共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Kreutzberger J 《Applied microbiology and biotechnology》2006,70(4):383-390
Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein–protein
interactions or protein–small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification
of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy
slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred
to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts
of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or
expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins
or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein
microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein
microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides)
are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard
to investigations of microorganisms. 相似文献
3.
The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.K. Kuroda, M. Kato and J. Mima contributed equally to this work. 相似文献
4.
Amita Nand Vikramjeet Singh Javier Batista Pérez Deependra TyagiZhiqiang Cheng Jingsong Zhu 《Analytical biochemistry》2014
In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10−13 M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields. 相似文献
5.
Protein flexibility lies at the heart of many protein–ligand binding events and enzymatic activities. However, the experimental
measurement of protein motions is often difficult, tedious and error-prone. As a result, there is a considerable interest
in developing simpler and faster ways of quantifying protein flexibility. Recently, we described a method, called Random Coil
Index (RCI), which appears to be able to quantitatively estimate model-free order parameters and flexibility in protein structural
ensembles using only backbone chemical shifts. Because of its potential utility, we have undertaken a more detailed investigation
of the RCI method in an attempt to ascertain its underlying principles, its general utility, its sensitivity to chemical shift
errors, its sensitivity to data completeness, its applicability to other proteins, and its general strengths and weaknesses.
Overall, we find that the RCI method is very robust and that it represents a useful addition to traditional methods of studying
protein flexibility. We have implemented many of the findings and refinements reported here into a web server that allows
facile, automated predictions of model-free order parameters, MD RMSF and NMR RMSD values directly from backbone 1H, 13C and 15N chemical shift assignments. The server is available at .
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
Dinesh C. Soares Paul N. Barlow David J. Porteous Rebecca S. Devon 《Journal of molecular modeling》2009,15(2):113-122
Defects in the human ALS2 gene, which encodes the 1,657-amino-acid residue protein alsin, are linked to several related motor neuron diseases. We created
a structural model for the N-terminal 690-residue region of alsin through comparative modelling based on regulator of chromosome
condensation 1 (RCC1). We propose that this alsin region contains seven RCC1-like repeats in a seven-bladed beta-propeller
structure. The propeller is formed by a double clasp arrangement containing two segments (residues 1–218 and residues 525–690).
The 306-residue insert region, predicted to lie within blade 5 and to be largely disordered, is poorly conserved across species.
Surface patches of evolutionary conservation probably indicate locations of binding sites. Both disease-causing missense mutations—Cys157Tyr
and Gly540Glu—are buried in the propeller and likely to be structurally disruptive. This study aids design of experimental
studies by highlighting the importance of construct length, will enhance interpretation of protein–protein interactions, and
enable rational site-directed mutagenesis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
7.
Srđan Đ. Stojanović Vesna B. Medaković Goran Predović Miloš Beljanski Snežana D. Zarić 《Journal of biological inorganic chemistry》2007,12(7):1063-1071
Searching structures of porphyrin-containing proteins from the Protein Data Bank revealed that the π system of every porphyrin ring is involved in XH/π interactions, with most of the porphyrins having several interactions. Both five-membered pyrrole rings and six-membered
chelate rings are involved in XH/π interactions; the number of interactions with five-membered rings is larger than the number of interactions with six-membered
rings. We found interactions with C–H and N–H groups as hydrogen-atom donors; however, the number of CH/π interactions is much larger than the number of NH/π interactions. The amino acids involved in the interactions show a high conservation score. Our results that every porphyrin
is involved in XH/π interactions and that amino acids involved in these interactions are highly conserved demonstrate that XH/π interactions play an important role in porphyrin–protein stability.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Jensen MR Ortega-Roldan JL Salmon L van Nuland N Blackledge M 《European biophysics journal : EBJ》2011,40(12):1371-1381
Protein–protein interactions occur with a wide range of affinities from tight complexes characterized by femtomolar dissociation
constants to weak, and more transient, complexes of millimolar affinity. Many of the weak and transiently formed protein–protein
complexes have escaped characterization due to the difficulties in obtaining experimental parameters that report on the complexes
alone without contributions from the unbound, free proteins. Here, we review recent developments for characterizing the structures
of weak protein–protein complexes using nuclear magnetic resonance spectroscopy with special emphasis on the utility of residual
dipolar couplings. 相似文献
9.
DNA DAMAGED BINDING PROTEIN 1 (DDB1) is a highly conserved protein of around 125 kDa. It serves as a substrate adaptor subunit
to a CUL4-based E3 ubiquitin ligase within the ubiquitin proteasome pathway. However, based on a set of three beta-propellers,
the protein is able to mediate various protein–protein interactions, suggesting that it participates in many developmental
and physiological processes in the plant. Arabidopsis encodes for two closely related DDB1 proteins, named DDB1a and DDB1b. While loss-of DDB1a does not severely affect development,
loss-of DDB1b has been reported to result in an embryo lethal phenotype. Here we describe two novel ddb1b T-DNA insertion mutants that are not embryo lethal, which we utilized as genetic tools to dissect DDB1b from DDB1a function.
Information generated by these studies showed that the C-terminal part of the DDB1 proteins is critical for specific protein–protein
interactions. In addition, we demonstrated that DDB1a, like DDB1b, is critical for embryo development, and that both proteins
have distinct functions in whole plant development. 相似文献
10.
Fabien Ferrage Kaushik Dutta Alexander Shekhtman David Cowburn 《Journal of biomolecular NMR》2010,47(1):41-54
Protein interactions are important for understanding many molecular mechanisms underlying cellular processes. So far, interfaces
between interacting proteins have been characterized by NMR spectroscopy mostly by using chemical shift perturbations and
cross-saturation via intermolecular cross-relaxation. Although powerful, these techniques cannot provide unambiguous estimates
of intermolecular distances between interacting proteins. Here, we present an alternative approach, called REDSPRINT (REDduced/Standard
PRoton density INTerface identification), to map protein interfaces with greater accuracy by using multiple NMR probes. Our
approach is based on monitoring the cross-relaxation from a source protein (or from an arbitrary ligand that need not be a
protein) with high proton density to a target protein (or other biomolecule) with low proton density by using isotope-filtered
nuclear Overhauser spectroscopy (NOESY). This methodology uses different isotropic labeling for the source and target proteins
to identify the source-target interface and also determine the proton density of the source protein at the interface for protein-protein
or protein-ligand docking. Simulation indicates significant gains in sensitivity because of the resultant relaxation properties,
and the utility of this technique, including a method for direct determination of the protein interface, is demonstrated for
two different protein–protein complexes. 相似文献
11.
Background
In structural genomics, an important goal is the detection and classification of protein–protein interactions, given the structures of the interacting partners. We have developed empirical energy functions to identify native structures of protein–protein complexes among sets of decoy structures. To understand the role of amino acid diversity, we parameterized a series of functions, using a hierarchy of amino acid alphabets of increasing complexity, with 2, 3, 4, 6, and 20 amino acid groups. Compared to previous work, we used the simplest possible functional form, with residue–residue interactions and a stepwise distance-dependence. We used increased computational ressources, however, constructing 290,000 decoys for 219 protein–protein complexes, with a realistic docking protocol where the protein partners are flexible and interact through a molecular mechanics energy function. The energy parameters were optimized to correctly assign as many native complexes as possible. To resolve the multiple minimum problem in parameter space, over 64000 starting parameter guesses were tried for each energy function. The optimized functions were tested by cross validation on subsets of our native and decoy structures, by blind tests on series of native and decoy structures available on the Web, and on models for 13 complexes submitted to the CAPRI structure prediction experiment. 相似文献12.
Protein crystallization is in part driven by the changes in the entropy of the system, but opinions differ as to whether the
solute (protein) or solvent (water) molecules make more of a contribution to the overall entropic change. Methylation of lysine
residues in proteins has been used to enhance protein crystallization. We investigated using molecular dynamics simulations
with explicit solvent molecules, the behavior of several native proteins and their methylated counterparts chosen from an
earlier large-scale study. Methylated lysines are capable of making a variety of interactions including H-bonds with protein
residues and solvent. We demonstrate that methylation on the lysine slightly increases its side chain conformational entropy
by about 3.5 J mol−1 K−1. Analysis of the radial and spatial distributions of the water molecules around the methylated lysine surface in oxidoreductase
from Streptococcus pneumoniae revealed a larger sphere of water molecules with low entropy, as compared with solvent associated with unmethylated lysine.
If methylated lysine were to make interactions at the protein–protein interface, the low-entropy water molecules associated
with methylated lysines would be released, resulting in a gain of entropy. We show that this gain more than compensates for
the loss of protein entropy. Therefore, we propose that lysine methylation favors the formation of crystals through solvent
entropic gain. 相似文献
13.
Thermophiles, mesophiles, and psychrophiles have different amino acid frequencies in their proteins, probably because of the
way the species adapt to very different temperatures in their environment. In this paper, we analyse how contacts between
sidechains vary between homologous proteins from species that are adapted to different temperatures, but displaying relatively
high sequence similarity. We investigate whether specific contacts between amino acids sidechains is a key factor in thermostabilisation
in proteins. The dataset was divided into two subsets with optimal growth temperatures from 0–40 and 35–102°C. Comparison
of homologues was made between low-temperature species and high-temperature species within each subset. We found that unspecific
interactions like hydrophobic interactions in the core and solvent interactions and entropic effects at the surface, appear
to be more important factors than specific contact types like salt bridges and aromatic clusters.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
14.
The interaction of a quercetin copper(II) complex with DNA was investigated using UV–vis spectra, fluorescence measurement,
viscosity measurement, agarose gel electrophoresis, and thiobarbituric acid reactive substances assay. The results indicate
that the quercetin copper(II) complex can promote the cleavage of plasmid DNA, producing single and double DNA strand breaks,
and intercalate into the stacked base pairs of DNA. Moreover, the complex can induce oxidative DNA damage involving generation
of reactive oxygen species such as H2O2 and Cu(I)OOH. In addition, the cytotoxicity experiments carried out with A549 cells confirmed its apoptosis-inducing activity.
And we also demonstrate that the levels of survivin protein expression in A549 cells decreased, and that relative activity
of caspase-3 increased significantly after treatment with the complex. So our results suggest that the antitumor mechanism
of the quercetin copper(II) complex involves not only its oxidative DNA damage with generation of reactive oxygen species
but also its specific interaction with DNA.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
Gabellieri E Cioni P Balestreri E Morelli E 《European biophysics journal : EBJ》2011,40(11):1237-1245
We evaluated the potential of tryptophan (Trp) phosphorescence spectroscopy for investigating conformational states of proteins
involved in interaction with nanoparticles. Characterization of protein–nanoparticle interaction is crucial in assessing biological
hazards related to use of nanoparticles. We synthesized glutathione-coated CdS quantum dots (GSH-CdS), which exhibited an
absorption peak at 366 nm, indicative of 2.4 nm core size. Chemical analysis of purified GSH-CdS suggested an average molecular
formula of GSH18S56Cd60. Investigations were conducted on model proteins varying in terms of isoelectric point, degree of burial of the Trp probe,
and quaternary structure. GSH-CdS fluorescence measurements showed improvement in nanoparticle quantum yield induced by protein
interaction. Trp phosphorescence was used to examine the possible perturbations in the protein native fold induced by GSH-CdS.
Phosphorescence lifetime measurements highlighted significant conformational changes in some proteins. Despite their small
size, GSH-CdS appeared to interact with more than one protein molecule. Rough determination of the affinity of GSH-CdS for
proteins was derived from the change in phosphorescence lifetime at increasing nanoparticle concentrations. The estimated
affinities were comparable to those observed for specific protein–ligand interactions and suggest that protein–nanoparticle
interaction may have a biological impact. 相似文献
16.
Bertini I Felli IC Gonnelli L Pierattelli R Spyranti Z Spyroulias GA 《Journal of biomolecular NMR》2006,36(2):111-122
The copper-mediated protein–protein interaction between yeast Atx1 and Ccc2 has been examined by protonless heteronuclear NMR and compared with the already available 1H–15N HSQC information. The observed chemical shift variations are analyzed with respect to the actual solution structure, available
through intermolecular NOEs. The advantage of using the CON-IPAP spectrum with respect to the 1H–15N HSQC resides in the increased number of signals observed, including those of prolines. CBCACO-IPAP experiments allow us
to focus on the interaction region and on side-chain carbonyls, while a newly designed CEN-IPAP experiment on side-chains
of lysines. An attempt is made to rationalize the chemical shift variations on the basis of the structural data involving
the interface between the proteins and the nearby regions. It is here proposed that protonless
13C direct-detection NMR is a useful complement to 1H based NMR spectroscopy for monitoring protein–protein and protein–ligand interactions.
Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at 相似文献
17.
The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15
relaxation using multiple-echo Carr–Purcell–Meiboom–Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features
extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears
to be much less mobile on this time scale. At 308 K, exchange rates k
ex = 500–2000 s−1 were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83–87. These residues occlude
an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family,
such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated,
suggesting local ‘breathing’ motions rather than a concerted global conformational change.
Electronic supplementary material The online version of this article
(doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Gyri Teien Haugland Claire R. Rollor Nils-Kåre Birkeland Zvi Kelman 《Extremophiles : life under extreme conditions》2009,13(1):81-88
Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown
that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are
studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3′ → 5′ helicase activity.
The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in
T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
19.
Fanyan Meng Chuanlai Shen Yong He Fengqin Miao Jianqiong Zhang 《Biotechnology letters》2009,31(6):831-836
The application of the peptide-linked β2-microglobulin (β2m) strategy is limited in some cases due to the incompatibility
between the sequences of the peptides and the restriction sites of the plasmid vectors. An isocaudamer technique was adapted
to overcome this restriction. Three peptide-linked β2m genes, HBc18–27-hβ2m gene, OVA257–264-mβ2m gene and HER2/neu369–377-mβ2m gene, were inserted into the pET28a vectors with this technique. The corresponding proteins were expressed in Escherichia coli with yields of over 50 mg/l culture and purities of over 80%. This strategy facilitates the construction of peptide-linked
β2m molecules and will simplify the preparation of major histocompatibility complex-peptide complexes.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
Sachiko Shiokai Hiroyasu Kitashiba Kenta Shirasawa Kuniaki Nagano Takeshi Nishio 《Molecular breeding : new strategies in plant improvement》2009,23(2):329-336
DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious.
In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly
usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better
than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using
the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates,
named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献