Protein biosynthesis: the codon-specific activation of amino acids.

At the start of protein biosynthesis the amino acids are chemically activated by esterification with transfer ribonucleic acids bearing the appropriate anticodons. The esterification is catalyzed with an exceptionally high specificity by aminoacyl-tRNA synthetases. Current knowledge of the structural properties of the reactants, the course of the reaction, and the reasons for its specificity are summarized.     

Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis

Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.     

Antibiotics as inhibitors of nucleic acid and protein synthesis

The antibiotics of the rifamycin, actinomycin, chromomycin, and anthracycline groups have been found to be specific inhibitors for the DNA-controlled synthesis of RNA in vitro. Streptomycin, chloramphenicol, and puromycin can specifically suppress certain steps in the biosynthesis of proteins. The investigation of the mode of action of such substances may help us to gain a better insight into the transmission of hereditary information.     

The 3′-End of tRNA and Its Role in Protein Biosynthesis

In the course of protein biosynthesis, the 3′-ends of aminoacyl-tRNA (aa-tRNA) and peptidyl-tRNA specifically interact with macromolecules of the protein biosynthesis machinery. The 3′-end of tRNA consists of an invariant C-C-A single strand. Interaction of the aminoacyl-tRNA 3′-end with elongation factor Tu (EF-Tu) containing bound GTP is necessary for the formation of the aa-tRNA·EF-Tu·GTP complex and, after the complex binds to the ribosome, for the GTP hydrolysis. This process is followed by the specific binding of the aminoacyl-tRNA 3′-end to the aminoacyl (A) site of the ribosome. In this review, a model is proposed that involves Watson-Crick base pairing of the C? C sequence of the aminoacyl-tRNA 3′-end with a specific G? G sequence of the ribosomal 23S RNA. Similarly, peptidyl-tRNA binds with its 3′-end to the peptidyl (P) site of the ribosome. This binding may also involve Watson-Crick base pairing of the C-C-A sequence with a complementary sequence of 23S RNA. It is proposed that peptide bond formation is catalyzed by a functional site of the 23S RNA located near the 3′-ends of aminoacyl-tRNA and peptidyl-tRNA. A model is suggested in which two loops of the 23S RNA, brought into close proximity via folding, are involved both in binding the 3′-ends of the tRNAs and in catalyzing peptide bond formation. This model presumes a dynamic structure for ribosomal RNA, which is modulated by interaction with elongation factors and ribosomal proteins.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than ∼10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than ∼650 nM of anti-HSA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

2DE‐based approach for estimation of number of protein species in a cell

Insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. But to go further, we need at least to know the proteome size, or how many different protein species compose this proteome. This is the task that could be at least partially realized by the method described in this article. The approach used in our study is based on detection of protein spots in 2DE after staining by protein dyes with various sensitivities. As the different protein spots contain different protein species, counting the spots opens a way for estimation of number of protein species. The function representing the dependence of the number of protein spots on sensitivity or LOD of protein dyes was generated. And extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) allowed to counting the number of different molecules (polypeptide species) at the concentration level of a single polypeptide per proteome. Using this approach, it was estimated that the minimal numbers of protein species for model objects, Escherichia coli and Pirococcus furiosus, are 6200 and 3400, respectively. We expect a single human cell (HepG2) to contain minimum 70 000 protein species.     

The DNA aptamers that specifically recognize ricin toxin are selected by two in vitro selection methods

Aptamers which specifically recognize cytotoxin ricin were successfully selected using the two different in vitro selection methods. One selection method was used to isolate aptamers by affinity chromatography. Another selection method, named CE-SELEX, was carried out using CE as a separation approach. The high separation efficiency of CE evidently improved the rate of enrichment and obviously shortened the selection rounds, with near 87.2% binding just after the fourth round of selection. The aptamers A3, C1, and C5, derived from the two selection methods, were found to possess high affinity and specificity for ricin with the Kd values in the low nanomolar range, and did not recognize abrin toxin similar to ricin in the structures and properties, or BSA. Among the aptamers selected, A3 isolated by affinity chromatography shared extensive sequence similarity with C1 and C5 derived from CE-SELEX. They differed by only one base from each other. Their stable secondary structures predicted also had very similar structure motifs, and all folded a long and internal loop-embedded loop stem structure by base pairing. The ELISA and dot-blot analysis also proved that the selected DNA aptamers had the high specificity to ricin toxin.     

Studies on the biosynthesis of secobotryane skeleton

The labelling and coupling patterns of secobotrytrienediol, biosynthesised from [1-¹³C] and [1,2-¹³C₂]-acetate by the fungus Botrytis cinerea, have been used to define the mode of formation and the biogenetic origin of secobotrytrienediol. [10-²H]-Botrydiol was not incorporated into the secobotryane skeleton. In addition, this feeding experiment led to the isolation of three new unlabelled derivatives possessing a secobotryane skeleton, secobotrydiene-3,10,15-triol, secobotrydiene-3,4,10,15-tetraol, and secobotrytriene-10,12,15-triol.     

Proteomics in cancer research: methods and application of array-based protein profiling technologies

With the human genome sequence now determined, the field of molecular medicine is moving beyond genomics to proteomics, the large-scale analysis of proteins.It is now possible to examine the expression of more than 1000 proteins using mass spectrometry technology coupled with various separation methods.Microarray technology is a new and efficient approach, for extracting relevant biomedical data and has a wide range of applications. It provides a versatile tool to study protein-protein, protein-nucleic acid, protein-lipid, enzyme-substrate and protein-drug interactions.This review paper will explore the key themes in proteomics and their application in clinical cancer research.     

Interpreting conformational effects in protein nano-ESI-MS spectra

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is employed here to describe equilibrium protein conformational transitions and to analyze the influence of instrumental settings, pH, and solvent surface tension on the charge-state distributions (CSD). A first set of experiments shows that high flow rates of N₂ as curtain gas can induce unfolding of cytochrome c (cyt c) and myoglobin (Mb), under conditions in which the stability of the native protein structure has already been reduced by acidification. However, it is possible to identify conditions under which the instrumental settings are not limiting factors for the conformational stability of the protein inside ESI droplets. Under such conditions, equilibrium unfolding transitions described by ESI-MS are comparable with those obtained by other established biophysical methods. Experiments with the very stable proteins ubiquitin (Ubq) and lysozyme (Lyz) enable testing of the influence of extreme pH changes on the ESI process, uncoupled from acid-induced unfolding. When HCl is used for acidification, Ubq and Lyz mass spectra do not change between pH~7 and pH 2.2, indicating that the CSD is highly characteristic of a given protein conformation and not directly affected by even large pH changes. Use of formic or acetic acid for acidification of Ubq solutions results in major spectral changes that can be interpreted in terms of protein unfolding as a result of the increased hydrophobicity of the solvent. On the other hand, Lyz, cyt c, and Mb enable direct comparison of protein CSD (corresponding to either the folded or the unfolded protein) in HCl or acetic acid solutions at low pH. The values of surface tension for these solutions differ significantly. Confirming indications already present in the literature, we observe very similar CSD under these solvent conditions for several proteins in either compact or disordered conformations. The same is true for comparison between water and water–acetic acid for folded cyt c and Lyz. Thus, protein CSD from water–acetic solutions do not seem to be limited by the low surface tension of acetic acid as previously suggested. This result could reflect a general lack of dependence of protein CSD on the surface tension of the solvent. However, it is also possible that the effect of acetic acid on the precursor ESI droplets is smaller than generally assumed.     

Advances in the analysis of dynamic protein complexes by proteomics and data processing

Signal transduction governs virtually every cellular function of multicellular organisms, and its deregulation leads to a variety of diseases. This intricate network of molecular interactions is mediated by proteins that are assembled into complexes within individual signaling pathways, and their composition and function is often regulated by different post-translational modifications. Proteomic approaches are commonly used to analyze biological complexes and networks, but often lack the specificity to address the dynamic and hence transient nature of the interactions and the influence of the multiple post-translational modifications that govern these processes. Here we review recent developments in proteomic research to address these limitations, and discuss several technologies that have been developed for this purpose. The synergy between these proteomic and computational tools, when applied together with global methods to the analysis of individual proteins, complexes and pathways, may allow researchers to unravel the underlying mechanisms of signaling networks in greater detail than previously possible.     

A comparative analysis of polyurethane hydrogel for immobilization of IgG on chips

Hydrogels are considered an optimum material for protein chip surfaces, since they provide a quasi-liquid environment which allows protein activity to be maintained and shows good spot morphology as well as excellent immobilization capacity. In the following, we present a polyurethane (PU) chip that electrostatically binds IgG. The PU surface is optimized with regard to layer thickness (∼200 nm), hydrogel (2%) and immobilized antibody concentration (0.5 mg mL⁻¹; 0.3 ng spot⁻¹), pH and ionic strength of the print buffer as well as to blocking solution. Evaluation is done in a direct IgG immunoassay using the Nexterion slide H as a reference. It is shown that higher IgG loading is achieved on the PU chip than on slide H, no matter whether 1× PBS (pH 7.2), Sörensen (pH 5.8) or Nexterion buffer was used as a spotting solution. Moreover, the crossreactivity with goat IgG, human IgG and monoclonal anti-CRP spotted in Nexterion buffer was as low as ≤0.74% (slide H: ≤3.34%).     

Immobilization of Protein A on SAMs for the elaboration of immunosensors

Binary mixtures of 11-mercaptoundecanoic acid (MUA) and other thiols of various lengths and terminal functions were chemisorbed on gold-coated surfaces via S–Au bonds to form mixed self-assembled monolayers (SAMs). Several values of the mole fraction of MUA in the thiol mixtures were tested and the structure and composition of the resulted thin films were characterized by X-ray photoelectron spectroscopy (XPS) and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The results made it clear that co-adsorption of MUA with thiols of similar chain length led to well-ordered monolayers whereas the co-adsorption of MUA with shorter thiols yielded less crystalline-like thin films, but with more reactive carboxylic acid terminal groups. This criterion appeared decisive for efficient covalent binding of Staphylococcus aureus Protein A (PrA), a protein that displays high affinity for the constant fragment (Fc) of antibodies of the IgG type from various mammal species. The ability of immobilized Protein A to recognize and bind a model IgG appeared to be optimal for the mixed SAM of MUA and the short-chain, ω-hydroxythiol 6-mercaptohexanol in the proportion 1–3.     

Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells

Resveratrol is a naturally occurring defense compound produced by a limited number of plants in response to stresses. Besides cardiovascular benefits, this health-promoting compound has been reported to extend life spans in yeasts, flies, worms, and fish. To biosynthesize resveratrol de novo, tyrosine ammonia lyase (TAL), 4-coumarate CoA-ligase (4CL), and stilbene synthase (STS) were isolated from Rhodobacter sphaeroides, Arabidopsis thaliana, and Vitis vinifera, respectively. Yeast cells expressing 4CL and STS produce resveratrol when fed with 4-coumaric acid, the substrate of 4CL. When a translational fusion protein joining 4CL and STS was used, yeast cells produced 15-fold more resveratrol than the cotransformed cells, suggesting that physical localization of 4CL and STS facilitate resveratrol production. When the resveratrol pathway was introduced into human HEK293 cells, de novo biosynthesis was detected, leading to intracellular accumulation of resveratrol. We successfully engineered an entire plant natural product pathway into a mammalian host.     

Controlling the orientation of immobilized proteins on an affinity membrane through chelation of a histidine tag to a chitosan-Ni surface

The ability of histidine-tagged proteins to chelate to Ni⁺⁺ ions that are coordinated to functionalized polymeric matrices is the basis of affinity column separations. In this study, affinity membranes based on a Ni⁺⁺-chelating chitosan surface were fabricated to immobilize C-terminus hexahistidine-tagged green fluorescent protein (his-GFP). The binding of GFP antibody (antiGFP) to the immobilized his-GFP was measured and compared to a membrane with a chitosan surface to which his-GFP was immobilized through amine-glutaraldehyde chemistry as a control. Both membranes had comparable amounts of his-GFP immobilized on the surface. However, the amount of antiGFP bound to the Ni⁺⁺-chelated his-GFP at saturation was higher than that bound to the glutaraldehyde-immobilized his-GFP by a factor of five. Furthermore, fitting the data to a single-site Langmuir model resulted in an affinity constant for the Ni⁺⁺-chelated his-GFP towards antiGFP that was 14 times higher than the glutaraldehyde-immobilized his-GFP. The higher affinity suggests that immobilizing a protein at its C-terminus results in the proper orientation for subsequent antibody binding. At low antibody concentrations, the sensitivity of the affinity membrane is 70 times that of the control.