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1.
The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (Delta DeltaG(o)) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex. 相似文献
2.
Kamata T Handa M Ito S Sato Y Ohtani T Kawai Y Ikeda Y Aiso S 《The Journal of biological chemistry》2010,285(49):38428-38437
Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs. 相似文献
3.
There is considerable evidence that the mechanism of glucose transport by the transporter of human erythrocytes is one in which the transporter oscillates between two conformations, To and Ti. Each conformer possesses a single glucose binding site that in vivo faces either the extracellular space (conformer To) or the cytoplasm (conformer Ti). In this study, the interconversions of these conformers in the absence and presence of D-glucose have been directly observed by means of the stopped-flow method with fluorescence detection. Nearly unidirectional conversion of one conformer to the other was accomplished by rapidly mixing purified transporter (a mixture of To and Ti) with either 4,6-ethylidene-D-glucose, which preferentially binds to To, or phenyl beta-D-glucoside, which preferentially binds to Ti. The values of the individual rate constants for the conversion of Ti to To and vice versa in the absence and presence of D-glucose at 10.0 degrees C have been obtained, and these show that the kinetics are consistent with the alternating conformation model for transport. Conformational change occurs much more rapidly with glucose bound to the transporter. Furthermore, the activation energy Ea for conformer interconversion is much less when glucose is bound than for unliganded transporter. For example, Ea is approximately 28 kcal/mol for Ti----To versus 17 kcal/mol for Ti + S----ToS, where S is glucose. The alpha-anomer of glucose was 37% more effective than the beta-anomer in speeding the interconversion.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
The conformational heterogeneity of several quinoxaline antibiotics, a class of naturally occurring quinoxaline peptides with antitumor properties, and their synthetic analogues was investigated in polar and nonpolar solvents by high performance liquid chromatography (HPLC) with uv photodiode array detection, uv-absorbance, low-temperature phosphorescence, and nmr techniques. Multiple peak formation and interconversion in the HPLC and 1H-nmr analysis of triostin A, its under-N-methylated synthetic analogues (des-N-tetramethyltriostin A [TANDEM] and [N-MeCys3, N-MeCys7]-TANDEM [MCTAN-DEM]), and echinomycin were examined as a function of temperature, solvent polarity, and residence time in solution prior to analysis. Slow interconversion between HPLC peaks, ascribed to the presence of multiple solution conformers, was exhibited by these peptides although at very different interconversion rates. Among the triostins, the rate of interconversion appeared to vary with the degree of N-methylation of the residues in the cyclic depsipeptide chain. Interconversion of the n and p conformers of triostin A in chloroform occurred on a chromatographic timescale (a few minutes with kn----p calculated to be 0.02 s-1 at 25 degrees C) while the solution conformers of TANDEM in methanol equilibrated very slowly to one preferred conformer over a period of several weeks at ambient temperature. MCTANDEM, a synthetic analogue of triostin A with an intermediate degree of N-methylation of the residues in the peptide ring, consisted of an equilibrium mixture of n and p conformers in methanol that interconverted on a chromatographic time scale. Two additional conformers of MCTANDEM developed within a few weeks' residence time in methanol at ambient temperature. Echinomycin was found to exist in methanol as an interconverting mixture of at least four minor conformers in addition to the major isoform (95% by peak area) of the peptide. The solution conformers of the quinoxaline peptides investigated in this report are most likely a consequence of hindered rotation about the N-methylated peptide bonds in the depsipeptide ring and/or intramolecular hydrogen bonding. 相似文献
5.
The decay of Trp phosphorescence of proteins in fluid solutions was shown to provide a sensitive tool for probing the conformational homogeneity of these macromolecules in the millisecond to second time scale. Upon examination of 15 single Trp emitting proteins multiexponential decays were observed in 12 cases, a demonstration that the presence of slowly interconverting conformers in solution is more the norm rather than an exception. The amplitude of preexponential terms, from which the conformer equilibrium is derived, was found to be a sensitive function of solvent composition (buffer, pH, ionic strength and glycerol cosolvent), temperature, and complex formation with substrates and cofactors. In many cases, raising the temperature, a point is reached at which the decay becomes practically monoexponential, meaning that conformer interconversion rates have become commensurate with the triplet lifetime. Estimation of activation free energy barriers to interconversion shows that the large values of DeltaG* are rather similar among polypeptides and that the protein substates involved are sufficiently long-lived to display individual binding/catalytic properties. 相似文献
6.
Helical junctions are common architectural features in RNA. They are particularly important in autonomously folding molecules, as exemplified by the hairpin ribozyme. We have used single-molecule fluorescence spectroscopy to study the dynamic properties of the perfect (4H) four-way helical junction derived from the hairpin ribozyme. In the presence of Mg(2+), the junction samples parallel and antiparallel conformations and both stacking conformers, with a bias towards one antiparallel stacking conformer. There is continual interconversion between the forms, such that there are several transitions per second under physiological conditions. Our data suggest that interconversion proceeds via an open intermediate with reduced cation binding in which coaxial stacking between helices is disrupted. The rate of interconversion becomes slower at higher Mg(2+) concentrations, yet the activation barrier decreases under these conditions, indicating that entropic effects are important. Transitions also occur in the presence of Na(+) only; however, the coaxial stacking appears incomplete under these conditions. The polymorphic and dynamic character of the four-way RNA junction provides a source of structural diversity, from which particular conformations required for biological function might be stabilised by additional RNA interactions or protein binding. 相似文献
7.
Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into flavonoids. The activity of CHI is essential for the biosynthesis of flavonoids precursors of floral pigments and phenylpropanoid plant defense compounds. In the present study, we explored the detailed binding structures and binding free energies for two different active site conformations of CHI with s-cis/s-trans conformers of three chalcone compounds by performing molecular dynamics (MD) simulations and binding free energy calculations. The computational results indicate that s-cis/s-trans conformers of chalcone compounds are orientated in the similar binding position in the active site of CHI and stabilized by the different first hydrogen bond network and the same second hydrogen bond network. The first hydrogen bond network results in much lower binding affinity of s-trans conformer of chalcone compound with CHI than that of s-cis conformer. The conformational change of the active site residue T48 from indirectly interacting with the substrate via the second hydrogen bond network to directly forming the hydrogen bond with the substrates cannot affect the binding mode of both conformers of chalcone compounds, but remarkably improves the binding affinity. These results show that CHI has a strong stereoselectivity. The calculated binding free energies for three chalcone compounds with CHI are consistent with the experimental activity data. In addition, several valuable insights are suggested for future rational design and discovery of high-efficiency mutants of CHI. Figure
Stereoselectivity of chalcone isomerase with chalcone derivatives 相似文献
8.
Infrared spectra of carbonyl hemoglobins: characterization of dynamic heme pocket conformers 总被引:3,自引:0,他引:3
The infrared spectra for carbon monoxide complexed to hemoglobins were examined in the C-O stretch region. Deconvolution of the spectra requires four bands and supports the presence of four distinct conformers at the ligand binding site. Most typical hemoglobins exhibit only one predominant conformer for each subunit represented by a band at 1951 cm-1 in contrast to myoglobins, which typically exist in two major conformations. Several hemoglobins with an enlarged heme pocket are shown to shift the C-O frequency into the higher frequency conformer regions. Many factors, including pH, temperature, solvents, and divalent metals, are also shown to be capable of expanding the heme pocket. Only very specific structural changes that can reduce the size of the heme pocket will result in the lower frequency conformers. The weighted averages of the multiple CO vibrational frequencies are linearly related to the single 13CO NMR chemical shift values and to the exponential of fast CO on-rates. Conformer interconversion occurs at a rate greater than 10(4) s-1. The infrared C-O stretch spectra provide qualitative and quantitative information on the structural dynamics, stability, and ligand binding properties of hemoglobins. 相似文献
9.
The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related
proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native
heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-α-L-idopyranosyluronic
acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers.
Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a 4C1 chair), the
idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type
and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature).
It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity.
In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in
iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1ρ measurements,
T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion
in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-peneta-O-acetyl-α-D-idopyranose
can be reasonably well described as existing in a two-state equilibrium consisting of the 4C1 and 0S2 conformers. 13C NMR
line shape analysis yields a ΔH+ of 40 kJ mol-1 and a ΔS‡ of 31 J mol-1 K-1 for the 4C1→0S2 interconversion and a ΔH‡ of 31
kJ mol-1 and a ΔS‡ of 13 J mol-1K-1 for the 0S2→4C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz,
respectively, at room temperature.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
10.
( ? )-Meptazinol is an analgesic with an additional acetylcholinesterase (AChE) inhibitory activity. In order to investigate the formation mechanism of its biological conformation observed in AChE-bis( ? )-meptazinol complex, two different and naturally stable conformers of ( ? )-meptazinol hydrochloride in solution were determined and identified by nuclear magnetic resonance (NMR) and molecular dynamic simulations. Moreover, ab initio calculations and NMR evidence showed the difficulties in conformer interconversion. In combination with the results of conformational comparison, it was proposed that the pharmacophoric conformer of ( ? )-meptazinol might come from the conformer with less favourable energy rather than the conformer with the lowest energy. 相似文献
11.
Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two codon-specific binding sites for transfer RNA. 总被引:3,自引:2,他引:1
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30S subunits were isolated capable to bind simultaneously two molecules of Phe-tRNAPhe (or N-Acetyl-Phe-tRNAPhe), both poly(U) dependent. The site with higher affinity to tRNA was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2 x 10(-5)M) and, on the other hand, N-Acetyl-Phe-tRNAPhe, initially prebound to the 30S.poly(U) complex in the presence of tetracycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNAPhe and N-Acetyl-Phe-tRNAPhe. Binding of two molecules of Phe-tRNAPhe to the 30S.poly(U) complex followed by the addition of 50S subunits resulted in the formation of (Phe)2-tRNAPhe in 75-90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formation of whole 70S ribosomes with usual donor and acceptor sites. 相似文献
12.
13.
A quinoxaline antibiotic triostin A has a bicyclic octadepsipeptide structure. Proton and carbon-13 nmr spectra showed the presence of two symmetrical conformations favoring polar and nonpolar solvents, respectively. They interconvert slowly on the nmr time scale, and this slow interconversion is due to the cooperative effects of the presence of the quinoxaline ring and the N-methyl peptide bonds. Reversal of the chirality of the disulfide bond as the origin of the slow exchange was excluded by the presence of two conformers for S-benzyltriostin A. Conformer 2, which favors the polar solvent, can form hydrogen-bonded complexes with purine nucleoside derivatives in organic solvents, but conformer 1 does not. The binding sites were elucidated and a mode of interaction with DNA proposed. 相似文献
14.
N Higuchi Y Kyogoku M Shin K Inouye 《International journal of peptide and protein research》1983,21(5):541-545
The n.m.r. pattern of triostin A in weakly polar solvents was explained by the presence of two symmetrical conformers. Its S-benzyl derivative still shows the n.m.r. pattern of two conformers, while des-N-tetramethyltriostin A, which lacks the N-methyl groups, gives one conformer. Therefore the slow interconversion of two conformers arises from the cis-trans isomerization of the N-methyl peptide bonds, but not from the reversal of the chirality around the S-S bond. Only one of the two conformers of triostin A interacts with adenosine and guanosine derivatives. Des-N-tetramethyltriostin A can also interact with the purine nucleosides, but more preferentially with the adenosine derivative. 相似文献
15.
Halothane, an inhaled anesthetic, destabilizes the folded structure of myoglobin. To determine whether this is due to preferential interactions with less stable folded conformers of myoglobin versus the completely unfolded state, we used photoaffinity labeling, hydrogen exchange, fluorescence spectroscopy, and circular dichroism spectroscopy. Apomyoglobin was used as a model of a less stable conformer of myoglobin. Halothane destabilizes myoglobin and binds with low affinity and stoichiometry but stabilizes and binds with higher affinity to apomyoglobin. The same halothane concentration has no effect on cytochrome c stability. The apomyoglobin/halothane complex is favored at pH 6.5 as compared to pH 4.5 or pH 2.5. Halothane photoincorporates into several sites in apomyoglobin, some allosteric to the heme pocket. Guanidinium unfolding of myoglobin, monitored by CD spectroscopy, shows destabilization at less than 1.3 M Gdm but stabilization at greater than 1.3 M Gdm, consistent with the hypothesis that less stable conformers of myoglobin bind halothane preferentially. We suggest the structural feature underlying preferential binding to less stable conformers is an enlarged cavity volume distribution, since myoglobin has several intermediate-sized cavities, while cytochrome c is more well packed and has no cavities detected by GRASP. Specific binding to less stable intermediates may underlie anesthetic potentiation of protein activity. 相似文献
16.
The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4). Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation. 相似文献
17.
Chelation of Fe from the Fe-protein component (Av2) of Azotobacter vinelandii nitrogenase has been investigated. The chelation, which requires MgATP binding by Av2, is best described as a two-exponential process. The rates for the two phases differed by approximately 10-fold and increased as the concentration of MgATP was increased. The rates for both phases were 50% of maximum at approximately 1.5 mM MgATP. At MgATP concentrations greater than 100 microM, the more rapid phase represented approximately 25% of the total Fe chelated from Av2. However, below 100 microM MgATP, the proportion of the faster phase decreased until at 20 microM MgATP, only a single phase could be detected. The properties of Av2 were studied at various stages of Fe chelation. The partially chelated protein was isolated from the reaction by gel filtration and was subjected to a second MgATP-dependent Fe chelation. Material isolated after the completion of the first phase regained biphasic kinetics in subsequent chelation reactions. However, if MgATP was present during the isolation of Av2, then only a single phase was observed in the subsequent chelation studies. In addition, the enzymatic activity of Av2 decreased concomitantly with total Fe chelation. To account for these observations, a model is presented in which Av2 exists in two conformers. Fe chelation is proposed to occur from either conformer but only when two MgATP are bound. Both conformers bind MgATP with the same affinity but are distinguished by a 10-fold difference in chelation rate. The two conformers are in equilibrium and can interconvert only in the absence of MgATP. That is, MgATP binding prevents the conversion of the two conformational states. 相似文献
18.
D M Gra?fer M A Zenkova G G Karpova A A Malygin N B Matasova 《Molekuliarnaia biologiia》1990,24(4):1076-1083
Binding of labelled oligouridylates--mRNA analogs--to human placenta 80S ribosomes in the presence of Phe-tRNAPhe has been studied. The single site for (pU)n (n = 6, 9, 13) binding on the ribosome was found; association constants for their tRNA-dependent binding were evaluated. In the presence of oligouridylates as templates [14C]Phe-tRNAPhe was found to be able to bind simultaneously at acceptor and donor ribosomal sites which resulted in diphenylalanine formation. The observed maximum Phe-tRNAPhe binding level was considerably lower than for the corresponding oligouridylate binding; the longer oligouridylate the higher Phe-tRNAPhe maximum binding level. To explain the results obtained we have proposed that (i) (Phe)2-tRNA produced from transpeptidation dissociates from the ribosomal A site to a significant extent and (ii) when oligouridylate length increases its interaction with 3'-side of mRNA binding center results in allosteric stabilization of the complex of peptidyl-tRNA with the ribosome at A site. 相似文献
19.
The thermally interconvertible diastereomers of the (5S)-methyl-3-(o-aryl)-2,4-oxazolidinediones were synthesized and their conformers studied by (1)H NMR and HPLC. The barriers to rotation about the N-C(aryl) bond were found to be very much solvent dependent. For the o-fluoro oxazolidinedione, difference in barriers to rotation in deuterated methanol and deuterated chloroform amounted to 34 kJ/mol. ortho-Bromo substitution increased the barrier to rotation up to 100 kJ/mol in ethanol, which enabled the analytical separation of the diastereomers and observation of the thermodynamic enrichment of the S-P conformer by HPLC. In CDCl(3) by (1)H NMR, on the other hand, a barrier of only 89 kJ/mol was determined. The S-M and S-P conformers of the diastereomers of o-methyl, alpha-naphthyl and o-iodo derivatives have been assigned by NOESY experiments and the kinetic and thermodynamic constants for the interconversion between the S-M and S-P conformers were determined. 相似文献
20.
Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation 总被引:1,自引:0,他引:1
M Schmitt U Neugebauer C Bergmann H G Gassen D Riesner 《European journal of biochemistry》1982,127(3):525-529
The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis. 相似文献