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1.
For base-paired nucleic acids, variations in 1
J
NH and the imino 1H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the 1
J
NH coupling for the imino proton then can be approximated by 1
J
NH = (1.21Hz/ppm)δH − 103.5 ± 0.6 Hz, where δH represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting
from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (1
J
NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNAVal and the alignment tensor determined from magnetic-field alignment of tRNAVal agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work. 相似文献
2.
A procedure is presented for refinement of a homology model of E. coli tRNAVal, originally based on the X-ray structure of yeast tRNAPhe, using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm
is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important
for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its
application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data
consisting of 24 imino N–HN RDCs measured with Pf1 phage alignment, and 20 imino N–HN RDCs obtained from magnetic field dependent alignment of tRNAVal. The refinement strategy aims to largely retain the local geometry of the 58% identical tRNAPhe by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved
base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic
symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments.
The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular
shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed
Q
free = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms
than observed in the X-ray structure of tRNAPhe, in agreement with previous NMR-based tRNAVal models.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
Residual dipolar couplings (RDCs) complement standard NOE distance and J-coupling torsion angle data to improve the local
and global structure of biomolecules in solution. One powerful application of RDCs is for domain orientation studies, which
are especially valuable for structural studies of nucleic acids, where the local structure of a double helix is readily modeled
and the orientations of the helical domains can then be determined from RDC data. However, RDCs obtained from only one alignment
media generally result in degenerate solutions for the orientation of multiple domains. In protein systems, different alignment
media are typically used to eliminate this orientational degeneracy, where the combination of RDCs from two (or more) independent
alignment tensors can be used to overcome this degeneracy. It is demonstrated here for native E. coli tRNAVal that many of the commonly used liquid crystalline alignment media result in very similar alignment tensors, which do not
eliminate the 4-fold degeneracy for orienting the two helical domains in tRNA. The intrinsic magnetic susceptibility anisotropy
(MSA) of the nucleobases in tRNAVal was also used to obtain RDCs for magnetic alignment at 800 and 900 MHz. While these RDCs yield a different alignment tensor,
the specific orientation of this tensor combined with the high rhombicity for the tensors in the liquid crystalline media
only eliminates two of the four degenerate orientations for tRNAVal. Simulations are used to show that, in optimal cases, the combination of RDCs obtained from liquid crystalline medium and
MSA-induced alignment can be used to obtain a unique orientation for the two helical domains in tRNAVal.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
5.
Lia Millucci Roberto Raggiaschi Davide Franceschini Georg Terstappen Annalisa Santucci 《Journal of biosciences》2009,34(2):293-303
The highly toxic Aβ(25–35) is a peculiar peptide that differs from all the other commonly studied β-amyloid peptides because of its extremely rapid aggregation properties and enhanced neurotoxicity. We investigated Aβ(25–35) aggregation in H2O at pH 3.0 and at pH 7.4 by means of in-solution analyses. Adopting UV spectroscopy, Congo red spectrophotometry and thioflavin
T fluorimetry, we were able to quantify, in water, the very fast assembling time necessary for Aβ(25–35) to form stable insoluble aggregates and their ability to seed or not seed fibril growth. Our quantitative results,
which confirm a very rapid assembly leading to stable insoluble aggregates of Aβ(25–35) only when incubated at pH 7.4, might be helpful for designing novel aggregation inhibitors and to shed light on the
in vivo environment in which fibril formation takes place. 相似文献
6.
The PsbH protein of cyanobacterium Synechocystis sp. PCC 6803 was expressed as a fusion protein with glutathione-S transferase (GST) in E. coli grown on a mineral medium enriched in 15N isotope. After enzymatic cleavage of the fusion protein, the 1H-15N-HSQC spectrum of PsbH protein in presence of the detergent β-D-octyl-glucopyranoside (OG) was recorded on a Bruker DRX 500 MHz NMR spectrometer equipped with a 5 mm TXI cryoprobe to enhance the sensitivity and resolution. Non-labelled protein
was used for secondary structure estimation by deconvolution from circular dichroism (CD) spectra. Experimental results were
compared with our results from a structural model of PsbH using a restraint-based comparative modelling approach combined
with molecular dynamics and energetic modelling. We found that PsbH shows 34–38% α-helical structure (Thr36-Ser60), a maximum
of around 15% of β-sheet, and 12–19% of β-turn. 相似文献
7.
The access to weak alignment media has fuelled the development of methods for efficiently and accurately measuring residual
dipolar couplings (RDCs) in NMR-spectroscopy. Among the wealth of approaches for determining one-bond scalar and RDC constants
only J-modulated and J-evolved techniques retain maximum resolution in the presence of differential relaxation. In this article, a number of J-evolved experiments are examined with respect to the achievable minimum linewidth in the J-dimension, using the peptide PA4 and the 80-amino-acid-protein Saposin C as model systems. With the JE-N-BIRD
d,X
-HSQC experiment, the average full-width at half height could be reduced to approximately 5 Hz for the protein, which allows
the additional resolution of otherwise unresolved peaks by the active (J+D)-coupling. Since RDCs generally can be scaled by the choice of alignment medium and alignment strength, the technique introduced
here provides an effective resort in cases when chemical shift differences alone are insufficient for discriminating signals.
In favorable cases even secondary structure elements can be distinguished. 相似文献
8.
Correlations between GABAA receptor (GABAA-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho)
extraction with β-cyclodextrin (β-CD). The mere pre-incubation (PI) at 37°C accompanying the β-CD treatment was an underlying
source of perturbations increasing [3H]-FNZ maximal binding (70%) and K
d (38%), plus a stiffening of SMs’ hydrocarbon core region. The latter was inferred from an increased compressibility modulus
(K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy
(A
DPH) in SMs submitted to a heating–cooling cycle (4–37–4°C) with A
DPH,heating < A
DPH,cooling. Compared with PI samples, the β-CD treatment reduced B
max by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased
A
TMA-DPH. PI, but not β-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with β-CD-, SM-partitioned,
and free in solution showed that, contrary to what is usually assumed, β-CD is not completely eliminated from the system through
centrifugation washings. It was concluded that β-CD treatment involves effects of at least three different types of events
affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual β-CD on SM organization, and
(c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head
groups, leading to a net increase in GABAA-R density, relative to untreated samples. 相似文献
9.
Kumar GR Sharma A Kumari M Jagannadham MV Debnath M 《European biophysics journal : EBJ》2011,40(8):923-935
Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH
using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions,
pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS
binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the
denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that
the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral
conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding
of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein,
suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition
from one molten globule like state (AMG1) into another (IMG2). 相似文献
10.
Rinaldo Montalvao Carlo Camilloni Alfonso De Simone Michele Vendruscolo 《Journal of biomolecular NMR》2014,58(4):233-238
Residual dipolar couplings (RDCs) can provide exquisitely detailed information about the structure and dynamics of proteins. It is challenging, however, to extract such information from RDC measurements in conformationally heterogeneous states of proteins because of the complex relationship between RDCs and protein structures. To obtain new insights into this problem, we discuss methods of calculating the RDCs that do not require the definition of an alignment tensor. These methods can help in particular in the search of effective ways to use RDCs to characterise disordered or partially disordered states of proteins. 相似文献
11.
Eletsky A Acton TB Xiao R Everett JK Montelione GT Szyperski T 《Journal of structural and functional genomics》2012,13(1):9-14
The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350
members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41–180
of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35–182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/β fold comprised of a four-stranded β-sheet, three α-helices packed against one side of the sheet, and
a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence
alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative
orientation of two of the four helices. Comparison with known protein structures reveals that the α/β architecture of CG2496(41–180)
and PG0361(35–182) has previously not been characterized. Moreover, calculation of surface charge potential and identification
of surface clefts indicate that the two domains very likely have different functions. 相似文献
12.
The individual components of the backbone 15N CSA tensor, σ11, σ22, σ33, and the orientation of σ11 relative to the NH bond described by the angle β have been determined for uniformly labeled 15N, 13C ubiquitin from partial alignment in phospholipid bicelles, Pf1 phage, and poly(ethylene glycol) by measuring the residue-specific residual dipolar couplings and chemical shift deviations. No strong correlation between any of the CSA tensor components is observed with any single structural feature. However, the experimentally determined tensor components agree with the previously determined average CSA principal components [Cornilescu and Bax (2000) J. Am. Chem. Soc. 122, 10143–10154]. Significant deviations from the averages coincide with residues in β-strand or extended regions, while α-helical residue tensor components cluster close to the average values.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . 相似文献
13.
Debasish Haldar Arindam Banerjee 《International journal of peptide research and therapeutics》2007,13(3):439-446
The terminally protected peptide Boc-Leu-Val-Phe-Phe-Ala-OMe bearing sequence similarity with the central hydrophobic cluster
(CHC) of Alzheimer’s Aβ17–21 peptide self-assembles to produce amyloid-like straight unbranched fibrils from organic solvents. The fibrils readily bind
with a physiological dye Congo red (CR) and exhibits green gold birefringence under polarized light, a characteristic feature
of amyloid plaque obtained from many neurodegenerative diseases. FTIR spectroscopy and in silico energy minimization study shed some light on the antiparallel supramolecular β-sheet aggregation of the peptide. 相似文献
14.
García-Hidalgo J Hormigo D Prieto MA Arroyo M de la Mata I 《Applied microbiology and biotechnology》2012,93(5):1975-1988
The phaZ
Sex
gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and
purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer.
Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2%
random coil, with a midpoint transition temperature (T
m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased
the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence
of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass
spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating
that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well
as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable
at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of
(R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics. 相似文献
15.
Yu Qiu Hai Niu Wen Huang Yang He Xiao-Hua Wu 《Biotechnology and Bioprocess Engineering》2011,16(5):858-866
A tannase with a molecular mass of 72 kDa was obtained from Penicillium herquei isolated from valonia acorns following fermentation in a 5 L bioreactor. This tannase showed optimum activity at pH 6.0 and
30°C. The enzyme was inhibited by Fe3+, Zn2+, dithiothrietol (DTT), β-mercaptoethanol, formaldehyde, and ethanol, and induced by K+, Mn2+, Tween 80, and Triton X-100. The Michaelis constant (K
m) and the second-order constant (k
cat/K
m) values of the tannase for propyl gallate (PG) were 0.62 mM and 174.1 mM/sec. The circular dichroism (CD) spectra indicated
that the secondary structure of the tannase contained 14% α helix, 32.4% anti-parallel β-sheet, 4.8% β-sheet, 18.8% β-turn,
and 30% random coil. Native tannase in ultrapure water manifested as spherical nano-particle aggregates with diameters ranging
from 50 to 300 nm determined by atomic force microscopy (AFM). 相似文献
16.
The presence of dipole-dipole cross-correlated relaxation as well as unresolved E.COSY effects adversely impacts the accuracy
of 1
J
NH splittings measured from gradient-enhanced IPAP-HSQC spectra. For isotropic samples, the size of the systematic errors caused
by these effects depends on the values of 2
J
NHα
, 3
J
NHβ
and 3
J
HNHα
. Insertion of band-selective 1H decoupling pulses in the IPAP-HSQC experiment eliminates these systematic errors and for the protein GB3 yields 1
J
NH splittings that agree to within a root-mean-square difference of 0.04 Hz with values measured for perdeuterated GB3. Accuracy
of the method is also highlighted by a good fit to the GB3 structure of the 1H-15N RDCs extracted from the minute differences in 1JNH splitting measured at 500 and 750 MHz 1H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of 2
J
NHα
couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the
15N–1H
α
or 15N–1H
β
dipolar interaction. As expected, we find that 2
J
NHα
≤ 2 Hz, with values in the α-helix (0.86 ± 0.52 Hz) slightly larger than in β-sheet (0.66 ± 0.26 Hz). Results indicate that under isotropic conditions, N–HN/N–H
β
cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve 3
J
HNHα
and J
NHα
, but when weakly aligned any aliphatic proton proximate to both N and HN can contribute.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
Guanghua Gao Jixun Dai Ming Ding Göran Hellekant Jinfeng Wang Dacheng Wang 《中国科学C辑(英文版)》1999,42(4):409-419
Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one α-helix (residues 21–29), one short 310-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus. A comparative analysis found that brazzein shares a so-called ‘cysteine-stabilized alpha-beta’ (CSαβ) motif with scorpion neurotoxins, insect defensins and plant γ - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed. 相似文献
18.
Marc Quinternet Chrystel Beaufils Fabrice Neiers Pascale Tsan Sandrine Boschi-Muller Marie-Christine Averlant-Petit Guy Branlant Manh-Thong Cung 《Biomolecular NMR assignments》2007,1(1):143-145
We report the nearly complete 1H, 13C and 15N resonance assignments of the oxidized form (Cys67–Cys70) of the N-terminal domain of PilB from Neisseria meningitidis. Secondary structure determination using CSI method and TALOS leads mainly to the prediction of 7 α-helical and 5 β-sheet
parts. 相似文献
19.
Abolghasem Abbasi Kejani Sayed Ali Hosseini Tafreshi Sayed Mojtaba Khayyam Nekouei Mohammad Reza Mofid 《Molecular biology reports》2010,37(2):797-800
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew
(Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation
were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform
extraction steps from the isolation procedure. Both spectrophotometric (A260/A280 and A260/A230 ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the
average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g
leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively. 相似文献
20.
C. P. J. Maury 《Origins of life and evolution of the biosphere》2009,39(2):141-150
The idea is advanced that under the extreme earth conditions for ~3.9 billions years ago, protein-based β-sheet molecular structures were the first self-propagating and information-processing biomolecules that evolved. The amyloid
structure of these aggregates provided an effective protection against the harsh conditions known to decompose both polyribonucleotides
and natively folded polypeptides. In the prebiotic amyloid world, both the replicative and informational functions were carried
out by structurally stable β-sheet protein aggregates in a prion-like mode involving templated self-propagation and storage of information in the β-sheet conformation. In this amyloid (protein)-first, hybrid replication-metabolism view, the synthesis of RNA, and the evolvement
of an RNA-protein world, were later, but necessary events for further biomolecular evolution to occur. I further argue that
in our contemporary DNA↔RNA→protein world, the primordial β-conformation-based information system is preserved in the form of a cytoplasmic epigenetic memory. 相似文献