Dimethylsulfoxide-stabilized conformer of guanine-adenine repeat strand of DNA

Circular dichroism spectroscopy and other methods are used to show that the addition of dimethylsulfoxide causes reversible folding of the (GA)(10) strand of DNA into an ordered single-stranded conformer. The ordered conformer melts in a cooperative way and it does not contain protonated adenine. The (TA)(10), (A)(20), and (G)(20) are all unstable in this conformer. To the best of our knowledge, this is the first known ordered conformer of DNA that is stabilized by dimethylsulfoxide. This conformer might be a DNA analog of the protein alpha helix, which is an interesting idea for thinking about the evolution of DNA.     

Stabilization of (dG-dC)n.(dG-dC)n in the Z conformation by a crosslinking reaction

(dG-dC)n.(dG-dC) was converted to the Z conformer by heating in the presence of Mn++n. Reaction of this preparation with the crosslinking reagent, DL-diepoxybutane (DEB), stabilized this conformer so that it retained its structure even when returned to conditions that favored reversion to the B conformation. Treatment of the crosslinked Z conformer with periodate caused scission of the crosslink, allowing reversion to the B conformer. Reaction of (dG-dC)n.(dG-dC)n in the B conformation with DEB did not prevent conversion to the Z conformer in 4M NaC1; dialysis of the high salt solution against low ionic strength buffer allowed return to the B conformer. The Z in equilibrium B transitions were followed by circular dichroism studies and immunochemical procedures. The results suggest the feasibility of stabilizing Z sequences of DNA in chromatin by crosslinking, so that they could then be identified after DNA isolation.     

Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.     

Chrysophanol anthrone: a theoretical study on the potential energy surface

The potential energy surface (PES) of chrysophanol anthrone, the active component of Goa Powder, was systematically explored and thoroughly scrutinised via density functional theory, in order to gain an understanding of its physicochemical properties. In particular, we focused on the rotations of the two hydroxyl-phenyl dihedral angles. A picture with a four stable rotamers emerged where only the most stable conformer has a planar structure and the less stable conformer has the maximum deviation from planarity. The computed PES shows that the energy barriers for the conformer interconversion are less than 15 kcal/mol. From the analysis of the calculated intramolecular hydrogen bond enthalpy, we conclude that the number of the intramolecular hydrogen bonds governs the conformer stability. Additionally, the conformational equilibrium was pursued by means of an analysis of the energy of OH internal rotation barriers. The total energy changes were decomposed in an electrostatic decomposition scheme in order to gain an insight into the effects governing the torsional barrier and preferred conformations. This analysis shows that the interplay between the repulsive and attractive potentials causes the conformer stability, where the attractive term dominates the conformer stabilisation.     

Non-toxic conformer of amyloid β may suppress amyloid β-induced toxicity in rat primary neurons: Implications for a novel therapeutic strategy for Alzheimer’s disease

The 42-mer amyloid β-protein (Aβ42) oligomers cause neurotoxicity and cognitive impairment in Alzheimer’s disease (AD). We previously identified the toxic conformer of Aβ42 with a turn at positions 22–23 (“toxic” turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25–26. G25P-Aβ42 and E22V-Aβ42 are non-toxic mutants that disfavor the “toxic” turn. Here we hypothesize that these non-toxic mutants of Aβ42 could suppress Aβ42-induced neurotoxicity, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-Aβ42 and E22V-Aβ42 suppressed the neurotoxicity and aggregation of Aβ42 as well as the formation of the toxic conformer. The neurotoxicity induced by Aβ42 was also significantly reduced by the treatment of 11A1, but not of Aβ-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that Aβ oligomers contain parallel β-sheet, the present results suggest that the non-toxic mutants of Aβ42 without the “toxic” turn could prevent the propagation process of the toxic conformer of Aβ42 resulting in suppression of the formation of the toxic oligomers. This could be a promising strategy for AD therapeutics.     

Theoretical and NMR investigations on the conformations of ( − )-meptazinol hydrochloride in solution

( ? )-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.     

Alkali cation effect on carbonyl-hemoglobin's and -myoglobin's conformer populations when exposed to freeze-concentration of their phosphate-buffered aqueous solutions

Freeze-concentrated aqueous phosphate-buffered (pH 6.8) solutions of carbonyl-hemoglobin (HbCO) and -myoglobin (MbCO) were investigated by Fourier-transform infrared spectroscopy for the effect of alkali cation on the population of conformers. When using sodium phosphates as buffer components, HbCO was transformed from conformer III (at approximately 1951 cm-1) which is the dominant form at ambient temperatures, into conformer IV (at buffer concentration at a given temperature. The conformational changes started slightly below the temperature where ice began to crystallize and the remaining solution became freeze-concentrated, and they were reversible for HbCO. For MbCO in 0.5 M sodium phosphate buffer solution, however, they were irreversible and MbCO denatured completely. When potassium phosphate salts were used for preparing the buffer at the same pH of 6.8, little or no transformation of conformer III into conformer IV was observed. The conformational changes induced by sodium salts are attributed to a decrease in pH and it is shown by infrared spectroscopy that during freeze concentration drastic changes in composition of the two buffer components H2PO4-/HPO(2)4- occur, the acid component increasing strongly relative to the base component. Supersaturation is also important because change from conformer III to IV requires a minimum concentration of sodium salts: whereas 0.1 M sodium phosphate buffer concentration shows a strong effect, 0.03 M concentration does not and therefore behaves like a potassium phosphate buffer.     

Two alternating structures of the HIV-1 leader RNA

In this study we demonstrate that the HIV-1 leader RNA exists in two alternative conformations, a branched structure consisting of several well-known hairpin motifs and a more stable structure that is formed by extensive long-distance base pairing. The latter conformation was first identified as a compactly folded RNA that migrates unusually fast in nondenaturing gels. The minimally required domains for formation of this conformer were determined by mutational analysis. The poly(A) and DIS regions of the leader are the major determinants of this RNA conformation. Further biochemical characterization of this conformer revealed that both hairpins are disrupted to allow extensive long-distance base pairing. As the DIS hairpin is known to be instrumental for formation of the HIV-1 RNA dimer, the interplay between formation of the conformer and dimerization was addressed. Formation of the conformer and the RNA dimer are mutually exclusive. Consequently, the conformer must rearrange into a branched structure that exposes the dimer initiation signal (DIS) hairpin, thus triggering formation of the RNA dimer. This structural rearrangement is facilitated by the viral nucleocapsid protein NC. We propose that this structural polymorphism of the HIV-1 leader RNA acts as a molecular switch in the viral replication cycle.     

Partial phase diagram of aqueous bovine carbonic anhydrase: analyses of the pressure-dependent temperatures of the low- to physiological-temperature nondenaturational conformational change and of unfolding to the molten globule state

At 1.0 atm pressure and in 150 mM sodium phosphate (pH = 7.0), bovine carbonic anhydrase undergoes a nondenaturational conformational change at 30.3 degrees C and an unfolding transition from the physiological conformer to the molten globule state at 67.4 degrees C. The pressure dependences of the temperatures of these transitions have been studied under reversible conditions for the purpose of understanding DeltaH degrees , DeltaS degrees , and DeltaV for each conformational change. Temperatures for the low-temperature to physiological-temperature conformational change T(L-->P) are obtained from physiologically relevant conditions using slow-scan-rate differential scanning calorimetry. Temperatures for the physiological-temperature conformation to molten globule state conversion T(P-->MG) are obtained from differential scanning calorimetry measurements of the apparent transition temperature in the presence of guanidine hydrochloride extrapolated to zero molar denaturant. The use of slow-scan-rate differential scanning calorimetry permits the calculation of the activation volume for the conversion of the low-temperature conformer to the physiological-temperature conformer DeltaV(double dagger)(L-->P). At 1.0 atm pressure, the transition from the low-temperature conformer to the physiological-temperature conformer involves a volume change DeltaV(L-->P) = 15 +/- 2 L/mole, which contrasts with the partial unfolding of the physiological-temperature conformer to the molten globule state (DeltaV(P-->MG) = 26 +/- 9 L/mole). The activation volume for this process DeltaV(double dagger)(L-->P) = 51 +/- 9 L/mole and is consistent with a prior thermodynamic analysis that suggests the conformational transition from the low-temperature conformation to the physiological-temperature conformation possesses a substantial unfolding quality. These results provide further evidence the structure of the enzyme obtained from crystals grown below 30 degrees C should not be regarded as the physiological structure (the normal bovine body temperature is 38.3 degrees C). These results should therefore have implications in any area that seeks to correlate the crystal structure of bovine carbonic anhydrase to physiological function.     

The interconversion of conformers of phenylalanyl-tRNA with different affinity to 70S ribosomes of Escherichia coli.

Earlier the existence of two conformers of Phe-tRNAPhe of E. coli was demonstrated because one of them yields complexes with 70S-poly(U) of extremely high affinity and the other with at least a 105 lower binding constant. We denote the first conformer as HAC (high affinity conformer) and the second as LAC (low affinity conformer). This high difference in binding constants was used for studying the process of reversible interconversion of conformers of Phe-tRNAPhe. The transition kinetics of LAC to HAC in conditions when the latter is stable (in the presence of magnesium ions) was studied and a high value of activation energy (35 kcal/mole) found. The interconversion is the first order reaction and equilibrium does not depend of overall Phe-tRNA concentration.     

Mutation of asparagine 52 to glycine promotes the alkaline form of iso-1-cytochrome c and causes loss of cooperativity in acid unfolding

The kinetics and thermodynamics of the alkaline and acid conformational transitions of a Lys 79 --> Ala/Asn 52 --> Gly (A79G52) variant of iso-1-cytochrome c are studied. The Lys 79 --> Ala mutation is designed to limit heme ligation in the alkaline conformer to Lys 73. The Asn 52 --> Gly mutation is intended to shift the population of the alkaline conformer to physiological pH based on the hierarchical nature of the cooperative substructures of this protein. The midpoint pH for formation of the alkaline conformer is approximately 7.45. The kinetics for the alkaline conformational transition of the A79G52 variant are consistent with the ionization constant, pK(H), for the trigger group controlling formation of the alkaline conformer being approximately 9.5. This pK(H) is low for alkaline conformers involving lysine-heme ligation but is consistent with the pK(a) of the highest of three ionizable groups which modulate formation of the histidine-heme alkaline conformer of a His 73 variant of iso-1-cytochrome c [Martinez, R. E., and Bowler, B. E. (2004) J. Am. Chem. Soc. 126, 6751-6758]. The acid transition of the A79G52 variant is split into two phases. Both the Lys 79 --> Ala and Asn 52 --> Gly mutations are expected to affect the buried hydrogen bond network of cytochrome c, suggesting that this network is an important modulator of the acid unfolding of cytochrome c.     

High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.     

Amino-terminal flanking residues determine the conformation of a peptide-class II MHC complex

The peptide spanning residues 48-62 of hen egg white lysozyme presented by I-A(k) molecules gives rise to two T cell populations, types A and B, that recognize distinct conformers of the complex generated in late and recycling endosomes. The class II-like accessory molecule H2-DM functions as a conformational editor, eliminating the type B conformer in late endosomes. Here, we show that the conformation of the complex, and its susceptibility to editing by H2-DM, are determined by peptide amino-terminal flanking residues. Elimination of these residues abolished editing, permitting formation of the type B conformer in late endosomes. Substitutions at P(-2) affected the stability of the type B conformer, preventing its formation and/or editing, without hindering peptide binding or formation of the type A conformer of the complex. We conclude that interactions involving amino-terminal flanking residues stabilize peptide-MHC conformers and confer resistance to editing by H2-DM, influencing the nature of the T cell repertoire.     

Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers

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)     

Enhancement of the chiroptical response of α-amino acids via N-substitution for molecular structure determination using vibrational circular dichroism

The chiroptical response in the form of vibrational circular dichroism (VCD) in the midinfrared region is found to be enhanced when a hydrogen of amino group of l -tryptophan is substituted with acetyl, acryloyl, or maleyl group. The order of preference for VCD enhancement is found to be acryloyl > acetyl > maleyl group. The resulting experimental VCD spectra are also found to be satisfactorily reproduced by the quantum mechanical (QM) predicted spectra. The QM predicted spectra were simulated using the conformer populations, (a) predicted by Gibbs energies and (b) optimized to maximize the similarity between experimental and predicted VCD spectra. It is found that the conformer populations predicted by Gibbs energies do not yield the maximum possible similarity between experimental and the QM predicted spectra. This work identifies the N-substitution of α-amino acids and determining the conformer populations that best reproduce the experimental spectra as two new approaches for molecular structure determination.     

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

Sensory rhodopsin-I as a bidirectional switch: opposite conformational changes from the same photoisomerization

The phototaxis receptor sensory rhodopsin I (SRI) exists in two protein conformations, each of which is converted to the other by light absorption by the protein's retinylidene chromophore. One conformer inhibits a histidine-kinase attached to its bound transducer HtrI and its formation induces attractant motility responses, whereas the other conformer activates the kinase and its formation induces repellent responses. We performed Fourier transform infrared spectroscopy with temperature, pH, and mutation-induced shifts in the conformer equilibrium, and found that both conformers when present in the unphotolyzed dark state contain an all-trans retinal configuration that is photoisomerized to 13-cis, i.e., the same photoisomerization causes the opposite conformational change in the photointerconvertible pair of conformers depending on which conformer is present in the dark. Therefore, switching between the protein global conformations that define the two conformers is independent of the direction of isomerization. Insights into this phenomenon are gained from analysis of the evolution of the receptor from light-driven proton pumps, which use similar conformers for transport. The versatility of the conformational changes of microbial rhodopsins, including conformer interexchangeability in the photocycle as shown here, is likely a significant factor in the evolution of the diverse functionality of this protein family.     

Sonication induced intermediate in prion protein conversion

We have observed that hamster prion protein (PrP(C)) undergoes conformational changes on exposure to heat or sonication. If a sonication induced new conformer is seeded with a small amount of its abnormal pathogenic isoform (PrP(Sc)) it undergoes a significant conversion to a proteinase-resistant isoform. This suggests the presence of a third stable PrP conformer, which may be intermediate in the conversion of PrP(C) to PrP(Sc).     

Structural dynamics of individual Holliday junctions

The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.     

Conformational investigation of alpha,beta-dehydropeptides. XV: N-acetyl-alpha,beta-dehydroamino acid N 'N '-dimethylamides: conformational properties from infrared and theoretical studies.

The FTIR spectra were analysed in the region of the nu(s)(N-H), AI(C=O) and nu(s)(Calpha=Cbeta) bands for a series of Ac-DeltaXaa-NMe2, where DeltaXaa = DeltaAla, (Z)-DeltaAbu, (Z)-DeltaLeu, (Z)-DeltaPhe and DeltaVal, to determine a predominant solution conformation of these alpha,beta-dehydropeptide-related molecules. Measurements were taken in CCl4, DCM and MeCN solutions. In the same way, spectra of saturated analogues Ac-Xaa-NMe2, where Xaa = Ala, Abu, Leu, Phe and Val, were investigated. To help interpret the spectroscopic results, conformational maps were calculated by the B3LYP/6-31+G** method. Also, the relative energies of all conformers of the dehydro compounds in vacuo as well as in the studied solvents in addition to the theoretical IR frequencies of these conformers were calculated. For comparison, molecules of two saturated analogues, Ac-L-Ala-NMe2 and Ac-L-Phe-NMe2, were calculated in a similar way. Both unsaturated and saturated compounds, which have an aliphatic side chain, occur in CCl4 and DCM mainly as a mixture of extended conformers with the C5 H-bond and open conformers. As solvent polarity increases, participation of the open conformers also increases, and in MeCN, the model amides are almost exclusively in the open form, except Ac-DeltaAla-NMe2, which shows a small amount of the H-bonded conformer. Ac-DeltaAla-NMe2 and Ac-DeltaAbu-NMe2 have stronger C5 hydrogen bonds than those of their saturated counterparts. As the calculations indicate, the open conformation of the unsaturated amides is conformer H/F with phi, psi -44 +/- 5 degrees, 127 +/- 4 degrees. This is the second lowest in energy conformer in vacuo and in CCl4 and the lowest one in more polar solvents. The open conformation of Ac-L-Ala-NMe2 constitutes conformer C with phi, psi -101.5 degrees, 112.7 degrees. For Ac-DeltaAla-NMe2 and Ac-DeltaAbu-NMe2, FTIR also reveals the presence of a third conformer. Calculations indicate that is the semiextended conformer D with the N1-H1...N2 hydrogen bond/contact. In all solvents, Ac-L-Phe-NMe2 and Ac-(Z)-DeltaPhe-NMe2 show only the extended E and the open H/F, respectively. In both there is an amide/pi(Ph) interaction.