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171.
Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca2+ homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn2+ or Ca2+ with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses. 相似文献
172.
Variations in the structure of d(GGGA)(5) oligonucleotide in the presence of Li(+), Na(+), and K(+) ions and its temperature stability were studied using electronic and vibrational circular dichroism, IR absorption, and ab initio calculations with the Becke 3-Lee-Yang-Parr functional at the 6-31G** level. The samples were characterized by nondenaturing gel electrophoresis. Oligonucleotide d(GGGA)(5) in the presence of Li(+) forms a nonplanar single tetramer, with angles of 102 degrees and 171 degrees between neighboring guanine bases. This tetramer changes its geometry at temperatures >50 degrees C, but does not form a quadruplex structure. In the presence of Na(+), the d(GGGA)(5) structure was optimized to almost planar tetramers with an angle of 177 degrees between neighboring guanines. The spectral results suggest that it stacks into a quadruplex helical structure. This quadruplex structure decayed to a single tetramer at temperatures >60 degrees C. The Hartree-Fock energies imply that d(GGGA)(5) prefers to form complexes with Na(+) rather than Li(+). The d(GGGA)(5) structure in the presence of monovalent ions is stabilized against thermal denaturation in the order Li(+) < Na(+) < K(+). 相似文献
173.
Group II introns are ribozymes occurring in genes of plants, fungi, lower eukaryotes, and bacteria. These large RNA molecular machines, ranging in length from 400 to 2500 nucleotides, are able to catalyze their own excision from pre-mRNA, as well as to reinsert themselves into RNA or sometimes even DNA. The intronic domain 1 contains two sequences (exon binding sites 1 and 2, EBS1 and EBS2) that pair with their complementary regions at the 3′-end of the 5′-exon (intron binding sites 1 and 2, IBS1 and IBS2) such defining the 5′-splice site. The correct recognition of the 5′-splice site stands at the beginning of the two steps of splicing and is thus crucial for catalysis. It is known that metal ions play an important role in folding and catalysis of ribozymes in general. Here, we characterize the specific metal ion requirements for the formation of the 5′-splice site recognition complex from the mitochondrial yeast group II intron Sc.ai5γ. Circular dichroism studies reveal that the formation of the EBS1 · IBS1 duplex does not necessarily require divalent metal ions, as large amounts of monovalent metal ions also promote the duplex, albeit at a 5000 times higher concentration. Nevertheless, micromolar amounts of divalent metal ions, e.g. Mg2+ or Cd2+, strongly promote the formation of the 5′-splice site. These observations illustrate that a high charge density independent of the nature of the ion is needed for binding EBS1 to IBS1, but divalent metal ions are presumably the better players. 相似文献
174.
Porschke D 《Journal of molecular biology》2003,331(3):667-679
Structure changes of purple membranes during the photocycle were analysed in solution by measurements of the electric dichroism. The D96N-mutant was used to characterize the M-state at neutral pH. The transition from the resting state to 61% photo-stationary M-state is associated with a strong reduction of the dichroism decay time constant by a factor of approximately 2. Because the change of the time constant is independent of the bacteriorhodopsin concentration, the effect is not attributed to light-induced dissociation but to light-induced bending of purple membranes. After termination of light-activation the dichroism decay of the resting state is restored with a time constant close to that of the M-state decay, which is more than two orders of magnitude slower than proton transfer to the bulk. Thus, bending is not due to asymmetric protonation but to the structure of the M-state. A very similar reduction of decay time constants at a corresponding degree of light-activation was found for wild-type bacteriorhodopsin at pH-values 7.8-9.3, where the lifetime of the M-state is extended. Light-induced bending is also reflected in changes of the stationary dichroism, whereas the overall permanent dipole moment remains almost constant, suggesting compensation of changes in molecular and global contributions. Bead model simulations indicate that disks of approximately 1 microm diameter are bent at a degree of photo-activation of 61% to a radius of approximately 0.25 microm, assuming a cylindrical bending modus. The large light-induced bending effect is consistent with light-induced opening of the protein on the cytoplasmic side of the membrane detected by electron crystallography, which is amplified due to coupling of monomers in the membrane. Bending may function as a mechanical signal. 相似文献
175.
We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m–2 s–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the QY band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl
bacteriochlorophyll
- CD
circular dichroism
- DAS
decay associated spectrum
- PMSF
phenylmethylsulfonyl fluoride 相似文献
176.
Gregory Genta‐Jouve Lionel Weinberg Vincent Cocandeau Yannick Maestro Olivier Paul Thomas Serge Holderith 《Chirality》2013,25(3):180-184
Coatline A ( 1 ) and α‐epi‐coatline A ( 4 ) co‐occur in the trunk extract of Andira coriacea. Inspection of their chiroptical properties led to intriguing results. After a careful examination of the experimental data used for the previously reported absolute configuration of these compounds, some uncertainties were identified. A combined theoretical approach including conformational analyses and calculation of electronic circular dichroism (ECD) spectra, in addition with experimental data obtained for schoepfin A ( 5 ) and the new schoepfin D ( 6 ) isolated from Senna quinquangulata, allowed the revision of the absolute configuration of coatlines A ( 1 ) and B ( 2 ). Chirality 25:180–184, 2013. © 2012 Wiley Periodicals, Inc. 相似文献
177.
The influence of lignin, lignin model compounds, and black liquor from the kraft pulping process on the hydrolysis of xylan by xylanase was investigated. Addition of vanillic acid, acetovanillone, and protocatechuic acid increased the rate of hydrolysis of xylan by as much as 18–50% at low concentrations, but reached maxima at about 0.05% concentration. Addition of vanillin caused a 15% improvement in xylan hydrolysis, while addition of guaiacol more than doubled the hydrolysis rate. Increasing concentrations of either lignin or black liquor also increased the hydrolysis rate of xylan. Circular dichroism spectroscopy indicated a change in the structure of xylanase in the presence of black liquor. 相似文献
178.
Saboury AA Atri MS Sanati MH Moosavi-Movahedi AA Haghbeen K 《International journal of biological macromolecules》2005,36(5):305-309
Thermodynamic analysis of calcium ions binding to human growth hormone (hGH) was done at 27 °C in NaCl solution, 50 mM, using different techniques. The binding isotherm for hGH-Ca2+ was obtained by two techniques of ionmetry, using a Ca2+-selective membrane electrode, and isothermal titration calorimetry. Results obtained by two ionmetric and calorimetric methods are in good agreement. There is a set of three identical and non-interacting binding sites for calcium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 52 μM and −17.4 kJ/mol, respectively. Temperature scanning UV–vis spectroscopy was applied to elucidate the effect of Ca2+ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Calcium ions binding increase the protein thermal stability by increasing of the alpha helix content as well as decreasing of both beta and random coil structures. 相似文献
179.
Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state 2H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific 2H labels have been introduced into the methyl groups of retinal and solid-state 2H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent 2H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu122 and Trp265 within the binding pocket. The solid-state 2H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism. 相似文献
180.
We report on the reversible association of anionic liposomes induced by an antimicrobial peptide (LAH4). The process has been characterized for mixed membranes of POPC and POPS at molar ratios of 1:1, 3:1, and 9:1. Although the vesicles remain in suspension in the presence of excess amounts of peptide, the addition of more lipids results in surface charge neutralization, aggregation of the liposomes, and formation of micrometer-sized structures that coexist in equilibrium with vesicles in suspension. At low ratios of anionic lipids, vesicle aggregation is a reversible process, and vesicle disassembly is observed upon inversion of the surface charge by further supplementation with anionic vesicles. In contrast, a different process, membrane fusion, occurs in the presence of high phosphatidylserine concentrations. Upon binding to membranes containing low POPS concentrations, the peptide adopts an in-plane α-helical structure, a secondary structure that is conserved during vesicle association and dissociation. Our finding that peptides are essential for vesicle aggregation contributes to a better understanding of the activity of antimicrobial peptides, and suggests an additional layer of complexity in membrane-protein lipid interactions. 相似文献