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1.
Julia Rumpf Bernd Simon Yvonne Groemping Michael Sattler 《Biomolecular NMR assignments》2008,2(1):55-58
EH domains are protein–protein interaction domains that function in vesicular trafficking and endocytosis. Here, we report
the NMR spectral assignments of the high-affinity complex between the second EH domain of Eps15 and a stonin 2 peptide—providing
the basis for the characterization of a two-site binding mode. 相似文献
2.
Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug
action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate
exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for
detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein
can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances
possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations.
Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations
are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence
of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular
NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we
employ methods related to driven NOE spectroscopy to investigate protein–ligand interactions in the intermediate exchange
regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable
NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples
selectively protonated at a few sites so that the 1D 1H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap
with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein–ligand NOEs that
allow calculation of initial complex structures, suitable for structure-based optimization of primary drug leads obtained
from high-throughput screening. The method was applied to measure individual intermolecular NOEs between the anti-apoptotic
protein Bcl-xL at 25 μM and a “first generation” small-molecule ligand, for which the spectrum is entirely broadened at stoichiometric
concentrations. This approach is general and can also be used to characterize protein–protein or protein–nucleic-acid complexes. 相似文献
3.
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
4.
Viivi Majava Chaozhan Wang Matti Myllykoski Salla M. Kangas Sung Ung Kang Nobuhiro Hayashi Peter Baumgärtel Anthony M. Heape Gert Lubec Petri Kursula 《Amino acids》2010,39(1):73-74
Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral
and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein
ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the
major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally
characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular
dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein
complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the
two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize
in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP
modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the
complex between full-length proteins. 相似文献
5.
Peroxiredoxin systems in plants were demonstrated involved in crucial roles related to reactive oxygenated species (ROS) metabolism and the linked cell signalling to ROS. Peroxiredoxins function as peroxidasic systems that combine at least a reactivating reductant agent like thioredoxins, and sometimes glutaredoxins and glutathion. In the past three years a number of peroxiredoxin structures were solved by crystallography in different experimental crystallisation conditions. The structures have revealed a significant propensity of peroxiredoxins for oligomerism that was confirmed by biophysical studies in solution using NMR and other methods as analytical ultra-centrifugation. These studies showed that quaternary structures of peroxiredoxins involve specific protein–protein interaction interfaces that rely upon the peroxiredoxin types and/or their redox conditions. The protein–protein interactions with the reactivating redoxins essentially lead to transient unstable complexes. We review herein the different protein–protein interactions characterized or deduced from those reports.VNM is recipient of a PhD fellowship of the French Ministère de l’Enseignement Supérieur de la Recherche et des Nouvelles Technologies for the year 2003–2006 and the Research Doctorate School of Chemistry of Lyon. 相似文献
6.
Lozinsky E Iametti S Barbiroli A Likhtenshtein GI Kálai T Hideg K Bonomi F 《The protein journal》2006,25(1):1-15
Binding sites for hydrophobic molecules on bovine β-lactoglobulin, and their susceptibility to temperature, were studied by
using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or
both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual
probes was found to be required for binding to
β-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible
denaturation. Binding became extremely tight and stable upon cooling of the protein–probe mixture. Comparison among the various
probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially—and reversibly—modified
form of β-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes
binding to β-lactoglobulin may be modulated by simple physical treatment of the protein. 相似文献
7.
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
8.
Because interactions between cisplatin and plasma proteins contribute to drug efficacy and side effects, it is important to
understand both the binding sites of cisplatin on the proteins and the formation of protein–cisplatin adducts. Previous results
suggest that cisplatin preferentially binds to residues on the protein surface. The present work employed electrospray ionization
mass spectrometry (MS) to identify such sites on both native and denatured ubiquitin (Ub). Fourier transform (FT) MS and tandem
MS (MS/MS and MS3) enable analysis of Ub–cisplatin adduct digests to locate specific cisplatin binding sites. Results indicate that there are
three such binding sites, i.e., M1, T12 and T14, and D32, on native Ub. The intensity of the relevant peaks in the FT-MS spectrum
of the native Ub adduct digest demonstrates that residues T12 and T14 comprise the primary cisplatin binding site under the
native conditions rather than residue M1 as reported in previous research studies. It is found in the present work, however,
that M1 is the primary binding site on denatured Ub. Comparison of cisplatin binding sites on native and denatured Ub in this
research demonstrates that the conformation of a protein significantly influences the preference of cisplatin for specific
binding sites. 相似文献
9.
We undertook this project in response to the rapidly increasing number of protein structures with unknown functions in the
Protein Data Bank. Here, we combined a genetic algorithm with a support vector machine to predict protein–protein binding
sites. In an experiment on a testing dataset, we predicted the binding sites for 66% of our datasets, made up of 50 testing
hetero-complexes. This classifier achieved greater sensitivity (60.17%), specificity (58.17%), accuracy (64.08%), and F-measure (54.79%), and a higher correlation coefficient (0.2502) than those of the support vector machine. This result can
be used to guide biologists in designing specific experiments for protein analysis. 相似文献
10.
Structural and physical properties of DNA provide important constraints on the binding sites formed on surfaces of DNA-targeting proteins. Characteristics of such binding sites may form the basis for predicting DNA-binding sites from the structures of proteins alone. Such an approach has been successfully developed for predicting protein–protein interface. Here this approach is adapted for predicting DNA-binding sites. We used a representative set of 264 protein–DNA complexes from the Protein Data Bank to analyze characteristics and to train and test a neural network predictor of DNA-binding sites. The input to the predictor consisted of PSI-blast sequence profiles and solvent accessibilities of each surface residue and 14 of its closest neighboring residues. Predicted DNA-contacting residues cover 60% of actual DNA-contacting residues and have an accuracy of 76%. This method significantly outperforms previous attempts of DNA-binding site predictions. Its application to the prion protein yielded a DNA-binding site that is consistent with recent NMR chemical shift perturbation data, suggesting that it can complement experimental techniques in characterizing protein–DNA interfaces. 相似文献
11.
Guharoy M Pal A Dasgupta M Chakrabarti P 《Journal of structural and functional genomics》2011,12(1):33-41
Residues in a protein–protein interface that are important for forming and stabilizing the interaction can usually be identified
by looking at patterns of evolutionary conservation in groups of homologous proteins and also by the computational identification
of binding hotspots. The PRICE (PRotein Interface Conservation and Energetics) server takes the coordinates of a protein–protein
complex, dissects the interface into core and rim regions, and calculates (1) the degree of conservation (measured as the
sequence entropy), as well as (2) the change in free energy of binding (∆∆G, due to alanine scanning mutagenesis) of interface
residues. Results are displayed as color-coded plots and also made available for download. This enables the computational
identification of binding hot spots, based on which further experiments can be designed. The method will aid in protein functional
prediction by correct assignment of hot regions involved in binding. Consideration of sequence entropies for residues with
large ∆∆G values may provide an indication of the biological relevance of the interface. Finally, the results obtained on
a test set of alanine mutants has been compared to those obtained using other servers/methods. The PRICE server is a web application
available at . 相似文献
12.
Ming Tang Gemma Comellas Leonard J. Mueller Chad M. Rienstra 《Journal of biomolecular NMR》2010,48(2):103-111
High resolution 13C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been
collected to show that all 15N, 13C′, 13Cα and 13Cβ sites are resolved in 13C–13C and 15N–13C spectra, with significant improvement in T
2 relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T
2 values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that
13Cα T
2′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength,
and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint
of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy
to selectively detect polar residues that are often important for protein function and protein–protein interactions. 相似文献
13.
We have developed a surface plasmon resonance (SPR)-based immunocapture approach to study multimeric protein–protein complexes.
A composition and spatial architecture of protein complexes that contained GST-tagged p53, p14ARF, and MDM2 was examined by
the developed approach. Obtained results verified that the p53 protein possesses two binding sites for MDM2. Ternary complexes
containing p14ARF, MDM2, and p53 proteins could only be formed when MDM2 protein functions as a bridging molecule. That was
confirmed by immunoprecipitation and immunostaining.
Andrej Savchenko and Mariya Yurchenko have contributed equally to this article. 相似文献
14.
The major barrier responsible for the slow pace of structure determination of integral membrane proteins is the difficulty
of crystallizing detergent-solubilized hydrophobic proteins, particularly hetero-oligomeric integral membrane proteins. For
the latter class of multi-subunit proteins, we have encountered the following problems in addition to the ubiquitous problem
of detergent compatibility: (i) instability caused by over-purification that results in delipidation; (ii) protease activity
degrading exposed loops and termini of subunits of the complex that could not be inhibited; (iii) poor protein–protein contacts
presumably arising from masking by the detergent micelle. Problem (i) could be ameliorated in crystallization of the cytochrome
b6f complex by augmenting the delipidated complex with synthetic lipid. Problem (ii) has not been solved. Problem (iii) has been
solved in other systems by the use of monoclonal antibodies (or other protein ligands) to increase the probability of protein–protein
contacts. In the case of the complex formed by the cobalamin and colicin receptor, BtuB, and the receptor binding domain of
colicin E3, the latter served as a ligand for protein–protein contacts that facilitated crystallization. 相似文献
15.
Biophysical studies of protein–anesthetic interactions using nuclear magnetic resonance (NMR) spectroscopy are often conducted
by the addition of micro amounts of neat inhaled anesthetic which yields much higher than clinically relevant (0.2–0.5 mM)
anesthetic concentrations. We report a 19F NMR technique to measure clinically relevant inhaled anesthetic concentrations from saturated aqueous solutions of these
anesthetics (halothane, isoflurane, sevoflurane, and desflurane). We use a setup with a 3-mm NMR tube (containing trifluoroacetic
acid as standard), coaxially inserted in a 5-mm NMR tube containing anesthetic solution under investigation. All experiments
are conducted in a 5-mm NMR probe. We also have provided standard curves for four inhaled anesthetics using NMR technique.
The standard curve for each of these anesthetics is helpful in determining the prerequisite amount of aqueous anesthetic solution
required to prepare clinically relevant concentrations for protein–anesthetic interaction studies.
Parts of the results to be presented at Society for Neuroscience meeting, 2008. 相似文献
16.
Linser R 《Journal of biomolecular NMR》2011,51(3):221-226
Proteins with excessive deuteration give access to proton detected solid-state NMR spectra of extraordinary resolution and
sensitivity. The high spectral quality achieved after partial proton back-exchange has been shown to start a new era for backbone
assignment, protein structure elucidation, characterization of protein dynamics, and access to protein parts undergoing motion.
The large absence of protons at non-exchangeable sites, however, poses a serious hurdle for characterization of side chains,
which play an important role especially for structural understanding of the protein core and the investigation of protein–protein
and protein–ligand interactions, e.g. This has caused the perdeuteration approach to almost exclusively be amenable to backbone
characterization only. In this work it is shown that a combination of isotropic 13C mixing with long-range 1H/13C magnetization transfers can be used effectively to also access complete sets of side-chain chemical shifts in perdeuterated
proteins and correlate these with the protein backbone with high unambiguity and resolution. COmbined POlarization from long-Range transfers And Direct Excitation (COPORADE) allows this strategy to yield complete sets of aliphatic amino acid resonances with reasonable sensitivity. 相似文献
17.
18.
We present a novel target function based on atomic coordinates that permits quaternary structural refinement of multi-domain
protein–protein or protein–RNA complexes. It requires that the high-resolution structures of the individual domains are known
and that small angle scattering (SAS) data as well as NMR orientational restraints from residual dipolar couplings (RDCs)
of the complex are available. We show that, when used in combination, the translational and rotational restraints contained
in SAS intensities and RDCs, respectively, define a target potential function that permits to determine the overall topology
of complexes made up of domains with low internal symmetry. We apply the target function on a modestly anisotropic model system,
the Barnase/Barstar complex, and discuss factors that influence the structural refinement such as data errors and the geometrical
properties of the individual domains. 相似文献
19.
Lindsay J. Sperling Andrew J. Nieuwkoop Andrew S. Lipton Deborah A. Berthold Chad M. Rienstra 《Journal of biomolecular NMR》2010,46(2):149-155
Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-13C,15N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has
advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution
in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7,
17.6 and 21.1 Tesla (1H frequencies of 500, 750, and 900 MHz). For two protein systems—GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline
protein—line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in
the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function
of field, and for DsbA, resolution in the C–C region increases by 42%, according to the number of peaks that can be uniquely
picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely
observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the
benefits of high-field MAS SSNMR spectroscopy for protein structural studies. 相似文献
20.
Shibasaki S Kuroda K Duc Nguyen H Mori T Zou W Ueda M 《Applied microbiology and biotechnology》2006,70(4):451-457
A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field
of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma
membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability
to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein
(ECFP)–protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop
it for use as a detection system for protein–protein interactions, the Fc fragment of IgG (Fc) was genetically fused with
the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP–protein A-anchored cell. A microscopic
analysis showed that fluorescence resonance energy transfer (FRET) between ECFP–protein A and EYFP–Fc occurred, and the change
in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein–protein interactions
at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides
a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such
as the two-hybrid method in the nuclei.
S. Shibasaki and K. Kuroda equally contributed to this work 相似文献