Aggregating Ability of Seed Storage Proteins from Cereals Differing in Gluten Quality

The effects of medium pH, ionic strength, and composition on the formation of macrocomplexes of seed storage proteins from wheat, rye, and barley have been studied. Various noncovalent interactions (electrostatic and hydrophobic interactions and hydrogen bonds) are involved in protein aggregation. Their combined action depends significantly on the biochemical nature of the storage proteins and on the medium.     

A mechanistic analysis of the increase in the thermal stability of proteins in aqueous carboxylic acid salt solutions

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, deltaCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.     

Inter-residue interactions in protein folding and stability

During the process of protein folding, the amino acid residues along the polypeptide chain interact with each other in a cooperative manner to form the stable native structure. The knowledge about inter-residue interactions in protein structures is very helpful to understand the mechanism of protein folding and stability. In this review, we introduce the classification of inter-residue interactions into short, medium and long range based on a simple geometric approach. The features of these interactions in different structural classes of globular and membrane proteins, and in various folds have been delineated. The development of contact potentials and the application of inter-residue contacts for predicting the structural class and secondary structures of globular proteins, solvent accessibility, fold recognition and ab initio tertiary structure prediction have been evaluated. Further, the relationship between inter-residue contacts and protein-folding rates has been highlighted. Moreover, the importance of inter-residue interactions in protein-folding kinetics and for understanding the stability of proteins has been discussed. In essence, the information gained from the studies on inter-residue interactions provides valuable insights for understanding protein folding and de novo protein design.     

Metabolic changes in ratskin stored in buffer medium at minus 3 degrees C

The incorporation of amino acids into the proteins of rat skin after storage in a buffer medium greatly depends on the animal's age, and on storage time.At —3 °C protein synthesizing activity is primarily impaired, accompanied by cell membrane damage, while DNA metabolism appears remarkably immune to preservation injury. The major injury to protein synthesis occurs during the first days and even hours of storage, followed by a longer period of storage where protein metabolism is reduced to a low level of activity. Addition of a mixture of amino acids to the medium protects and stimulates the subsequently tested amino acid incorporation into the proteins of rat skin.     

Sorting of proteins to storage vacuoles: how many mechanisms?

Vacuoles receive their proteins through the secretory pathway, this requires protein sorting signals and molecular machineries that, until recently, have been believed to be markedly distinct for lytic and storage vacuoles. However, new biochemical, morphological and genetic data indicate that the only known class of vacuolar sorting receptors, believed to be specific for lytic vacuoles, might also be involved in the sorting of certain storage proteins. Furthermore, storage vacuoles can have a complex multimembrane structure that is difficult to explain based on a single trafficking mechanism. A new array of possible molecular interactions is thus emerging that no longer supports a clear-cut distinction between the two types of vacuoles based on sorting signals and putative receptors.     

Influence of Medium and Long Range Interactions in Different Structural Classes of Globular Proteins

An analysis of the dependence known three dimensional structure ofglobular proteins on their residue contacts and their interactions providesmuch information about their folding and stability. In this work, we analysethe residue-residue contacts and the role of medium and long rangeinteractions in globular proteins belonging to different structural classes.The results show that while medium range interactions predominate in allalpha class proteins, long range interactions predominate in all beta class.The residues Pro and Gly are found to have lowest medium range contacts,probably due to their helix breaking tendency. The hydrophobic residues Ile,Val and Tyr have higher long range contacts, and hence may serve as goodnucleation centres. Further, the role of charged residues and disulfidebridges in these interactions are also discussed.     

Computation of non-covalent interactions in TNF proteins and interleukins

The roles played by the non-covalent interactions have been investigated for a set of six TNF proteins and nine Interleukins. The stabilizing residues have been identified by a consensus approach using the concepts of available surface area, medium and long-range interactions and conservation of amino acid residues. The cation-pi interactions have been computed based on a geometric approach such as distance and energy criteria. We identified an average of 1 energetically significant cation-pi interactions in every 94 residues in TNF proteins and 1 in every 62 residues in Interleukins. In TNF proteins, the cationic groups Lys preferred to be in helix while Arg preferred to be in strand regions while in Interleukins the Arg residues preferred to be in helix and Lys preferred to be in strand regions. From the available surface area calculations, we found that, almost all the cation and pi residues in TNF proteins and Interleukins were either in buried or partially buried regions and none of them in the exposed regions. Medium and long-range interactions were predominant in both TNF proteins and Interleukins. It was observed that the percentage of stabilizing centers were more in TNF proteins as compared to the Interleukins, while the percentage of conserved residues were more in Interleukins than in TNF proteins. In the stabilizing residues Lys was observed to be a stabilizing residue in both TNF proteins and Interleukins. Among the aromatic group, Phe was seen to be a stabilizing residue in both TNF and Interleukins. We suggest that this study on the computation of cation-pi interactions in TNF proteins and Interleukins would be very helpful in further understanding the structure, stability and functional similarity of these proteins.     

Dye-Ligand Affinity Chromatography: The Interaction of Cibacron Blue F3GA® with Proteins and Enzyme

Abstract

The dye Cibacron Blue F3GA has a high affinity for many proteins and enzymes. It has therefore been attached to various solid supports such as Sephadex, Sepharose, polyacrylamide, and the like. In the immobilized form the dye has rapidly been exploited as an affinity chromatographic medium to separate and purify a variety of proteins including dehydrogenases, kinases, serum albumin, interferons, several plasma proteins, and a host of other proteins. Such a diversity shown by the blue dye in binding several unrelated classes of proteins has generated considerable work in terms of studies of the chromophore itself and also the immobilized ligand. As a prelude to realizing the full potential of the immobilized Cibacron Blue F3GA, an understanding of the basic interactions of the dye with its surroundings must be gained. It has been recognized that the dye is capable of hydrophobic and/or electrostatic interactions at the instance of the ambient conditions. The study of interactions of the dye with salts, solvents, and other small molecules indicates the nature of the interactions of the dye with different kinds of groups at the interacting sites of proteins. The review will cover such interactions of the dye with the proteins, the interactions of the proteins with the immobilized ligand, and the media used to elute the bound protein in several cases, and thus consolidate the available information on such studies into a cogent and comprehensive explanation.     

Accumulation pattern and identification of seed storage proteins in zygotic embryos of Pinus strobus and in somatic embryos from different maturation treatments

Somatic embryogenesis (SE) of Pinus strobus L. has been greatly improved over the last few years with respect to both the initiation frequencies from a number of seed families and production of mature somatic embryos that readily convert to plants. However, there are no data on biochemical characterization of somatic embryos in relation to zygotic embryos of eastern white pine and on the optimal duration of the maturation stage. It is believed that somatic embryos closely resembling zygotic embryos not only morphologically but biochemically would display more vigorous growth. Hence, in this study the accumulation pattern of the most abundant seed storage proteins in zygotic and somatic embryos were characterized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and identified by amino acid sequencing and tandem mass spectrometry (MS/MS). This showed that somatic embryos accumulated storage proteins in a similar manner to zygotic embryos and that the most abundant were the buffer‐insoluble 11S‐ globulins MW 59.6 kDa, which dissociated under reduced conditions to 38.2–40.0 and 22.5–23.5 kDa range polypeptides, and buffer‐soluble 7S vicilin‐like proteins MW 46.0–49.0 kDa, which did not separate under reduced conditions. Other relatively abundant soluble proteins were in the ranges of 25–27 and 27–29 kDa. The only group of proteins that showed different migration profiles in the presence of β‐mercaptoethanol (ME) were the low molecular mass proteins of 14.6–16.5 kDa. Somatic embryos that matured for 9 weeks on medium with 6% sucrose accumulated more storage proteins than those matured on medium with 3% sucrose and the extension of the maturation period to 12 weeks resulted in significant reduction of the storage proteins on both media. As expected, somatic embryos matured on medium with 6% sucrose had lower water potential (Ψ) than those from medium with 3% sucrose. Nonetheless, the somatic embryos matured under the best of tested conditions (6% sucrose for 9 weeks) had slightly higher water content; 1.35 ± 0.28 g H₂O g^?1 DM (mean ± sd ) than the mature non‐dried zygotic embryos; (1.16 ± 0.09 g H₂O g^?1 DM), and accumulated less storage proteins, whose amounts were either similar to (7S‐vicilins) or below (11S‐globulins) those found in the immature zygotic embryos collected 2 weeks prior to the usual cone collection. The implications of these results for further research and development of viable artificial seed is discussed.     

Thermodynamic analysis of the impact of the surfactant-protein interactions on the molecular parameters and surface behavior of food proteins

This paper reports on the thermodynamics of the interactions between surfactants (anionic, CITREM, SSL; nonionic, PGE; zwitterionic, phospholipids) and food proteins (sodium caseinate, legumin) depending on the chemical structure and molecular state (individual molecules, micelles) of the surfactants and the molecular parameters (conformation, molar mass, charge) of the proteins under changes of pH in the range from 7.2 to 5.0 and temperature from 293 to 323 K. The marked effect of the protein-surfactant interactions on the molecular parameters (the weight-average molar mass, the gyration and hydrodynamic radii) and the thermodynamic affinity of the proteins for an aqueous medium were determined by a combination of static and dynamic laser light scattering. Thermodynamically justified schematic sketches of the molecular mechanisms of the complex formation between like-charged proteins and surfactants have been proposed. In response to the complex formation between the proteins and the surfactants, the more stable and fine foams have been detected generally.     

The role of mRNA and protein sorting in seed storage protein synthesis, transport, and deposition.

Rice synthesizes and accumulates high levels of 2 distinct classes of seed storage proteins and sorts them to separate intracellular compartments, making it an ideal model system for studying the mechanisms of storage protein synthesis, transport, and deposition. In rice, RNA localization dictates the initial site of storage protein synthesis on specific subdomains of the cortical endoplasmic reticulum (ER), and there is a direct relation between the RNA localization site and the final destination of the encoded protein within the endomembrane system. Current data support the existence of 3 parallel RNA localization pathways leading from the nucleus to the actively synthesizing cortical ER. Additional pathways may exist for the synthesis of cytoplasmic and nuclear-encoded proteins targeted to organelles, the latter located in a stratified arrangement in developing endosperm cells. The study of rice mutants, which accumulate unprocessed glutelin precursors, indicates that these multiple pathways prevent nonproductive interactions between different classes of storage proteins that would otherwise disrupt protein sorting. Indeed, it appears that the prevention of disruptive interactions between different classes of storage proteins plays a key role in their biosynthesis in rice. In addition to highlighting the unique features of the plant endomembrane system and describing the relation between RNA and protein localization, this minireview will attempt to address a number of questions raised by recent studies on these processes.     

Protein patterns associated with <Emphasis Type="Italic">Pisum sativum</Emphasis> somatic embryogenesis

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.     

Role of arginine in protein refolding, solubilization, and purification

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.     

Peripheral membrane proteins mediate binding of vacuolar storage proteins to membranes of the secretory pathway of developing pea cotyledons

In developing pea cotyledons, storage proteins are sorted viadense vesicles into the protein storage vacuole. Formation ofthese unique transport vesicles is characterized by aggregationof their cargo proteins. Protein sorting into dense vesiclesis pH dependent. In order to gain insight into the molecularbasis of storage protein sorting, a membrane binding assay wasdeveloped which allows for a detailed biochemical analysis ofbinding events. Employing this assay it was possible to showthat storage proteins bind in a pH-dependent manner to the membranesof the secretory pathway with a pH optimum in the range of thelumenal pH of the Golgi cisternae. Through reconstitution experiments,it was possible to demonstrate further that this recruitmentoccurs via the interaction of peripheral rather than intrinsicmembrane proteins. Results of co-immunoprecipitation experimentspoint to interactions between different storage proteins inthe secretory system. These results are discussed in terms ofthe aggregation-mediated sorting of storage proteins into maturingdense vesicles. Key words: Dense vesicles, Golgi apparatus, legumin, pea, receptor, sorting Received 22 January 2008; Revised 22 January 2008 Accepted 23 January 2008     

Protein SUMOylation in spine structure and function

The active regulation of spine structure and function is of fundamental importance for information storage in the brain. Many proteins involved in spine development and activity-dependent remodelling are potential or validated substrates for modification by the Small Ubiquitin-like Modifier (SUMO). The functional consequences of neuronal protein SUMOylation appear diverse and, in many cases, have not yet been determined. However, for several proteins SUMOylation has been shown to be a key regulator, which has a profound impact on spine dynamics and protein trafficking and function. Here we provide an overview of neuronal SUMOylation and discuss how greater understanding of this relatively recently discovered posttranslational modification will provide insight into the complexity of protein interactions that control synaptic activity and dysfunction.     

Influence of medium and long range interactions in protein folding.

Protein structures are stabilized by both local and long range interactions. In this work, we analyze the residue-residue contacts and the role of medium- and long-range interactions in globular proteins belonging to different structural classes. The results show that while medium range interactions predominate in all-alpha class proteins, long-range interactions predominate in all-beta class. Based on this, we analyze the performance of several structure prediction methods in different structural classes of globular proteins and found that all the methods predict the secondary structures of all-alpha proteins more accurately than other classes. Also, we observed that the residues occurring in the range of 21-30 residues apart contributes more towards long-range contacts and about 85% of residues are involved in long-range contacts. Further, the preference of residue pairs to the folding and stability of globular proteins is discussed.     

Efficient recovery of recombinant proteins from cereal endosperm is affected by interaction with endogenous storage proteins

Cereal seeds are versatile platforms for the production of recombinant proteins because they provide a stable environment for protein accumulation. Endogenous seed storage proteins, however, include several prolamin-type polypeptides that aggregate and crosslink via intermolecular disulfide bridges, which could potentially interact with multimeric recombinant proteins such as antibodies, which assemble in the same manner. We investigated this possibility by sequentially extracting a human antibody expressed in maize endosperm, followed by precipitation in vitro with zein. We provide evidence that a significant proportion of the antibody pool interacts with zein and therefore cannot be extracted using non-reducing buffers. Immunolocalization experiments demonstrated that antibodies targeted for secretion were instead retained within zein bodies because of such covalent interactions. Our findings suggest that the production of soluble recombinant antibodies in maize could be enhanced by eliminating or minimizing interactions with endogenous storage proteins.     

A general system for studying protein-protein interactions in Gram-negative bacteria

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.     

Control of membrane structure and organization through chemical recognition

Cell membranes consist of a fluidic medium of lipids and proteins that organize into specific submicron scale structures for signaling and molecular trafficking processes. These organized molecular assemblies form as a result of the structure and chemistry of the membrane components as well as the interactions of those components with analytes from solution. Although considerable research has focused on the structure and chemistry of membrane components and their ability to form organized assemblies, less attention has been paid toward the influence that chemical recognition has upon membrane reorganization. This review focuses on the recognition and binding of metal ions, small molecules, polyelectrolytes, and proteins on model membrane systems to assess the effects of long- and short-range interactions upon the molecular organization of the membrane. Chemical recognition can induce dramatic changes on the membrane's phase transition temperature and the clustering or dispersion of membrane components.