Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm⁻¹) than a viral protein-capsid coat and sustains deformations of one-third of the virion''s diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.     

Enzymatic Iodination of Sindbis Virus Proteins

Sindbis virus was iodinated by using the enzyme lactoperoxidase, an iodination technique which labels only surface proteins. By this technique, the two viral glycoproteins are labeled, and the internal viral protein is not. The two glycoproteins are iodinated to strikingly different extents. This difference in susceptibility to iodination apparently is due to the position or conformation of the glycoproteins in the envelope spikes of the virion and not to differing contents of tyrosine, the amino acid substrate of lactoperoxidase. Both viral glycoproteins are iodinated by lactoperoxidase on the surface of Sindbis-infected chicken cells. Here, as in the virion, the glycoproteins are iodinated unequally, with the smaller glycoprotein again being preferentially iodinated. Another virus-specific protein found in large amounts in infected cells, and from which the preferentially iodinated virion glycoprotein is produced by a proteolytic cleavage, is not iodinated by lactoperoxidase. Thus it appears that the viral glycoproteins are present on the cell surface and that the precursor protein is not.     

Enzymatic Modification of Proteins in Foodstuffs

Peptic hydrolyzate of soy protein was submitted to the plastein reaction with α-chymotrypsin under the following condition: substrate concentration, 20%; enzyme-substrate ratio by weight, 1/100; reaction pH, 7.0; and reaction temperature, 37°C. The plastein yield resulting from the plastein reaction for 24 hr was found to depend on the degree of hydrolysis of the substrate (per cent ratio between nitrogen amount in 10% trichloroacetic acid soluble nitrogen and that in whole hydrolyzate); the optimum degree of hydrolysis for the highest plastein yield seemed to lie around 80%. A turbidity appeared in the process of the plastein reaction, whose intensity was correlative to the plastein yield. The peptic hydrolyzate of soy protein per se had bitterness and its magnitude decreased with increasing plastein yield.

As a result of the plastein reaction applied for 24 hr to the hydrolyzate whose degree of hydrolysis was 80%, the average molecular weight estimated by the change in amino nitrogen content increased by approximately three times. The molecular weight distribution pattern obtained by gel filtration supported the above result. The total amount of amino acids liberated from the plastein reaction product by its treatment with either leucine aminopeptidase or carboxypeptidase A was significantly less than that liberated from the original hydrolyzate by its similar treatment. This result also supports the formation of higher-molecular protein-like substances by the plastein reaction. Deuteration study followed by IR spectrometry showed the occurrence of peptide bond formation, i.e. decrease in ionized carboxyl group at 1575 cm^?1 and increase in deuterated amide at 1450 cm^?1, even at the earlier stages of the plastein reaction.     

Mechanical Properties of Thermo-moulded Biofilms in Relation to Proteins/Starch Interactions

The effects of hydrophilic and hydrophobic characteristics of proteins on the interactions with corn starch were investigated in this study. The model system included corn starch and proteins, i.e. zein, gliadin, gluten, soy protein and rapeseed protein. The blend films were prepared by thermo-moulding in gentle conditions at 70 °C in order to avoid starch gelatinization, with respect to water content, and avoid protein denaturation. The effects of different kinds of proteins on structure and mechanical behaviour of blend biomaterials were characterised by scanning electron microscopy (SEM) and tensile test, respectively. The effects of different kinds of proteins on intermolecular interactions between proteins and starch were investigated by dynamical mechanical thermal analysis. Based on the solubility measurement results, almost all protein films showed the similar solubility to the natural protein powders, resulting from the weak influence of mild thermo-moulding treatment on protein inner structure. Different morphologies were observed for different proteins and corresponding blends, which are relatively loose protein architecture that appeared for hydrophobic protein and blend films, and uniform and densely packed architecture for hydrophilic ones. Moreover, different mechanical behaviours were obtained for different proteins and corresponding blends. No significantly increased strength for hydrophilic protein blends with starch added can be explained that there is weak intermolecular interaction between both components based on SEM observation. However, the addition of corn starch granules in hydrophobic protein networks was assumed that starch destroyed or weakened the protein network, resulting in the decrease of mechanical strength.     

Enzymatic Detachment of Endplate Acetylcholinesterase from Muscle

AT the vertebrate neuromuscular junction acetylcholinesterase catalyses the hydrolysis of the transmitter, acetylcholine, which is released from presynaptic nerve terminals^1,2. The enzyme is present in high concentration at the endplate, where it can be located by histochemical³ and autoradiographic⁴ methods. Electron microscopic studies of the endplate region show most of the histochemical reaction product to be in the synaptic cleft or associated with the nerve and muscle membranes^5–9. We report here that enzymatic treatment of intact muscle causes the detachment of active endplate acetylcholinesterase from the muscle into the bathing fluid.     

The Mechanical Properties of Drosophila Jump Muscle Expressing Wild-Type and Embryonic Myosin Isoforms

Transgenic Drosophila are highly useful for structure-function studies of muscle proteins. However, our ability to mechanically analyze transgenically expressed mutant proteins in Drosophila muscles has been limited to the skinned indirect flight muscle preparation. We have developed a new muscle preparation using the Drosophila tergal depressor of the trochanter (TDT or jump) muscle that increases our experimental repertoire to include maximum shortening velocity (V_slack), force-velocity curves and steady-state power generation; experiments not possible using indirect flight muscle fibers. When transgenically expressing its wild-type myosin isoform (Tr-WT) the TDT is equivalent to a very fast vertebrate muscle. TDT has a V_slack equal to 6.1 ± 0.3 ML/s at 15°C, a steep tension-pCa curve, isometric tension of 37 ± 3 mN/mm², and maximum power production at 26% of isometric tension. Transgenically expressing an embryonic myosin isoform in the TDT muscle increased isometric tension 1.4-fold, but decreased V_slack 50% resulting in no significant difference in maximum power production compared to Tr-WT. Drosophila expressing embryonic myosin jumped <50% as far as Tr-WT that, along with comparisons to frog jump muscle studies, suggests fast muscle shortening velocity is relatively more important than high tension generation for Drosophila jumping.     

Enzymatic Production of Novel European Eel Proteins Hydrolysates: Biological Activities,Techno-Functional Properties and Maltodextrin-Hydrolysates Efficient Electrosprayability

International Journal of Peptide Research and Therapeutics - European eel proteins hydrolysates (EPHs) were produced from A. anguilla muscle protein and protein isolate using Purafect®, goby...     

Enzymatic Phosphorylation of Soybean Proteins by Protein Kinase

Soybean proteins were subjected to phosphorylation with cyclic adenosine monophosphate- dependent protein kinase (A-kinase). As a result, acidic subunits of the 11S fraction were found to be phosphorylated by A-kinase. To estimate the effect of the phosphorylation, 11S acidic subunits were isolated and subjected to A-kinase phosphorylation. The optimal enzyme amount and Mg^{2 +} concentration for the phosphorylation of 11S acidic subunits were determined to be 1.5U/ml and 1.6 mm, respectively. The rate of phosphorylation was 2mol/mol acidic subunits (MW 38,000) under the above conditions. The protein structures of 11S acidic subunits, as determined from UV and CD spectra, were slightly affected by the enzymatic phosphorylation.     

Mechanical Properties of the Sarcolemma and Myoplasm in Frog Muscle as a Function of Sarcomere Length

The elastimeter method was applied to the single muscle fiber of the frog semitendinosus to obtain the elastic moduli of the sarcolemma and myoplasm, as well as their relative contributions to resting fiber tension at different extensions. A bleb which was sucked into a flat-mouthed pipette at the fiber surface separated into an external sarcolemmal membrane and a thick inner myoplasmic region. Measurements showed that the sarcolemma does not contribute to intact fiber tension at sarcomere lengths below 3 µ. It was estimated that the sarcolemma contributed on the order of 10% to intact fiber tension at sarcomere lengths between 3 and 3.75 µ, and more so with further extension. Between these sarcomere lengths, the sarcolemma can be linearly extended and has a longitudinal elastic modulus of 5 x 10⁶ dyne/cm² (assuming a thickness of 0.1 µ). Resistance to deformation of the inner bleb region is due to myoplasmic elasticity. The myoplasmic elastic modulus was estimated by use of a model and was used to predict a fiber length-tension curve which agreed approximately with observations.     

Mechanical Resistance in Unstructured Proteins

Single-molecule pulling experiments on unstructured proteins linked to neurodegenerative diseases have measured rupture forces comparable to those for stable folded proteins. To investigate the structural mechanisms of this unexpected force resistance, we perform pulling simulations of the amyloid β-peptide (Aβ) and α-synuclein (αS), starting from simulated conformational ensembles for the free monomers. For both proteins, the simulations yield a set of rupture events that agree well with the experimental data. By analyzing the conformations occurring shortly before rupture in each event, we find that the mechanically resistant structures share a common architecture, with similarities to the folds adopted by Aβ and αS in amyloid fibrils. The disease-linked Arctic mutation of Aβ is found to increase the occurrence of highly force-resistant structures. Our study suggests that the high rupture forces observed in Aβ and αS pulling experiments are caused by structures that might have a key role in amyloid formation.     

Mechanical Resistance in Unstructured Proteins

Single-molecule pulling experiments on unstructured proteins linked to neurodegenerative diseases have measured rupture forces comparable to those for stable folded proteins. To investigate the structural mechanisms of this unexpected force resistance, we perform pulling simulations of the amyloid β-peptide (Aβ) and α-synuclein (αS), starting from simulated conformational ensembles for the free monomers. For both proteins, the simulations yield a set of rupture events that agree well with the experimental data. By analyzing the conformations occurring shortly before rupture in each event, we find that the mechanically resistant structures share a common architecture, with similarities to the folds adopted by Aβ and αS in amyloid fibrils. The disease-linked Arctic mutation of Aβ is found to increase the occurrence of highly force-resistant structures. Our study suggests that the high rupture forces observed in Aβ and αS pulling experiments are caused by structures that might have a key role in amyloid formation.     

Postmortem Changes in Regulatory Proteins of Rabbit Muscle

The procedures for complete extraction of regulatory proteins, particularly troponin and tropomyosin from the myofibrils of fresh and stored muscles were developed and the effect of postmortem storage of muscle on the properties of the regulatory proteins was studied. The ATPase enhancing ability and the sedimentation behavior of α-actinin from post-rigor muscle did not differ from those of α-actinin from pre-rigor muscle. The amounts of both troponin and tropomyosin decreased during the storage of muscle. Troponin decreased more rapidly than tropomyosin. These results were interpreted to support our hypothesis that the structural alteration of myofibril, which is mainly due to the change in the troponin-tropomyosin complex of thin filaments, proceeds during the postmortem storage of muscle.     

Studies on the Metabolic Activity of Muscle Proteins

The activities of glutamic oxaloacetic transaminase and Ca⁺⁺ ion-activated ATPase of muscle in the adult rats fed a protein-free diet for 8, 16 and 24 days were measured in order to clarify their metabolic responses with respect to reserve proteins. It was found that these enzyme activities, or presumably their enzyme proteins, decreased at the stage as early as the 8th day of protein depletion following the same pattern as seen in reserve proteins. Their responses, particularly those in unit activity, were somewhat different from each other. The metabolic significance of those responses was discussed in relation to protein nutrition.     

Studies on the Metabolic Activity of Muscle Proteins

The time-course of changes in total amount of proteins of sarcoplasmic, myofibrillar and stromal fractions in muscle of the rats fed a protein-free diet for 8, 16, 24 and 32 days, together with the referential data of those changes in the rats fed a protein-free diet up to time of death and a 60% casein diet for 12 days was determined respectively. The results were as follows: (1) The sarcoplasmic and the myofibrillar fractions decreased much more than the stromal fraction in the earlier stages of protein depletion following the same pattern as seen in reserve proteins. (2) The sarcoplasmic fraction decreased slightly more than the myofibrillar fraction as early as 8 days of the depletion, but the relative proportion between these two fractions was thereafter almost the same as that of the standard diet group. (3) In rats fed a 60% casein diet, the sarcoplasmic fraction increased markedly than the others.     

Some Properties of Caldesmon and Calponin and the Participation of These Proteins in Regulation of Smooth Muscle Contraction and Cytoskeleton Formation

The interaction of caldesmon with different Ca²⁺-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of caldesmon. The arrangement of caldesmon and Ca²⁺-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B" of caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of caldesmon. The complex of calmodulin and caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with caldesmon. Phosphorylation of caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actinbinding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and a-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton.     

Enzymatic Radioiodination of the Envelope Proteins of Avian Myeloblastosis Virus

Purified avian myeloblastosis virus was iodinated by the lactoperoxidase method. Disruption of the labeled virions and chromatography of the viral proteins showed that radioiodine was associated only with the glycoproteins of the viral envelope. Reaction of the radiolabeled virus with antiviral antibody followed by mild detergent treatment and subsequent recovery of immune complexes showed this technique to be useful for the isolation of viral envelope antigens.     

Enzymatic Hydrolysis of Proteins from Crustaceans of the Barents Sea

Enzymatic preparations from king crab hepatopancreas were shown to be capable, in principle, of producing protein hydrolysates. Hydrolysis of protein-containing waste of deep-water prawn and king crab occurs most successfully at pH 8.0–8.5 and 50–55°C for 5–6 h in the presence of 6 g enzyme per kg substrate. The total chemical composition of the hydrolysates, the molecular weight distributions of proteins and polypeptides, and the contents of free amino acids were studied in dry hydrolysates.