Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.     

Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution.

Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer-aided densitometers have become available as well as recipes for sensitive and background-free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time-dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.     

Carbon monoxide binding by de novo heme proteins derived from designed combinatorial libraries.

Carbon monoxide binding was studied in a collection of de novo heme proteins derived from combinatorial libraries of sequences designed to fold into 4-helix bundles. The design of the de novo sequences was based on the previously reported "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains are varied extensively.(1) The combinatorial mixture of amino acids included histidine and methionine, which ligate heme iron in natural proteins. However, no attempt was made to explicitly design a heme binding site. Nonetheless, as reported previously, approximately half of the binary code proteins bind heme.(2) This collection of novel heme proteins provides a unique opportunity for an unbiased assessment of the functional potentialities of heme proteins that have not been prejudiced either by explicit design or by evolutionary selection. To assess the capabilities of the de novo heme proteins to bind diatomic ligands, we measured the affinity for CO, the kinetics of CO binding and release, and the resonance Raman spectra of the CO complexes for eight de novo heme proteins from two combinatorial libraries. The CO binding affinities for all eight proteins were similar to that of myoglobin, with dissociation constants (K(d)) in the low nanomolar range. The CO association kinetics (k(on)) revealed that the heme environment in all eight of the de novo proteins is partially buried, and the resonance Raman studies indicated that the local environment around the bound CO is devoid of hydrogen-bonding groups. Overall, the CO binding properties of the de novo heme proteins span a narrow range of values near the center of the range observed for diverse families of natural heme proteins. The measured properties of the de novo heme proteins can be considered as a "default" range for CO binding in alpha-helical proteins that have neither been designed to bind heme or CO, nor subjected to genetic selections for heme or CO binding.     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

Properties of (Mg2 + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: influence of Mg2+, Ca2+, ATP, and protein activator.

Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated to different extents by a Ca2+-dependent protein activator isolated from hemolysates. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2-200 microM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed "high" and "low" affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strength, or membranes prepared by the EDTA (1-10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins.

The effects of internal motions on residual dipolar NMR couplings of proteins partially aligned in a liquid-crystalline environment are analyzed using a 10 ns molecular dynamics (MD) computer simulation of ubiquitin. For a set of alignment tensors with different orientations and rhombicities, MD-averaged dipolar couplings are determined and subsequently interpreted for different scenarios in terms of effective alignment tensors, average orientations of dipolar vectors, and intramolecular reorientational vector distributions. Analytical relationships are derived that reflect similarities and differences between motional scaling of dipolar couplings and scaling of dipolar relaxation data (NMR order parameters). Application of the self-consistent procedure presented here to dipolar coupling measurements of biomolecules aligned in different liquid-crystalline media should allow one to extract in a "model-free" way average orientations of dipolar vectors and specific aspects of their motions.     

Natural and biotech-derived therapeutic proteins: what is the future?

A myriad of novel proteins and ligands of unknown function will be generated by the Human Genomic Project. Due to differences in post-translational processing, proteins produced by recombinant DNA technology may not possess proper biological activity. One way to find their function is to search for their natural counterparts. Proteins are produced in the tissues, and many of them are secreted into plasma and excreted into urine. There is a virtually "unlimited" array of human proteins in our plasma and urine, many of them in a fully active form. They include small molecules like steroids, peptides, and large glycoproteins like human menopausal gonadotropin. A library of plasma and urinary proteins could be developed to serve as a reference for the novel proteins generated by the functional genomic projects.     

Solidified liquid layer model makes quartz crystal microbalance a convenient molecular ruler

The applications of quartz crystal microbalance (QCM) in biointerfaces are limited by its quantitative ambiguities caused by viscoelasticity and solution effects. Although many studies clearly indicated that the quantitative interpretation of QCM data needed caution, none of those studies provided a practical solution that enabled general and quantitative interpretation of QCM data. Recently we proposed a "solidified liquid layer" model that enabled QCM to be used as a biomolecular ruler. Here we applied five kinds of proteins with significant differences in their sizes and shapes to further validate this model. The effective thickness (T(eff)) of surface immobilized, hydrated proteins were 10.2, 4.7, 1.8 and 4.8 nm for rabbit IgG, streptavidin, lysozyme, and bovine serum albumin, respectively. The critical number of stakes needed for the formation of a solidified liquid layer was found to be protein dependent. We believed this "solidified liquid layer" model will facilitate the popularization of QCM as a valuable tool in biointerface studies, such as protein adsorption process or the conformational change on surface.     

A sequence and structural study of transmembrane helices

A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the -branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline, residue types normally associated with `helix-breaking' propensity are found to be relatively more common in membrane helices. Three dimensional nearest neighbour contacts along the helix, preferred rotamers, and peptide bond angles are very similar in the three sets of proteins as far as can be ascertained within the limits of the relatively low resolution of the membrane proteins dataset. Crossing angles for helices in the membrane protein set resemble the four helix bundle set more than the general non-redundant set, but in contrast to both sets they have smaller crossing angles consistent with the dual requirements for the helices to form a compact structure while having to span the membrane. In addition to the pairwise packing of helices we investigate their global packing and consider the question of helix supercoiling in helix bundle proteins.     

Comparison of the structural and chemical composition of giant T-even phage heads.

A study has been made of the structure of the capsids of T4D giant phage produced from mutants in gene 23 and temperature-sensitive mutants in gene 24, and T4D and T2L giant phage formed by the addition of L-canavanine followed by an Larginine chase in the growth medium. All the giant phage capsids have been shown to be built according to the same geometrical architecture. This consists of a near-hexagonal surface net, lattice constant 129.5 A, folded into a left-handed T = 13 prolate icosahedron elongated along one of its fivefold symmetry axes. Their only apparent difference from wild-type T-even phage capsids is their abnormally elongated tubular part. A comparison of the capsomere morphologies and protein compositions of the giant phage capsids showed that all T4D giants are identical but differ from T2L: The T4D capsomere has a complex (6 + 6 + 1)-type morphology, whereas the T2L has a simple 6-type. T2L phage, however, lack two capsid proteins, "soc" and "hoc", present in T4D. The difference in capsomere morphology can therefore be related to the difference in the protein compositions of these two phage. Possible differences between the initiation and means of length regulation of giant phage heads and the aberrant polyheads are discussed.     

Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients.

Two cultivars ("Alexis" and "Lenka") of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band "B" (isoelectric point approximately 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this "B" isoenzyme band, but exhibit the pronounced "A" isoenzyme band (isoelectric point approximately 6.5) instead, suggesting that these isoenzymes (which we suppose to be beta-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, "final attenuation" is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar "Lenka", may also have a role in determining quality.     

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins.

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.     

Proteomic analysis of chicken eggshell cuticle membrane layer

The eggshell is a barrier that plays an important role in the defense of the egg against microbial and other infections; it protects the developing bird against unfavorable impacts of the environment and is essential for the reproduction of birds. The avian eggshell is a complex structure that is formed during movement along the oviduct by producing a multilayered mineral-organic composite. The extractable proteins of avian eggshells have been studied extensively and many of them identified, however, the insoluble (non-extractable) proteins have been sparsely studied. We studied the EDTA-insoluble proteinaceous film from the cuticle layer of eggshell. This film consists of three main areas: spots (cca 300 μm diameter), blotches (small spots with diameter only tens of μm), and the surroundings (i.e., the area without spots and blotches) where spots contain a visible accumulation of pigment. These areas were cut out of the membrane by laser microdissection, proteins were cleavaged by trypsin, and the peptides were analyzed by nLC/MS (Q-TOF). This study has identified 29 proteins and a further eight were determined by less specific “cleavage” with semitrypsin. The relative abundances of these proteins were determined using the exponentially modified protein abundance index (emPAI) where the most dominant proteins were eggshell-specific ones, such as ovocleidin-17 and ovocleidin-116. Individual areas of the cuticle membrane differ in their relative proportions of 14 proteins, where significant differences between the three quantification criteria (direct, after normalization to ovocledin-17, or to ovocledin-116) were observed in four proteins.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Studies of DNA-protein interactions by gel electrophoresis

The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.     

Prestaining method as a useful tool for the agarose gel electrophoretic detection of polymerase chain reaction products with a fluorescent dye SYBR gold nucleic acid gel stain.

Three staining methods using SYBR Gold Nucleic Acid Gel Stain (SYBR Gold) as a fluorescent dye were evaluated for the agarose gel electrophoretic detection of DNA. The methods involve prestain, in-gel stain, and poststain methods. DNA markers and polymerase chain reaction (PCR) products obtained by minisatellite variant repeat-PCR (MVR-PCR) amplification in a D1S8 locus were used as model DNA and practical samples, respectively. Among the three methods tested under the usual electrophoretic conditions, a prestain method using a 10000-fold diluted SYBR Gold solution showed most excellent features regarding cost and rapidity to use with good stainability and resolution over loaded DNA amounts of about 98 ng to 300 ng. The prestain method was found to be applicable to the analysis of DNA in MVR-PCR products from a human hair root.     

A study of the water-soluble proteins of the seeds of the cotton plants Gossypium hirsutum and G. barbadense

It has been shown by electrophoresis in polyacrylamide gel that different enzymes with similar electrophoretic mobilities may be localized in one and the same zone of water-soluble cottonseed proteins. Some zones not stained by the usual protein dyes also possess enzymatic activity. It has been established that the majority of the electrophoretic fractions consist of a series of polypeptides with different molecular weights, mainly of low-molecular-weight nature. The results are given of an investigation of the peroxidase, α-amylase, lipase, NADH-DCPIP oxidoreductase, and NADPH-DCPIP oxidoreductase activities of the water soluble fraction of the proteins of the seeds of the species mentioned.     

Reduction of protein concentration range difference followed by multicolumn fractionation prior to 2-DE and LC-MS/MS profiling of serum proteins

This article is concerned with the reduction of protein concentration range differences by the peptide beads library technology (ProteoMiner? or "equalizer" technology), which in principle allows the enrichment of proteins to the same concentration level (i.e. protein equalizer) regardless of the original protein abundance in a given biological fluid such as human serum, which is the subject of our investigation. After the equalization step, the captured proteins from human serum were fractionated on a series of tandem monolithic columns with surface-bound iminodiacetic acid ligands to which three different metal ions, namely, Zn2+, Ni2+ and Cu2+ were immobilized to yield the so-called immobilized metal affinity chromatography columns. These three monolithic columns were connected to a reversed-phase column packed with polystyrene divinyl benzene beads. Aliquots taken from the four collected fractions from the four tandem columns were subsequently fractionated by 2-DE. Also, aliquots from the four collected fractions were tryptically digested and analyzed by LC-MS/MS. The strategy of subsequent fractionation on the four tandem columns after equalization allowed the identification of more proteins than simply using the equalization by ProteoMiner? . The equalizer technology was compared to the immuno-subtraction approach. While the ProteoMiner? technology is superior in terms of the overall number of captured proteins, it only complements the immuno-subtraction approach since the latter can capture the proteins that were not captured by the former.     

D/H amide isotope effect in model alpha-helical peptides

The contribution of amide related hydrogen bonds to protein stability has recently been evaluated using the "Cm experiment", which measures the D/H amide isotope effect in proteins. We show here using isolated alpha-helical peptides that there is a significant effect of denaturant concentration on the measured D/H isotope effect, and that valid comparison of different proteins requires correcting for differences in denaturant (GdmHCl) concentration. Finally our results suggest that H-bonds in an isolated alpha-helix may contribute more to helix stability because of less strain compared to those in helical proteins and that the buried helical H-bonds in helical proteins are not necessarily energetically more favorable than solvent exposed H-bonds in isolated helices.