Investigating the Functional Role of Hypothetical Proteins From an Antarctic Bacterium Pseudomonas sp. Lz4W: Emphasis on Identifying Proteins Involved in Cold Adaptation.

Exploring the molecular mechanisms behind bacterial adaptation to extreme temperatures has potential biotechnological applications. In the present study, Pseudomonas sp. Lz4W, a Gram-negative psychrophilic bacterium adapted to survive in Antarctica, was selected to decipher the molecular mechanism underlying the cold adaptation. Proteome analysis of the isolates grown at 4°C was performed to identify the proteins and pathways that are responsible for the adaptation. However, many proteins from the expressed proteome were found to be hypothetical proteins (HPs), whose function is unknown. Investigating the functional roles of these proteins may provide additional information in the biological understanding of the bacterial cold adaptation. Thus, our study aimed to assign functions to these HPs and understand their role at the molecular level. We used a structured insilico workflow combining different bioinformatics tools and databases for functional annotation. Pseudomonas sp. Lz4W genome (CP017432, version 1) contains 4493 genes and 4412 coding sequences (CDS), of which 743 CDS were annotated as HPs. Of these, from the proteome analysis, 61 HPs were found to be expressed consistently at the protein level. The amino acid sequences of these 61 HPs were submitted to our workflow and we could successfully assign a function to 18 HPs. Most of these proteins were predicted to be involved in biological mechanisms of cold adaptations such as peptidoglycan metabolism, cell wall organization, ATP hydrolysis, outer membrane fluidity, catalysis, and others. This study provided a better understanding of the functional significance of HPs in cold adaptation of Pseudomonas sp. Lz4W. Our approach emphasizes the importance of addressing the "hypothetical protein problem" for a thorough understanding of mechanisms at the cellular level, as well as, provided the assessment of integrating proteomics methods with various annotation and curation approaches to characterize hypothetical or uncharacterized protein data. The MS proteomics data generated from this study has been deposited to the ProteomeXchange through PRIDE with the dataset identifier-PXD029741.

Mass spectral analysis of acetylated peptides: Implications in proteomics.

Sequence determination of peptides using mass spectrometry plays a crucial role in the bottom-up approaches for the identification of proteins. It is crucially important to minimise false detection and validate sequence of the peptides in order to correctly identify a protein. Chemical modification of peptides followed by mass spectrometry is an option for improving the spectral quality. In silico-derived tryptic peptides with different N-terminal amino acids were designed from human proteins and synthesized. The effect of acetylation on the fragmentation of peptides was studied. N-terminal acetylation of the tryptic peptides was shown to form b1-ions, improve the abundance and occurrence of b-ions. In some cases, the intensity and occurrence of some y-ions also varied. Thus, it is demonstrated that acetylation plays an important role in improving the de novo sequencing efficiency of the peptides. The acetylation method was extended to tryptic peptides generated from the proteome of an Antarctic bacterium Pseudomonas syringae Lz4W using the proteomics work flow and mass spectra of the peptides were analysed. Comparison of the MS/MS spectra of the acetylated and unacetylated peptides revealed that acetylation helped in improving the spectral quality and validated the peptide sequences. Using this method, 673 proteins of the 1070 proteins identified were validated.

Characterization of acetylated histidine b1-ion structure: A competition between oxazolone and side chain imidazole moiety.

The detection of post-translational modifications of proteins is an important comprehensive research area. Over the years, proteomic studies involving protein acetylation have attracted a great deal of attention. In the present study, we have focussed on the acetylation of histidine and the intrinsic stability of b1-ion of oxazolone ring and/or with side chain imidazole bicyclic product. The formation of oxazolone structure may occur when an amino moiety undergoes acetylation reaction and when it is present in the vicinity of the side chain imidazole moiety. Tryptic peptides generated from the proteins of Acenitobacter radioresistens MMC5-containing N-terminal histidine were explored in a standard proteomic workflow. Formation of [Formula: see text] ion with an oxazolone ring in these peptides has been supported by a tandem mass spectrometric study of a synthetic peptide and density functional theory calculations. The results obtained from this study have implications in understanding the fragmentation of the peptides generated in the proteomic workflows.

Evidence for a molten globule state in Cicer α-galactosidase induced by pH, temperature, and guanidine hydrochloride.

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and ß structures in Cicer α-galactosidase and showed that its secondary structure belongs to α + ß class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH 2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH 2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.

Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase.

Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Šin the trigonal space group P3(2)21, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known.

Deciphering the molecular structure of cryptolepain in organic solvents.

Solvent composition plays a major role in stabilizing/destabilizing the forces that are responsible for the native structure of a protein. Often, the solvent composition drives the protein into non-native conformations. Elucidation of such non-native structures provides valuable information about the molecular structure of the protein, which is unavailable otherwise. Inclusion of methanol (non-fluorinated alcohol) or TFE (fluorinated alcohol) in the solvent composition drove cryptolepain, a serine protease and an all-ß-protein, into a non-native structure with an enhanced ß-sheet or induction of α-helix. These solvents did not much affect cryptolepain under neutral conditions, even at higher concentrations, but the effects were predominant at lower pH, when the protein molecule is under stress. The organic solvent-induced state is partially unfolded with similar characteristics to the molten globule state seen with protein under a variety of conditions. Chemical- or temperature-induced unfolding of cryptolepain in the presence of organic solvent is distinctly different from that in the absence of organic solvent. Such different unfolding provided evidence of two structural variants in the molecular structure of the protein as well as the differential stabilization/destabilization of such structural variants and their sequential unfolding.

Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical characterisation.

A dimeric serine protease Neriifolin S of molecular mass 94kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (Km of 0.195mM) and physiological efficiency (Kcat/Km of 144.5mMs). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45°C. Its proteolytic activity is strongly stimulated in the presence of Ca+2 ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent Tm, was found to be 65°C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54kJmol-1, 24.89kJmol-1, -82.34kJmol-1 and 337.20Jmol-1K-1.

Crystallization and preliminary X-ray analysis of crinumin, a chymotrypsin-like glycosylated serine protease with thrombolytic and antiplatelet activity.

Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure-function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, α = γ = 90, ß = 114.19°. The Matthews coefficient was 2.81 Å(3) Da(-1), corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress.

Identification of outer membrane proteins from an Antarctic bacterium Pseudomonas syringae Lz4W.

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.

SDS induced molten globule state of heynein; a new thiol protease: Evidence of domains and their sequential unfolding.

The molten globule state can be an intermediate in the protein-folding pathway and its detailed description can help understand the protein folding and an insight into the molecular structure of a protein. Sodium dodecyl sulfate (SDS), an anionic surfactant is known to induce molten globule sate in some proteins. SDS-induced changes in heynein were monitored by CD, fluorescence, 8-anilino-1-napthalenesulfonic acid (ANS) binding and proteolytic activity measurements. An enhancement in the α-helicity of protein with increasing concentration of SDS along with exposure of tryptophans is seen. At a concentration of SDS (∼2mM) heynein loses activity and rigid tertiary structure but possesses considerable amount of secondary structure along with strong ANS binding, indicating the presence of an intermediate state, which is like molten globule state seen in the case heynein. Chemical and temperature induced unfolding of SDS-induced state of heynein is non-cooperative contrary to the protein in the absence of detergent. Further, the cooperative unfolding transition curve of heynein in the absence of SDS intersects at a point where the second transition of SDS-induced state starts suggesting that the molecule of heynein consist of at least two structural domains which are stabilized differentially and unfolds sequentially. Enhancement of α-helicity of heynein in the presence of SDS suggests the α-rich domain of the protein was stabilized and unfold later as compared with ß-rich domain during temperature and chemical induced denaturation. Equilibrium unfolding pathway of heynein in SDS-induced state provides knowledge of the structure and stability of this plant cysteine protease at domain level.

Decolorization of crude latex by activated charcoal, purification and physico-chemical characterization of religiosin, a milk-clotting serine protease from the latex of Ficus religiosa.

The crude latex of Ficus religiosa is decolorized by activated charcoal. Decolorization follows the Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to homogeneity from the decolorized latex using anion exchange chromatography. Religiosin is a glycoprotein with a molecular mass of 43.4 kDa by MALDI-TOF. Religiosin is an acidic protein with a pI value of 3.8 and acts optimally at pH 8.0-8.5 and temperature 50 degrees C. The proteolytic activity of religiosin is strongly inhibited by PMSF and chymostatin indicating that the enzyme is a serine protease. The extinction coefficient (epsilon(1%)(280)) of religiosin is 29.47 M(-1) cm(-1)with 16 tryptophan, 26 tyrosine, and 11 cysteine residues per molecule. The enzyme shows broad substrate specificity against natural as well as synthetic substrates with an apparent K(m) of 0.066 mM and 6.25 mM using casein and Leu-pNA, respectively. MS/MS analysis confirms the novelty of the enzyme. Religiosin is highly stable against denaturants, metal ions, and detergents as well as over a wide range of pH and temperature. In addition, the enzyme exhibits milk-clotting as well as detergent activity.

Characterization of chemical modification of tryptophan by matrix-assisted laser desorption/ionization mass spectrometry.

Tert- butylation of tryptophan (2', 5', 7'- tri tertiary butyl tryptophan), formed during acidolytic cleavage of synthetic peptides Ac-KLVYWAE-CONH(2) (A-YW) and Ac-KLVWWAE-CONH(2) (A-WW), that are analogs of the fragment of Alzheimer's beta-amyloid peptide Ac-KLVFFAE-CONH(2), during solid-phase peptide synthesis, was characterized by matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI TOF/TOF) mass spectrometry. Crude peptide was fractionated by high performance liquid chromatography. Peptide fractions were sequenced and modified tryptophan was determined with the help of MALDI TOF/TOF mass spectra. Thus, it is possible to pinpoint the particular tryptophan residue that undergoes modification during synthesis of peptides containing multiple tryptophan residues.

Benghalensin, a highly stable serine protease from the latex of medicinal plant Ficus benghalensis.

A serine protease was purified to homogeneity from the latex of medicinal plant Ficus benghalensis by a single step procedure using anion exchange chromatography. The enzyme, named benghalensin, has a molecular mass of 47 kDa (MALDI-TOF and SDS-PAGE). The purified protein is a glycoprotein, and the enzymatic activity is solely inhibited by PMSF and chymostatin, indicating that the enzyme belongs to the serine protease class. The isoelectric point of the enzyme is pH 4.4 with optimum pH and temperature of pH 8.0 and 55 degrees C respectively. The extinction coefficient (epsilon(1%)(280)) of the enzyme is 29.25, and the molecular structure consists of 17 tryptophan, 31 tyrosine and 09 cysteine residues. Peptide mass fingerprinting and de novo sequencing of tryptic-digested fragments of the protein did not find any putative conserved domains in BLAST analysis. The enzyme is stable and retains full activity over a broad range of pH and temperature or prolonged storage at 4 degrees C. Simple purification, high yield and stability enable exploration of the protein for structure-function relationship studies as well as other applications.

Biophysical characterization and folding studies of plant protease, wrightin: identification of folding intermediate under different conditions.

Wrightin, a serine protease from Wrightia tinctoria, has been used as model system to examine structure-function and stability. Our studies show high stability of the enzyme with major elements of secondary structure being beta-sheets. Under neutral conditions the enzyme is stable in 8 M urea and high temperature. GuHCl induced unfolding of wrightin at lower pH cannot be satisfactorily fit to a two state model for unfolding. Multiple intermediates were identified during unfolding of wrightin. Further, two intermediates, early and late are identified in the urea induced unfolding pathway at pH 3.0. Spectroscopic properties of intermediate states are analyzed and interpreted.

Effect of alkyl alcohols on partially unfolded state of proteinase K: Differential stability of alpha-helix and beta-sheet rich regions of the enzyme.

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to alpha/beta class of proteins and maintains structural integrity in the range of pH 7.0-3.0. Enzyme acquires partially unfolded conformation (U(P)) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from alpha/beta to beta-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U(P) state occurred in two steps with the formation of beta state at low alcohol concentration followed by stabilization of beta state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.

A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties.

Milin, a potent molluscicide from the latex of Euphorbia milii, holds promise in medicinal biochemistry. Electrophoresis, size exclusion chromatography, mass spectrometry and other biochemical characteristics identify milin as a homodimeric, plant subtilisin-like serine protease, the first of its kind. The subunits of milin are differentially glycosylated affecting dimer association, solubility and proteolytic activity. The dimeric dissociation is SDS-insensitive and strongly temperature dependent but does not appear to be linked by disulfide bridges. N-terminal sequence of acid hydrolyzed peptide fragments shows no homology to known serine protease. Peptide mass fingerprinting and de novo sequencing of the tryptic fragments also did not identify putative domains in the protein. Milin seems to be a novel plant enzyme with subunit association partly similar to human herpes virus serine proteases and partly to penicillin binding proteins. Its behaviour on SDS-PAGE gels and other properties is like "kinetically stable" proteins. Such subunit association and properties might play a critical role in its physiological function and in controlling Schistosomiasis.

Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids.

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.

Modeled structure of trypanothione reductase of Leishmania infantum.

Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.

Physiological changes and molluscicidal effects of crude latex and Milin on Biomphalaria glabrata.

Euphorbian latex is commonly used as molluscicides and the Euphorbia milii latex was reported as most powerful molluscicidal agents. The physiological and lethal effects of the latex components of Euphorbia milii, on the intermediate host Biomphalaria spp., of the human liver parasite Schistosoma mansoni were described in this study. The standard methodologies for testing plant derived molluscicides formulated by World Health Organisation (WHO) were followed with some modifications. The young specimen of fresh water snails showed altered physiological and physical response towards latex components. The working concentration of non-proteinaceous fraction (up to 0.1%) of the latex reduced the active physiological behaviour but was non-lethal to young specimen of snails. However, proteinaceous fractions (0.1mg/l) of the latex were found lethal to snail population, and lethality was enhanced with small amount of the non-proteinaceous fraction (0.01%) of the latex. Milin, a serine protease(up to 0.1mg/l), isolated from the latex of Euphorbia milii significantly reduced the growth and feeding activity but was not lethal to young specimen of snails. With an addition of 0.01% of non-proteinaceous fractions to Milin, lethality result was similar to that of crude latex. Milin is likely to be responsible for alteration of normal physiological functions and lethality of snails, thus it may be used as a molluscicide to control transmission of the endemic disease schistosomiasis.

A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties.

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.