Characterization of the high-resolution ESR spectra of superoxide radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Analysis of conformational exchange

It has been previously reported that the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) can form stable radical adducts with superoxide radical. However, the presence of diastereomers of DEPMPO radical adducts and the appearance of superhyperfine structure complicates the interpretation of the ESR spectra. It has been suggested that the superhyperfine structure in the ESR spectrum of DEPMPO/^⋅OOH is a result of conformational exchange between conformers. The analysis of the temperature dependence of the ESR spectrum of DEPMPO/^⋅OOH and of its structural analog DMPO/^⋅OOH have demonstrated that both ESR spectra contain exchange effects resulting from conversion between two conformers. Computer simulation calculates a conformer lifetime on the order of 0.1 μs for DMPO/^⋅OOH at room temperature. However, temperature dependence of the ESR spectrum of DEPMPO/^⋅OOH suggests that superhyperfine structure does not depend on the conformational exchange. We have now found that the six-line ESR spectrum with superhyperfine structure should be assigned to a DEPMPO-superoxide-derived decomposition product. Therefore, ESR spectra previously assigned to DEPMPO/^⋅OOH contain not only the two diastereomers of DEPMPO/^⋅OOH but also the decomposition product, and these spectra should be simulated as a combination of four species: two conformers of the first diastereomer, one conformer of the second diastereomer and the superoxide-derived decomposition product. The presence of four species has been supported by the temperature dependence of the ESR spectra, nucleophilic synthesis of radical adducts, and isotopic substitution experiments. It is clear that to correctly interpret DEPMPO spin trapping of superoxide radicals, one must carefully consider formation of secondary radical adducts.     

Conformational preferences and binding to neurophysins of oxytocin analogs with sarcosine or N-methylalanine in position 7

The 600 MHz proton n.m.r. spectra of (sarcosyl7)-oxytocin and (N-methylalanyl7) oxytocin in 2H2O solution have been recorded and completely assigned. In each case the spectrum indicates the presence of two slowly interconverting conformers, which are the cis-trans isomers about the peptide bond between residues six and seven. The trans isomer is energetically favored in both cases. When neurophysin is added to a solution of (N-methylalanyl7) oxytocin or (sacrosyl7)-oxytocin at pH 3.0, the proportion of minor conformer remains constant, indicating that the cis and trans conformers are equally tightly bound to the protein.     

Characterization of the high-resolution ESR spectra of superoxide radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Analysis of conformational exchange

It has been previously reported that the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) can form stable radical adducts with superoxide radical. However, the presence of diastereomers of DEPMPO radical adducts and the appearance of superhyperfine structure complicates the interpretation of the ESR spectra. It has been suggested that the superhyperfine structure in the ESR spectrum of DEPMPO/^?OOH is a result of conformational exchange between conformers. The analysis of the temperature dependence of the ESR spectrum of DEPMPO/^?OOH and of its structural analog DMPO/^?OOH have demonstrated that both ESR spectra contain exchange effects resulting from conversion between two conformers. Computer simulation calculates a conformer lifetime on the order of 0.1?μs for DMPO/^?OOH at room temperature. However, temperature dependence of the ESR spectrum of DEPMPO/^?OOH suggests that superhyperfine structure does not depend on the conformational exchange. We have now found that the six-line ESR spectrum with superhyperfine structure should be assigned to a DEPMPO-superoxide-derived decomposition product. Therefore, ESR spectra previously assigned to DEPMPO/^?OOH contain not only the two diastereomers of DEPMPO/^?OOH but also the decomposition product, and these spectra should be simulated as a combination of four species: two conformers of the first diastereomer, one conformer of the second diastereomer and the superoxide-derived decomposition product. The presence of four species has been supported by the temperature dependence of the ESR spectra, nucleophilic synthesis of radical adducts, and isotopic substitution experiments. It is clear that to correctly interpret DEPMPO spin trapping of superoxide radicals, one must carefully consider formation of secondary radical adducts.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.     

CD spectrum and conformational distribution of cyclotuftsin in solution

CD spectra for low-energy conformations of the tuftsin cycloanalogue, , were calculated. A theoretical spectrum obtained as the weighted average of calculated spectra for individual peptide backbone conformers is qualitatively consistent with an experimental CD spectrum in aqueous solution. The conformational distribution allows one to achieve agreement between calculated and experimental values of structural parameters of the cyclotuftsin molecule investigated by NMR spectroscopy.

CD spectrum calculation Theoretical conformational analysis Tuftsin cycloanalog Peptide conformation     

Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide lesion

One- and two-dimensional NMR spectroscopy has been used combined with molecular dynamics to determine the fine structure of the DNA duplex 5'-d(AGGAGCCACG).d(CGTGGFTCCT) where F is the N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue which is a ring fragmentation product of thymine. The formamide deoxyribose exists as two isomers with respect to the orientation about the peptide bond. The two isomers (trans and cis) are observed in a ratio 3:2 in solution. For both species, the oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. The formamide residue, whatever the isomer, is intrahelical and can pair with the guanine on the opposite strand with one hydrogen bond. For the cis isomer, the residue adopts a syn orientation and is able to form a second hydrogen bond with the guanine on the 5' side on the same strand. Off-resonance ROESY experiments have been used to investigate the chemical exchange observed at low temperature of the duplex. Conformational exchange has only been found for the oligonucleotide with the formamide residue in the trans conformation.     

Pulse fluorometry of cyclo-(glycyl-L-tryptophyl).

The fluorescence of cyclo-(glycyl-L-tryptophyl) in trimethyl phosphate has been studied in a temperature range varying from room temperature to -85 degrees C. At room temperature, the fluorescence decay is the sum of two exponentials, the relative amplitude of which depends on the emission wavelength. This can be explained by the presence of the two following emitting molecular states: on one hand the unfolded state, the fluorescence decay time and the emission spectrum of which are close to these of skatole; on the other hand the folded state which has a shorter decay time and a blue-shifted spectrum. By lowering the temperature, the fluorescence spectrum shifts to the blue, while the skatole spectrum shifts to the red. This behavior corresponds to an increase of the folded conformation concentration in agreement with the NMR results. Furthermore the rate of exchange between the folded and the unfolded conformations decreases. Accordingly the wavelength dependence of the fluorescence decay lessens. There are two possible values of the conformational angle x2 differing by 180 degrees, which correspond to the folded state; due to the indole asymmetry, the interactions between the indole and diketopiperazine rings differ in these conformers. Consequently the fluorescence decay remains biexponential even at -85 degrees C.     

High‐resolution crystal structures reveal a mixture of conformers of the Gly61‐Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox‐active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one‐electron reduced state. Here, we present high‐resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one‐electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single‐crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild‐type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.     

Structure-activity relationships on adrenoceptors and imidazoline-preferring binding sites (I(1,2)-PBSs). Part 1: Weak intramolecular H-bond and conformational flexibility in a new I1-PBS-selective imidazoline analogue, trans1-(4',5'-dihydro-1'H-imidazol-2'-yl)methyl-2-hydroxyindane (PMS 952)

The highly selective I1-PBS imidazoline analogue PMS 952 has been selected to study the incidence of intramolecular hydrogen bond and molecular flexibility on its biological activity. On one hand, the weak energy difference between three calculated conformers does not support the stabilization of one conformer by an internal hydrogen bond. The 3-D electrostatic map confirms this feature and the solvent effect does not significantly modify the relative energy of these conformers. On the other hand, the conformational spaces of the neutral and ionized forms present a great number of equilibrium structures, in a short energetic range (20 Kcal). The results are representative of an exceptional conformational flexibility due to a cooperative effect between several parts of the molecule.     

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Fluorescence spectra of luteinizing hormone releasing hormone,a decapeptide containing histidyl,tryptophyl and tyrosyl residues: Analysis by derivative spectroscopy

Fluorescence spectra have been obtained for luteinizing hormone releasing hormone, a decapeptide containing His, Trp and Tyr, and analogs lacking one or more of these residues. The second derivatives of these spectra were used to examine the contributions of the three residues to the spectrum of the hormone. Tyr influences the excitation spectrum when fluorescence is monitored at an emission wavelength of 305 nm but makes little or no contribution to the emission spectrum when the compound is excited at 275 nm. His and Trp influence both excitation and emission spectra.     

The coordination of the isomerization of a conserved non-prolyl cis peptide bond with the rate-limiting steps in the folding of dihydrofolate reductase

The propensity for peptide bonds to adopt the trans configuration in native and unfolded proteins, and the relatively slow rates of cis-trans isomerization reactions, imply that the formation of cis peptide bonds in native conformations are likely to limit folding reactions. The role of the conserved cis Gly95-Gly96 peptide bond in dihydrofolate reductase (DHFR) from Escherichia coli was examined by replacing Gly95 with alanine. The introduction of a beta carbon at position 95 is expected to increase the propensity for the trans isomer and perturb the isomerization reaction required to reach the native conformation. Although G95A DHFR is 1.30 kcal mol(-1) less stable than the wild-type protein, it adopts a well-folded structure that can be chemically denatured in a cooperative fashion. The mutant protein also retains the complex refolding kinetic pattern attributed to a parallel-channel mechanism in wild-type DHFR. The spectroscopic response upon refolding monitored by Trp fluorescence and the absence of a Trp/Trp exciton coupling apparent in the far-UV CD spectrum of the wild-type protein, however, indicated that the tertiary structure of the folded state for G95A DHFR is altered. The addition of methotrexate (MTX), a tight-binding inhibitor, to folded G95A DHFR restored the exciton coupling and the fluorescence properties through five slow kinetic events whose relaxation times are independent of the ligand and the denaturant concentrations. The results were interpreted to mean that MTX-binding drives the formation of the cis isomer of the peptide bond between Ala95 and Gly96 in five compact and stable but not wild-type-like conformations that contain the trans isomer. Folding studies in the presence of MTX for both wild-type and G95A DHFR support the notion that the cis peptide bond between Gly95 and Gly96 in the wild-type protein forms during four parallel rate-limiting steps, which are primarily controlled by folding reactions, and lead directly to a set of native, or native-like, conformers. The isomerization of the cis peptide bond is not a source of the parallel channels that characterize the complex folding mechanism for DHFR.     

Conformational energy analysis of retro-all-Dmethionine enkephalin

Empirical conformational energy calculations have been carried out on the molecule retro-all-D -methionine enkephalin. Low-energy conformers were found by energy minimization and conformational search procedures. The lowest energy conformers wee found toi have some stereochemical relationship to the calculated normal met-enkephalin conformers, but they were not retro-all-D -equivalent to the Met-enkephalin structures. The retro-all-D -equivalent conformations were ～10 kcal/mol higher energy than the low-energy conformers found here. A structural comparison between the retro-all-D -conformers and the met-enkephalin conformers shows hat one cannot rely solely on topochemical analysis to predict biological activity for linear retro-all-D -peptides.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.     

Identification of dynamical hinge points of the L1 ligase molecular switch

The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5′ to 3′ phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive–active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.     

Changes of the antenna of photosystem I induced by short-term heating

Changes in low-temperature fluorescence spectra of pea chloroplasts induced by the short-term heating were studied. Excitation spectra of the long-wavelength fluorescence were studied as well. Heating was carried out at 45°C for 5 min in the darkness or in the presence of white light sourced with intensities of 260 or 1400 μmol/m² s. All variants of heating decreased the intensity of the long-wavelength fluorescence band. The integral of the excitation spectrum decreased after the exposure to heating in the darkness and increased after the exposure to heating in the presence of light. The observed changes in most intensive components — 726, 729 and 731 nm — of the long-wavelength fluorescence band, induced by various modes of heating, were similar. The changes in the fourth intensive component at 735 nm were different. Twenty-five components were found in the fine structure of the excitation spectrum of the long-wavelength fluorescence. Positions of most of peaks corresponded to the absorption peaks of Lhca proteins. Heat-induced changes in the excitation spectrum in the regions corresponding to the absorption of chl b and short-wavelength forms of chl a have been shown to correlate with changes in the intensities of the 726-, 729-, and 731-nm components of the long-wavelength fluorescence. This allows one to assign them to the emission of the outer antenna of Photosystem I. Changes in the intensity of the component at 735 nm correlated only with changes in excitation spectrum in the long-wavelength region that corresponded to the absorption of the long-wave-length forms of chlorophyll a. Therefore, the 735-nm component could be assigned to the emission of the Photosystem I inner antenna. Analysis of the changes induced by heating in the emission and excitation spectra of fluorescence revealed changes in the energy transfer in the outer and the inner antennas of Photosystem I. Heating in the darkness lowered the energy transfer in the outer and in the inner antennas. Both modes of heating in the presence of light increased the energy transfer in the outer antenna. For the inner antenna, presence of the light promotes an efficient of energy transfer at the levels close to the control one. It is proposed that illumination during heating exposure causes a specific state of the antenna complex in Photosystem I that provides an increase in funneling of the energy toward the reaction centers.     

Molecular dynamics studies of the conformation of sorbitol

Molecular dynamics simulations of a 3 molal aqueous solution of d-sorbitol (also called d-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2–C-3 torsion (spanned by the O-2–O-4 hydrogen bond), where the NMR data support a more bent structure.