability of proteins, might also affect the structure and energy states of folding intermediates. This was examined using cytochrome c (Cyt) as a model protein which forms a spectroscopically detectable intermediate during thermal denaturation transition. Most of the secondary structure and the native heme-ligation were intact in the intermediate state of the protein. Denaturants, urea and guanidinium hydrochloride, and ionic salt destabilizes the intermediate and drive the protein to follow two-state transition. The effect of polyol class of osmolytes, glycol, glycerol, erythritol, xylitol and sorbitol (with OH-groups two to six), on the intermediate was studied using Soret absorbance and far-UV circular dichroism. With the increasing concentration of any of the polyols, the transition-midpoint temperature (Tm) and the enthalpy change (ΔH) for native to intermediate transition were decreased. This indicated that the intermediate was destabilized by the polyols. However, the polyols increased the overall stability of the protein by increasing Tm and ΔH for intermediate to unfolded transition, except for glycol which destabilized the protein. These results show that the polyols could alter the energy state of the intermediate, and the effect of lower and higher polyols might be different on the stability and folding pathway of the protein.Communicated by Ramaswamy H. Sarma.

Cryo vs Thermo: Duality of Ethylene Glycol on the Stability of Proteins.

Osmolytes are known to stabilize proteins under stress conditions. Thermal denaturation studies on globular proteins (ß-lactoglobulin, cytochrome c, myoglobin, α-chymotrypsin) in the presence of ethylene glycol (EG), a polyol class of osmolyte, demonstrate a unique property of EG. EG stabilizes proteins against cold denaturation and destabilizes them during heat-induced denaturation. Further, chemical denaturation experiments performed at room temperature and at a sub-zero temperature (-10 °C) show that EG stabilizes the proteins at subzero temperature but destabilizes them at room temperature. The experiments carried out in the presence of glycerol, however, showed that glycerol stabilizes proteins against all of the denaturing conditions. This differential effect has not been reported for any other polyol class of osmolyte and might be specific to EG. Moreover, molecular dynamics simulations of all of the four proteins were carried out at three different temperatures, 240, 300, and 340 K, in the absence and presence of EG (20 and 40%). The results suggest that EG preferably accumulates around the hydrophobic residues and reduces the hydrophobic hydration of the proteins at a low temperature leading to stabilization of the proteins. At 340 K, the preferential hydration of the proteins is significantly reduced and the preferential binding of EG destabilizes the proteins like common denaturants.

Surface hydration and preferential interaction directs the charged amino acids-induced changes in protein stability.

In the present study, we investigate the interaction of amino acid osmolytes, Arg, Lys, Asp and Glu, and a denaturant, guanidinium chloride (Gdm) with proteins. To achieve this, molecular dynamics (MD) simulation of RNase A and α-lactalbumin was performed in the presence of three charged amino acids Arg, Lys, and Asp and the molecular mechanism of amino acid-induced (de)stabilization of the proteins was examined by combining with our earlier report on Glu. As Arg has the side chain similar to that of Gdm and destabilizes the proteins, MD simulation was carried out in the presence of Gdm as well. Radial distribution function and hydration fraction around the protein surface reveals that preferential hydration increases upon the addition of any of the cosolvent; however, the extent of increase is more in the presence of stabilizing cosolvents (stAAs: Lys, Asp and Glu) compared to destabilizing cosolvents (Arg and Gdm). Moreover, the preferential interaction of Arg and Gdm with the proteins is higher than that of stAAs. Residue-level interaction analysis suggests that stAAs preferably interacts with charged amino acids of the proteins whereas Arg and Gdm interactions could be found on almost all the surface exposed residues which might provide higher preferential interaction for these residues. From the results, we propose that the net outcome of preferential hydration versus preferential interaction of the amino acids might determine their effect on the stability of proteins.

Counteracting Effect of Charged Amino Acids Against the Destabilization of Proteins by Arginine.

Studies on osmolyte-induced effects on proteins help in enhancing protein stability under stressed conditions for various applications. Using mixtures of osmolytes could indeed widen their applications. The combinatorial effects of osmolytes with methylamines are majorly found in the literature; however, such studies are limited on the amino acid class of osmolytes. The present study examines the effect of charged amino acids Arg, Asp, and Lys on the stability of RNase A and α-LA. The thermal stabilities of the proteins in the presence of osmolytes are monitored by absorption changes, and the structural changes are analyzed using fluorescence quenching and near-UV circular dichroism (CD). These results are compared with our previous report on the effect of Glu. Arg destabilizes both the proteins whereas Asp, Lys, and Glu stabilize the proteins. The extent of stability provided by Asp and Glu is almost same and higher than Lys in RNase A. However, the stability acquired in the presence of Asp and Lys is comparable for α-LA and Glu provides higher stability. Further, the quenching and CD results suggest that the addition of amino acids do not alter the structure of the proteins significantly. The counteracting abilities of the stabilizing amino acids (stAAs) against Arg are then investigated. The results show that Glu could counteract Arg at the lowest fraction in the mixture. Lys requires nearly equimolar concentration whereas Asp needs almost double the concentration to counteract Arg induced destabilization of the proteins. At higher concentrations, the counteracting ability of Asp and Lys is similar for both the proteins. The counteracting ratio might slightly vary among the proteins, and it is not necessary that the amino acid providing higher stability to the protein could more effectively counteract Arg. This could be due to the change in the extent of preferential hydration of the proteins by stAAs in the presence of Arg. The results suggest that the addition of stAAs could be an effective strategy to increase the protein stability in biotechnology and biopharma applications.

Homology modelling, molecular docking, and molecular dynamics simulations reveal the inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase enzyme by Withaferin-A.

OBJECTIVE: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite's DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. RESULTS: A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

Analysing the microenvironment of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) in solvents and in different conformational states of proteins in relation to its fluorescence properties: a computational study.

Characterization of different conformational states of proteins is essential to understand their stability and activity. Biophysical techniques aid in analysing these conformational states and molecular fluorescence is one of the most reliable and quickly accessible methods. Apart from the intrinsic fluorescence of proteins, external fluorescence dyes such as TNS, ANS, nile red and thioflavin are also used to characterize partially unfolded, aggregated and fibrillar states of proteins, though their exact molecular-level interactions with proteins are yet to be completely unravelled. The present study attempts to investigate the binding of TNS molecules on different conformational states of proteins. Unconstrained molecular dynamics simulation of 50 molecules of TNS with the native state of BSA, native and two partially unfolded states of RNase A and α-lactalbumin in water was carried out. Dynamics simulation of TNS alone in different solvents such as water, ethanol, DMF and DMSO was also performed. Binding environments in all the proteins and the solvents were analysed in terms of H-bonding interactions, order of contacts, amino acid specificity and conformational changes of TNS, and correlated with experimentally observed fluorescence changes of the dye. The results suggest that TNS forms aggregates in water whereas in non-aqueous solvents the order of aggregates is lower which might result in an enhancement of its fluorescence intensity. Further, TNS preferably interacts with basic and aromatic amino acid residues of the proteins. In RNase A and α-lactalbumin, most of the TNS molecules tend to form aggregates even with the unfolded conformations of the proteins. However in BSA, the number of aggregated TNS molecules is less and TNS molecules in monomeric form are found in the hydrophobic crevices of the protein. This might result in an enhancement of the fluorescence in BSA compared to the other proteins. The distributions of angles and dihedrals of TNS in different environments suggest that the bending movement between the naphthyl and tolyl rings is constrained whereas significant planar rotations could be observed both in solvents and in protein-bound states.

Kinetics of protein fibril formation: Methods and mechanisms.

Amyloid fibril formation is a self-assembly reaction induced by favourable conformational changes of proteins leading to a stable, structurally organized aggregates. The deposition of stable protein fibrils in organs and tissues results in many diseases which are generally referred as amyloidosis. Though different disease conditions originate from sequentially and structurally different proteins, their fibrillar forms share common structural features. In vitro, fibril structure and kinetic pathway are investigated by using spectroscopic (fluorescence, circular dichroism, crystallography and solid state-NMR) and microscopic techniques. The kinetics of fibril formation is analysed using different mechanisms to understand the microscopic processes involved in the fibrillation reaction. This review discusses the assumptions, mechanisms, and limitations of some of the widely applied kinetic equations. Understanding of these equations would help to quantify the effect of the different microscopic process on the overall fibrillation kinetics which could aid in designing appropriate molecules to intervene in the aggregation process at different stages.

In silico screening for identification of novel ß-1,3-glucan synthase inhibitors using pharmacophore and 3D-QSAR methodologies.

The enzyme ß-1,3-glucan synthase, which catalyzes the synthesis of ß-1,3-glucan, an essential and unique structural component of the fungal cell wall, has been considered as a promising target for the development of less toxic anti-fungal agents. In this study, a robust pharmacophore model was developed and structure activity relationship analysis of 42 pyridazinone derivatives as ß-1,3-glucan synthase inhibitors were carried out. A five-point pharmacophore model, consisting of two aromatic rings (R) and three hydrogen bond acceptors (A) was generated. Pharmacophore based 3D-QSAR model was developed for the same reported data sets. The generated 3D-QSAR model yielded a significant correlation coefficient value (R (2) = 0.954) along with good predictive power confirmed by the high value of cross-validated correlation coefficient (Q (2) = 0.827). Further, the pharmacophore model was employed as a 3D search query to screen small molecules database retrieved from ZINC to select new scaffolds. Finally, ADME studies revealed the pharmacokinetic efficiency of these compounds.

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents.

BACKGROUND: Understanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood. METHODS: Dynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPLmut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed. RESULTS: PPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPLmut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents. CONCLUSIONS: Only the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid. GENERAL SIGNIFICANCE: This study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue-residue interactions during lid opening movement in water and in octanol.

Lid closure dynamics of porcine pancreatic lipase in aqueous solution.

BACKGROUND: Pancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water. METHODS: Molecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated. RESULTS: The lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around ß5- and ß9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation. CONCLUSIONS: PPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure. GENERAL SIGNIFICANCE: Conformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.

Modeling of babesipain-1 and identification of natural and synthetic leads for bovine babesiosis drug development.

Babesiosis is a tick-borne, zoonotic disease caused by species of the intraerythrocytic protozoan Babesia. It is distributed all around the world and affects various domestic and wild animals, mainly cattle. Recently, the cysteine protease enzyme, babesipain-1 from Babesia bigemina has been identified as a potential target for designing new anti-babesiosis drugs. In the present study, a three-dimensional structural model of babesipain-1 was developed. An active site with three pockets (S1, S2, and S3), which is congruent with its homolog, falcipain-3, was also identified. Moreover, the conservation of active site residues was consistent with the cysteine protease family. In order to identify potential inhibitors, a virtual screening workflow was employed with a chemical library containing natural and synthetic compounds. Potential inhibitors interacting with all the three subsites were identified. Further, molecular dynamic simulations were carried out to assess the interactions and stability of the inhibitors. The informatics approach, and the findings presented in this study will assist researchers in further development of potential anti-babesiosis molecules.

Sodium dodecyl sulphate (SDS) induced changes in propensity and kinetics of α-lactalbumin fibrillation.

Understanding surfactants induced changes on protein folding, aggregation, and fibrillation has a lot of implications in their laboratory and industrial applications. The effect of an anionic surfactant, sodium dodecyl sulphate (SDS), on fibrillation of an acidic protein α-lactalbumin (α-LA) at neutral pH condition was investigated. SDS at lower concentrations increased the lag time by nearly two-fold whereas the fibril elongation rate was not significantly altered. At the concentrations above 0.2mM, SDS lengthened the lag time by many-fold (∼60), but fibril elongation was accelerated by 3-6 fold. At the concentrations above 2mM, SDS inhibited α-LA fibrillation and led it to the formation of amorphous aggregates. These results were compared with the effect of SDS on the fibrillation of lysozyme, a basic protein. Though fibril inhibition was observed on both the proteins at the micellar concentrations of SDS, there were differences in the effect on lag time and elongation rate at the lower concentrations of SDS. This suggests that the inhibition of protein fibrillation by SDS-micelles might be a common mechanism irrespective of the surface charges on protein.

Differential effects of ionic and non-ionic surfactants on lysozyme fibrillation.

Fibril formation is a common property of many proteins, though not all are associated with diseases. Protein surface charges and the added co-solvents play vital roles in determining fibrillation pathways and kinetics. In order to understand these phenomena, the effects of anionic, cationic and non-ionic surfactants on lysozyme fibrillation were studied. Lysozyme forms fibrils in 2 M and 4 M urea solutions following nucleation-dependent and nucleation-independent pathways, respectively, at neutral pH. Under these conditions, the effects of sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and triton X-100 (Tx) were investigated on the lysozyme structure and fibrillation. The results indicate that there are differential effects of ionic and non-ionic surfactants on fibrillation. In the presence of SDS and CTAB, above their critical micelle concentrations (CMC), lysozyme could not form fibrils. However, non-ionic Tx does not inhibit fibril formation at all concentrations. Note that the time for complete fibril formation is increased by Tx. All of the surfactants are found to increase the initial nucleation phase; however, the extent of increase is less at near the CMC of the ionic surfactants and at above the CMC of Tx. The rates of fibril elongation show varying effects in the presence of different surfactants. The results suggest that the nucleation phase of lysozyme fibrillation is primarily controlled by charge interactions and micellation of the surfactants, but multiple factors might influence the fibril elongation. Furthermore, the surfactants do not alter the fibrillation pathway from nucleation-dependent to nucleation-independent or vice versa in the studied conditions.

Stability and activity of porcine lipase against temperature and chemical denaturants.

Lipases are the class of hydrolases with wide industrial applications. The present study analyses the stability of porcine pancreatic lipase (PPL) against urea, guanidine hydrochloride (Gdn), sodium dodecyl sulphate (SDS), and temperature using different spectroscopic techniques. Interestingly, this two-domain protein shows a two-state unfolding transition against urea and Gdn. The free energy of unfolding of PPL calculated from global analysis of the unfolding transitions obtained from different spectroscopic techniques is ~2.2 kcal/mol. In the presence of SDS, PPL shows a cooperative loss of secondary and tertiary structures above 0.2 mM of SDS. At above 2 mM of SDS, PPL forms irreversible, non-native, thermally stable structure. PPL loses its activity even at lower concentrations of urea (3 M), Gdn (0.5 M), and SDS (0.8 mM). Thermal denaturation of PPL shows an irreversible unfolding, and the protein lost its activity even by increasing the temperature to 45 °C. Though PPL in higher concentrations of SDS (>5 mM) shows stable conformation against temperature, its activity is completely lost. The results suggest that the structure and activity of PPL are more sensitive against chemical denaturants and temperature, and forms irreversible, non-native (in SDS) or completely unfolded (in urea, Gdn, and at higher temperature) conformations in different denaturing conditions.

CyanoPhyChe: a database for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins.

CyanoPhyChe is a user friendly database that one can browse through for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins. We downloaded all the protein sequences from the cyanobacterial genome database for calculating the physico-chemical properties, such as molecular weight, net charge of protein, isoelectric point, molar extinction coefficient, canonical variable for solubility, grand average hydropathy, aliphatic index, and number of charged residues. Based on the physico-chemical properties, we provide the polarity, structural stability and probability of a protein entering in to an inclusion body (PEPIB). We used the data generated on physico-chemical properties, structure and biochemical pathway information of all cyanobacterial proteins to construct CyanoPhyChe. The data can be used for optimizing methods of expression and characterization of cyanobacterial proteins. Moreover, the 'Search' and data export options provided will be useful for proteome analysis. Secondary structure was predicted for all the cyanobacterial proteins using PSIPRED tool and the data generated is made accessible to researchers working on cyanobacteria. In addition, external links are provided to biological databases such as PDB and KEGG for molecular structure and biochemical pathway information, respectively. External links are also provided to different cyanobacterial databases. CyanoPhyChe can be accessed from the following URL: http://bif.uohyd.ac.in/cpc.