Chymotryptic Digestion Research Articles

Identification of lanthionine and lysinoalanine in heat-treated wheat gliadin and bovine serum albumin using tandem mass spectrometry with higher-energy collisional dissociation.

The present manuscript reports on the identification of various dehydroamino acid-derived bonds and cross-links resulting from thermal treatment (excess water, 240 min, 130 °C) of two model food proteins, bovine serum albumin, and wheat gliadin. S-Carbamidomethylated tryptic and chymotryptic digests of unheated (control) and heated serum albumin and gliadin, respectively, were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS/MS) with higher-energy collisional dissociation (HCD). Heat-induced β-elimination of cystine, serine and threonine, and subsequent Michael addition of cysteine and lysine to dehydroalanine and 3-methyl-dehydroalanine were demonstrated. Lanthionine, lysinoalanine, 3-methyl-lanthionine, and 3-methyl-lysinoalanine were identified. The detection of inter-chain lanthionine in both bovine serum albumin and wheat gliadin suggests the significance of these cross-links for food texture.

A Study of the Relaxed Mechanisms Induced by Novokinin in the Isolated Porcine Coronary Artery Ring Segments.

Novokinin is a vasorelaxing peptide designed according to the structure of ovokinin(2-7) that is released from ovalbumin by chymotryptic digestion. It has attracted much attention due to its variety of pharmacological and biological characteristics. The purpose of this research was to judge the effect and the mechanism of novokinin on porcine coronary arteries. The isometrical tension of coronary arterial rings getted from porcine hearts was measured and its reaction to novokinin (10(-12)-10(-5) mol/L) was observed. It was found that novokinin inhibited the vasocontractivity to KCl and CaCl2 and evidently decreased the isomeric tensile force of both quiescent and prostaglandin F2α (PGF2α) precontracted porcine coronary arterial ring segments. This relaxing effect of novokinin on porcine coronary arteries was apparently cutted down by getting rid of endothelium, and by the addition of methylene blue, N-nitro-L-arginine (L-NNA) or indomethacin, but not by propranolol. Novokini also inhibited the KCl- and CaCl2-induced vasocontraction. The experimental results show that relaxed effect of novokinin on porcine coronary arteries might relate to the function of nitric oxide (NO), cyclic guanosine monophosphate (cGMP) and the synthesis of prostaglandin, but not involve adrenergic β-receptor.

Gel-free mass spectrometry analysis of Drosophila melanogaster heads.

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under-represented group in proteomics datasets. Herein, a bottom-up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter-aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed-phase (RP) LC at high pH in the first dimension and acidic RP-LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 (http://proteomecentral.proteomexchange.org/dataset/PXD001712).

Interaction of β-Lactoglobulin with Small Hydrophobic Ligands - Influence of Covalent AITC Modification on β-LG Tryptic Cleavage

Covalent modification of proteins with bioactive organosulfur compounds is suggested to reduce the smell and increase the stability of the compound. Aside from these effects the covalent modification may also alter the physiochemical properties of the protein. In this study, the whey protein β-lactoglobulin (β-LG) was covalently modified with bioactive organosulfur compound allyl isothiocyanate (AITC), originating from cabbage. Native and AITC modified β-LG were subjected to tryptic and chymotryptic digestion to assess the influence of the covalent modification on peptide formation. AITC was shown to modify at least 13 different amino acid residues, containing thiol- or amino groups, in a concentration dependent manner. Therefore, AITC modification can be controlled to some extent. Besides cysteine thiols, the most accessible amino groups for AITC modification were found at the N-terminal end of the protein (residues L1 and K8) along with lysine residues K91, K77 and K83. Higher amount of AITC addition resulted in a significant blocking of several tryptic cleavage sites of β-LG (for example residue K14) resulting in longer peptides, influencing the concentration of certain bioactive peptides following tryptic cleavage.

Myosin denaturation in “Burnt” Bluefin tuna meat

A quantitative conversion of tuna meat into muscle homogenate made it possible to study myosin denaturation in Bluefin tuna meat. Myosin denaturation was accessed by measuring Ca2+-ATPase activity, salt-solubility with and without Mg-ATP, monomeric myosin content, and amount of subfragment-1 (S-1) and rod produced by chymotryptic digestion. Commercially available tuna used in this study showed a pH around 5.4–5.7. Myosin in the meat lost the salt-solubility measured in the absence of Mg-ATP; however, such myosin showed full salt-solubility when released from actin in the presence of Mg-ATP. Incubation of tuna meat at 30 °C for up to 90 min caused obvious myosin denaturation. However, the tuna meat dialyzed against neutral pH buffer showed practically no myosin denaturation by the same heating. It was suggested that exposure to lowered pH to around 5.5 and increased temperature of 30 °C led myosin denaturation. Myosin denaturation in the “Burnt” Bluefin tuna sample was analyzed. A significant myosin denaturation was observed with the part showing the “Burning” symptom, the inner part of the tuna meat near the spine. Myosin in that part showed almost no Ca2+-ATPase activity, no salt-solubility even with Mg-ATP, no recovery of monomeric myosin, and almost no production of S-1 by chymotryptic digestion. However, myosin denaturation was not detectable for the meat taken from outer parts of the same tuna near the skin with normal appearance. It was demonstrated that “Burning” of tuna meat occurring in the deep part of the body is accompanied by myosin denaturation. The above results suggested that insufficient cooling of the deep part of body would be the reason for “Burning” of tuna meat.

Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry.

The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization - tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT.

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

Characterization of Myosin Subfragment‐1 of Summer and Winter Silver Carp (Hypophthalmichthys molitrix) Muscle

Myosin subfragment-1 (S1) was prepared from myofibrils of summer and winter silver carp by chymotryptic digestion in the presence of ethylenediaminetetraacetic acid (EDTA). Two S1 heavy chain isoforms with different molecular sizes of 91 kDa and 95 kDa were detected in the fast skeletal muscle from summer and winter silver carp, respectively. ATPase inactivation assay indicated that winter S1 was about 20-fold unstable comparing to summer S1. Matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry (MALDI-TOF MS) further confirmed that summer and winter myosin S1 heavy chain isoforms were homologous to myosin high-temperature type and myosin low-temperature type S1 heavy chain, respectively. Moreover, both types of myosin S1 heavy chain isoforms were identified at the intermediate stage. The results indicated that myosin was expressed in a season-specific manner; different types of myosin isomer expressed in different seasons, showing different thermostabilities. Silver carp, Hypophthalmichthys molitrix, is one of the most abundant freshwater fish species in China. The structure thermal stability of myosin rod from silver carp was affected by season change. The gel-forming abilities of surimi prepared in different seasons were different. This study investigated the seasonal differences in structure thermal stability of myosin S1 which is vital for gel formation of myosin. The results of this study will aid understanding of the relationship between the structure and function of myosin, and effective production of surimi from freshwater fish species in different seasons.

The impact of alpha-N-acetylation on structural and functional status of parvalbumin

The effect of alpha-N-acetylation (Nt-acetylation) on the properties of parvalbumin (PA), a Ca2+-binding relaxing factor of skeletal muscles and major food allergen, has been explored. Intact PA contains an N-terminal acetyl group which is absent in the protein expressed in Escherichia coli (rWT), as confirmed by mass spectrometry. Compared to intact pike α-PA, its rWT form exhibits essentially altered profile of thermal unfolding, lowered α-helicity, and decreased affinities to Ca2+ and Mg2+. The structural destabilization of the rWT protein results in lowered resistance to chymotryptic digestion and increased propensity to oligomerization. The rate constants of Ca2+ dissociation from the rWT PA are markedly increased, which indicates that Nt-acetylation modifies functional status of the protein. Rat α-PA demonstrates similar properties for intact and rWT forms. The drastic difference in the effects induced by Nt-acetylation in the PA orthologs can be rationalized by higher disorder level of AB domain in pike PA. Though evolution of PA's genes resulted in the protein sequences with highly divergent properties, Nt-acetylation unifies their functional properties. The structural stability conferred to PA by Nt-acetylation may contribute to its allergenicity. Overall, Nt-acetylation is shown to be a prerequisite for maintenance of structural and functional status of some parvalbumins.

Biochemical properties of muscle proteins between summer and winter silver carp by chymotryptic digestion

Summer(Aug.) and winter(Dec.) silver carp,Hypophthalmichthys molitrix were studied.Because chymotrypsin catalyzes the hydrolysis of peptide bonds adjacent aromatic amino acids,it can be used to detect the difference in the protein internal structure combining with SDS-PAGE technique.The objective of the study is to characterize the structural stability and thermal stability of myosin from summer and winter silver carp by using chymotryptic digestion.The results showed that there was a longer subfragment of myosin S-1 produced by chymotrytic digestion for winter myosin,compared with that of summer one.If myosin was cleaved into HMM and LMM,the winter sample was liable to be cleaved into a shorter fragment as 135 ku HMM,while summer myosin still produced long 165 ku HMM even if digested at high temperatures(30 ℃).These results indicated the instability of winter myosin.Heating summer and winter myofibril at different temperatures,and then measuring the remaining ATPase activity and the amount of S-1 produced by chymotyptic digestion,it was found that the decreasing rate of ATPase activity and S-1 produced was similar to decreasing temperature.This indicated that S-1 produced by chymotryptic digestion only originated from native myosin.Furthermore,as the denaturation temperature for summer myosin was 6 ℃ higher than winter myosin,it indicated that summer myosin was more stable than winter one.This paper introduced chmotrypsin digestion as a very useful method to study the biochemical properties of muscle proteins such as myosin,which is a very important protein for gel formation.The information will be very useful for producing surimi from freshwater fish species in different seasons.

Type III Effector VopC Mediates Invasion for Vibrio Species

Vibrio spp. are associated with infections caused by contaminated food and water. A type III secretion system (T3SS2) is a shared feature of all clinical isolates of V. parahaemolyticus and some V. cholerae strains. Despite its being responsible for enterotoxicity, no molecular mechanism has been determined for the T3SS2-dependent pathogenicity. Here, we show that although Vibrio spp. are typically thought of as extracellular pathogens, the T3SS2 of Vibrio mediates host cell invasion, vacuole formation, and replication of intracellular bacteria. The catalytically active effector VopC is critical for Vibrio T3SS2-mediated invasion. There are other marine bacteria encoding VopC homologs associated with a T3SS; therefore, we predict that these bacteria are also likely to use T3SS-mediated invasion as part of their pathogenesis mechanisms. These findings suggest a new molecular paradigm for Vibrio pathogenicity and modify our view of the roles of T3SS effectors that are translocated during infection.

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.

Thermal denaturation profiles of catfish and tilapia myofibrils as affected by pH for heating

Thermal denaturation profiles of catfish myosin when heated as myofibrils (Mf) were compared with those of tilapia. The Ca2+-ATPase inactivation rate of catfish myofibrils was the same as that of tilapia myofibrils. The conclusion was the same with isolated myosin. Catfish Mf was clearly distinguished from tilapia Mf in terms of subfragment-1 (S-1) and rod denaturation. Quick denaturation of rod relative to S-1 was characteristic of catfish Mf, while slower denaturation of rod relative to S-1 was the pattern for tilapia Mf. These patterns were greatly affected by the pH for heating. Rod denaturation was accelerated with increasing pH for heating and oppositely suppressed by lowering the pH, for both Mf. Tilapia Mf showed a S-1 and rod denaturation pattern similar to that for catfish Mf, but at 1 pH unit higher; for example, the pattern of catfish Mf at pH 7.5 was similar to that for tilapia Mf at pH 8.5. Less rigid filament structure of catfish Mf than tilapia Mf was demonstrated by studying chymotryptic digestion at various pH values. Accordingly, the difference in the S-1/rod denaturation patterns between the two fish species can be explained by the different rigidity of their myosin filaments.

Calcium-sensitive Activity and Conformation of Caenorhabditis elegans Gelsolin-like Protein 1 Are Altered by Mutations in the First Gelsolin-like Domain

The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1-G4) and still exhibits Ca(2+)-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca(2+) activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca(2+) sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca(2+) sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca(2+)-free and Ca(2+)-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.

Structural Stability of Myosin Rod from Silver Carp as Affected by Season

Chymotrypsin digestion and Circular dichroism (CD) spectroscopy were used to study the structural stability of myosin rods prepared from silver carp in summer and winter. Differences in the thermal stability were clearly demonstrated by the chymotryptic digestion patterns and CD data obtained at pH 7.5 in 0.5 M KCl for myosin rods in both seasons, indicating the structural properties differed seasonally. In winter, the myosin rods were more susceptible to be cleaved into short (40 kDa) subfragments when digested at high temperature (30 °C). The major peaks in unfolding profiles showed that transition temperatures for the rods were 35 °C in winter, and 36 and 41 °C in summer. Rod subfragments (S-2 and LMM) were isolated and further analyzed by CD. In both winter and summer, the LMM fragments showed an unfolding transition at around 35.5 °C, but the S-2 fragments showed large seasonal difference; in winter, they showed a major unfolding peak around 35 °C and a minor peak at 40.5 °C, while in summer, they unfolded mostly at 44.5 °C, which was 8 °C higher than that for the LMM fragments. Short (40 kDa) S-2 subfragments isolated from winter rods gave a single unfolding transition at 41 °C. These results suggest that differences in the thermal stability of myosin rods between winter and summer were due to differences in the stability of S-2 region. The aim of this study was to detect the differences in the structure thermal stability of myosin rods between summer and winter, which is a very important portion for gel formation. The information will be very useful for producing surimi from freshwater fish species in different seasons.

The modified-bead stretched sample method: development and application to MALDI-MS imaging of protein localization in the spinal cord.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used to create spatial distribution maps from lipids, peptides, and proteins in a variety of biological tissues. MALDI-MSI often involves trade-offs between the extent of analyte extraction and desired spatial resolution, compromises that can adversely affect detectability. For example, increasing the extraction time can lead to unwanted analyte spatial redistribution. With the stretched sample method (SSM), the extraction period can be extended, resulting in reduced analyte redistribution while suppressing detection of cationic salt adducts. The SSM involves thaw-mounting a thin tissue section onto a substrate of small glass beads embedded in Parafilm M and then stretching the membrane to fragment the tissue into thousands of bead-sized pieces. Here, we applied the SSM method to MALDI-MSI using rat spinal cord as a model. We used surface-modified beads coated with trypsin or chymotrypsin in order to facilitate controlled digestion and detection of proteins. The enzymatic reactions were maintained by repeatedly condensing water on the stretched sample surface. As a result, new peptides formed by tryptic or chymotryptic protein digestion were detected and identified using a combination of MALDI-MSI and offline liquid chromatography tandem mass spectrometric analysis. Localization of these peptides indicated the distribution of their proteins of origin, including myelin basic protein, actin beta, and tubulin alpha chain. Additionally, we used uncoated beads to create distribution maps of many endogenous lipids and small peptides. The extension of the SSM using modified beads resulted in the creation of mosaic bead surfaces where adjacent beads were coated with different enzymes or other reactive chemicals, permitting investigation of the distributions of a wider range of analytes in biological samples within a single experiment.

9.3 KDa components of the injected venom of Conus purpurascens define a new five‐disulfide conotoxin framework

The 83-residue conopeptide (p21a) and its corresponding nonhydroxylated analog were isolated from the injected venom of Conus purpurascens. The complete conopeptide sequences were derived from Edman degradation sequencing of fragments from tryptic, chymotryptic and cyanogen bromide digestions, p21a has a unique, 10-cystine/5-disulfide 7-loop framework with extended 10-residue N-terminus and a 5-residue C-terminus tails, respectively. p21a has a 48% sequence homology with a recently described 13-cystine conopeptide, con-ikot-ikot, isolated from the dissected venom of the fish-hunting snail Conus striatus. However, unlike con-ikot-ikot, p21a does not form a dimer of dimers. MALDI-TOF mass spectrometry suggests that p21a may form a noncovalent dimer. p21a is an unusually large conotoxin and in so far is the largest isolated from injected venom. p21a provides evidence that the Conus venom arsenal includes larger molecules that are directly injected into the prey. Therefore, cone snails can utilize toxins that are comparable in size to the ones commonly found in other venomous animals.

Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA

BackgroundHuman cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain.ResultsWe delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA.ConclusionsThe results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.

Extensive Phosphorylation Flanking the C-Terminal Functional Domains of the Measles Virus Nucleoprotein

The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC-ESI-ion trap MS(1), MS(2) (neutral loss), MS(3) (phosphosite). Both recombinant NP (rNP) and vNP were α-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications.

Discovery and characterization of a distinctive exo-1,3/1,4-{beta}-glucanase from the marine bacterium Pseudoalteromonas sp. strain BB1.

Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-β-glucanase (ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., was found to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significant amounts of the storage polysaccharide laminarin (1,3-β-D-glucan with some 1,6-β branching). Four chromatographic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences for primer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50 bp downstream from a putative lichenase (endo-1,3-1,4-β-glucanase) gene, suggesting possible cotranscription of these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidases with high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-β-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins is an extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does not correlate with any known functional domain, but ExoP was not active against laminarin when this region was removed. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzyme was found to possess similar levels of exoglucanase activity against both 1,4-β linkages and 1,3-β linkages, and so ExoP is designated an exo-1,3/1,4-β-exoglucanase, the first such bacterial enzyme to be characterized. This broader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmic laminarin.