共查询到20条相似文献,搜索用时 296 毫秒
1.
Gath J Habenstein B Bousset L Melki R Meier BH Böckmann A 《Biomolecular NMR assignments》2012,6(1):51-55
Parkinson’s disease is amongst the most frequent and most devastating neurodegenerative diseases. It is tightly associated
with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous
system. The principal protein involved in this process is α-synuclein which is a structural component of the Lewy bodies observed
in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this protein, the
first one with a well-ordered and rigid N-terminal part. 相似文献
2.
NMR structures of ζ-subunits, which are recently discovered α-proteobacterial F1F0-ATPase-regulatory proteins representing a Pfam protein family of 246 sequences from 219 species (PF07345), exhibit a four-helix bundle, which is different from all other known F1F0-ATPase inhibitors. Chemical shift mapping reveals a conserved ADP/ATP binding site in ζ-subunit, which mediates long-range conformational changes related to function, as revealed by the structure of the Paracoccus denitrificans ζ-subunit in complex with ADP. These structural data suggest a new mechanism of F1F0-ATPase regulation in α-proteobacteria. 相似文献
3.
Croke RL Patil SM Quevreaux J Kendall DA Alexandrescu AT 《Protein science : a publication of the Protein Society》2011,20(2):256-269
The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding. 相似文献
4.
Wang C 《Protein and peptide letters》2011,18(4):354-361
Aβ is widely recognized as a key molecule in Alzheimer's disease, causing neurotoxicity through Aβ aggregates such as Aβ oligomers and fibrils. Aβ40 and Aβ42, composed of 40 and 42 residues, respectively, are the major Aβ species in human brain. Aβ42 aggregates much faster than Aβ40 but the mechanism of such difference in aggregation propensity is poorly understood. Using NMR spin relaxation, we have shown that Aβ40 and Aβ42 monomers have different dynamics in both backbone and sidechain on the ps-ns time scale. Aβ42 is more rigid in C-terminus in both backbone and sidechain while Aβ40 has more rigid methyl groups in the central hydrophobic cluster (CHC: Aβ17-21). These observations are consistent with differences in the major conformations of Aβ40 and Aβ42 monomers derived from replica exchange MD (REMD). To further demonstrate the relevance of dynamics in aggregation mechanism, a perturbation was introduced to Aβ42 in the form of M35 oxidation. After M35 side chain oxidation to sulfoxide, Aβ42 experiences Aβ40-like changes in dynamics. At the same time, M35 oxidation causes dramatic reduction in Aβ42 aggregation rate. These data have thus established an important role for protein dynamics in the mechanism of Aβ aggregation. 相似文献
5.
Gui-Fang Wang Conggang Li Gary J Pielak 《Protein science : a publication of the Protein Society》2010,19(9):1686-1691
α‐Synuclein function is thought to be related to its membrane binding ability. Solution NMR studies have identified several α‐synuclein‐membrane interaction modes in small unilamellar vesicles (SUVs), but how membrane properties affect binding remains unclear. Here, we use 19F NMR to study α‐synuclein‐membrane interactions by using 3‐fluoro‐L‐tyrosine (3FY) and trifluoromethyl‐L‐phenylalanine (tfmF) labeled proteins. Our results indicate that the affinity is affected by both the head group and the acyl chain of the SUV. Negatively charged head groups have higher affinity, but different head groups with the same charge also affect binding. We show that the saturation of the acyl chain has a dramatic effect on the α‐synuclein‐membrane interactions by studying lipids with the same head group but different chains. Taken together, the data show that α‐synuclein's N‐terminal region is the most important determinate of SUV binding, but its C‐terminal region also modulates the interactions. Our data support the existence of multiple tight phospholipid‐binding modes, a result incompatible with the model that α‐synuclein lies solely on the membrane surface. 相似文献
6.
Gordon C. K. Roberts 《Neurochemical research》1996,21(9):1117-1124
NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The
kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in
drug metabolism. Cytochromes P450 play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the
haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate
complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance
to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme
A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined,
providing valuable information on the catalytic mechanism.
Special issue dedicated to Dr. Herman Bachelard. 相似文献
7.
Kloepper KD Zhou DH Li Y Winter KA George JM Rienstra CM 《Journal of biomolecular NMR》2007,39(3):197-211
The protein α-synuclein (AS) is the primary fibrillar component of Lewy bodies, the pathological hallmark of Parkinson’s disease.
Wild-type human AS and the three mutant forms linked to Parkinson’s disease (A53T, A30P, and E46K) all form fibrils through
a nucleation-dependent pathway; however, the biophysical details of these fibrillation events are not yet well understood.
Atomic-level structural insight is required in order to elucidate the potential role of AS fibrils in Parkinson’s disease.
Here we show that low temperature acquisition of magic-angle spinning NMR spectra of wild type AS fibrils-greatly enhances
spectral sensitivity, enabling the detection of a substantially larger number of spin systems. At 0 ± 3°C sample temperature,
cross polarization (CP) experiments yield weak signals. Lower temperature spectra (−40 ± 3°C) demonstrated several times greater
signal intensity, an effect further amplified in 3D 15N–13C–13C experiments, which are required to perform backbone assignments on this sample. Thus 3D experiments enabled assignments
of most amino acids in the rigid part of the fibril (approximately residues 64 to 94), as well as tentative site-specific
assignments for T22, V26, A27, Y39, G41, S42, H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were
not observed in 2D or 3D spectra at 0 ± 3°C. Spectra acquired at low temperatures therefore permitted more complete chemical
shift assignments. Observation of the majority of residues in AS fibrils represents an important step towards solving the
3D structure. 相似文献
8.
NMR spectroscopy has established itself as one of the main techniques for the structural study of integral membrane proteins. Remarkably, over the last few years, substantial progress has been achieved in the structure determination of increasingly complex polytopical α-helical membrane proteins, with their size approaching ~100kDa. Such advances are the result of significant improvements in NMR methodology, sample preparation and powerful selective isotope labelling schemes. We review the requirements facilitating such work based on the more recent solution NMR studies of α-helical proteins. While the majority of such studies still use detergent-solubilized proteins, alternative more native-like lipid-based media are emerging. Recent interaction, dynamics and conformational studies are discussed that cast a promising light on the future role of NMR in this important and exciting area. 相似文献
9.
Nuclear magnetic resonance (NMR) spectroscopy is the only experimental technique that can determine the structures and dynamics of biological macromolecules and their complexes in solution and with atomic resolution. The award of the 2002 Nobel Prize in Chemistry to Kurt Wüthrich of the Swiss Federal Institute of Technology and The Scripps Research Institute honors his pioneering efforts in developing and applying this technique. Wüthrich shared the prize with John B. Fenn and Koichi Tanaka, who were recognized for the development of ionization methods for the analysis of proteins using mass spectrometry. 相似文献
10.
Dynamics of amyloid β fibrils revealed by solid-state NMR 总被引:1,自引:0,他引:1
Scheidt HA Morgado I Rothemund S Huster D 《The Journal of biological chemistry》2012,287(3):2017-2021
We have investigated the site-specific backbone dynamics of mature amyloid β (Aβ) fibrils using solid-state NMR spectroscopy. Overall, the known β-sheet segments and the turn linking these two β-strands exhibit high order parameters between 0.8 and 0.95, suggesting low conformational flexibility. The first approximately eight N-terminal and the last C-terminal residues exhibit lower order parameters between ~0.4 and 0.8. Interestingly, the order parameters increase again for the first two residues, Asp(1) and Ala(2), suggesting that the N terminus could carry some structural importance. 相似文献
11.
Bondarenko V Mowrey D Tillman T Cui T Liu LT Xu Y Tang P 《Biochimica et biophysica acta》2012,1818(5):1261-1268
The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators. 相似文献
12.
Laurent J. Catoire Manuela Zoonens Carine van Heijenoort Fabrice Giusti Éric Guittet Jean-Luc Popot 《European biophysics journal : EBJ》2010,39(4):623-630
The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established
by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane
β-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution
solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine.
Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used,
some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the
dynamics of the barrel. 相似文献
13.
Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts. 相似文献
14.
15.
A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative 13C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic
acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped
with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH)
were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement
in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages.
The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds.
Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl
glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl
ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower
amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood. 相似文献
16.
This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the
University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are
mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and
proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with
human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other
eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly
suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of
low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and
their dynamic properties during protein recognition. Several examples are given in this paper. 相似文献
17.
18.
Detection of protein–ligand interactions by NMR using reductive methylation of lysine residues 总被引:1,自引:1,他引:0
We show that reductive methylation of proteins can be used for highly sensitive NMR identification of conformational changes induced by metal- and small molecule binding, as well as protein-protein interactions. Reductive methylation of proteins introduces two (13)C-methyl groups on each lysine in the protein of interest. This method works well even when the lysines are not actively involved in the interaction, due to changes in the microenvironments of lysine residues. Most lysine residues are located on the protein exterior, and the exposed (13)C-methyl groups may exhibit rapid localized motions. These motions could be faster than the tumbling rate of the molecule as a whole. Thus, this technique has great potential in the study of large molecular weight systems which are currently beyond the scope of conventional NMR methods. 相似文献
19.
Simon P. Skinner Mois Moshev Mathias A. S. Hass Marcellus Ubbink 《Journal of biomolecular NMR》2013,55(4):379-389
The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure. 相似文献
20.
The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin
family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes,
and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as
a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold
and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear
magnetic resonance (NMR) studies of p7 in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that
are tilted approximately 10° and 25° relative to the bilayer normal. A truncated construct corresponding to the second transmembrane
domain of p7 is shown to have properties similar to those of the full-length protein, and was used to determine that the helix
segment tilted at 10° is in the C-terminal portion of the protein. The addition of the channel blocker amantadine to the full-length
protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs
targeted to p7. 相似文献