Petra Imhof

Guanine tautomerism revealed by UV–UV and IR–UV hole burning spectroscopy

The vibronic spectrum of laser desorbed and jet cooled guanine consists of bands from three different tautomers of guanine as revealed by UV–UV and IR–UV double resonance spectroscopy. 1-methylguanine, in which the Keto–Enol tautomerism is blocked, shows hole burning spectra from the 9H-and 7H-Keto form. A comparison of the vibronic pattern of the different tautomers demonstrates that the vibronic spectrum built on the redmost guanine band at 32 870 cm−1 (electronic origin 0) can be traced back to the 9H-Enol tautomer, while the spectra built on the origins at 0+404 cm−1 and 0+1044 cm−1 stem from the two Keto tautomers. The IR–UV double resonance spectra of the OH-and NH-stretch vibrations of the different tautomers support this assignment. The UV and IR spectra can be partly assigned by comparison with ab initio calculated vibrational frequencies and with the help of deuteration experiments.

Pairing of the nucleobases guanine and cytosine in the gas phase studied by IR–UV double-resonance spectroscopy and ab initio calculations

We present R2PI, IR–UV and UV–UV double resonance measurements of the guanine–cytosine (G–C) dimer formed in a supersonic jet. We show that there is only one isomer of G–C in the investigated wavelength range from 33200 to 34100 cm−1. We assigned the observed G–C isomer to a specific structure, based on comparisons of the IR spectra of the G and C monomers with the G–C dimer in the range of the OH and NH stretching vibrations and ab initio-calculated vibrational frequencies and dimer stabilities. The cluster exhibits an HNH⋯O/NH⋯N/CO⋯HNH bonding similar to the Watson–Crick G–C base pair bonding but with C as the enol tautomer. We did not observe any keto–keto or enol–enol G–C dimers in the investigated wavelength region.

REMPI Spectroscopy of Laser Desorbed Guanosines

To observe fundamental properties of DNA building blocks it is desirable to study individual nucleosides in the gas phase without interference from solvent molecules, or macromolecular structure. As a first step, we have recently reported the first vibronic spectrum of the nucleobase guanine, obtained by a combination of laser desorption, jet cooling, and resonance enhanced multiphoton ionization (REMPI).1 Although guanine is important as a chromophore in DNA, it is more realistic for understanding the photochemistry of DNA to study the nucleosides. Those are even harder to vaporize intact because they are thermally more labile and, with their larger molecular weights, have still lower vapor pressures. Using laser desorption, we have now succeeded in forming a molecular beam of nucleosides, and we report the first REMPI spectra of a series of individual guanosines, namely guanosine (Gs), 2′deoxyguanosine (2′deoxyGs), and 3′deoxyguanosine (3′deoxyGs). We compare our results with computations at the HF 6-31G(d,p) level. The results suggest the occurrence of two different conformations, each probably stabilized by internal hydrogen bonds. One of those two conformations is absent in 2′deoxyGs implying that the 2′ hydroxyl group is required for its stabilization. Spectroscopic properties of guanosines have been studied primarily by Raman techniques in solution.2-9 A great deal of attention has been given to potential Raman markers for hydrogen bonding and for structural conformation. Observation of hydrogen bonding by Raman spectroscopy requires identification of vibrations that depend strongly on those specific atoms in guanine, that serve as either proton donor or acceptor. However, most vibrations involve the concerted motion of multiple atoms, and therefore correlation of marker frequencies with specific hydrogen bonding sites is not straightforward. Guanosine vibrations involving motion along the glycosidic bond may provide conformational markers if their frequencies are sensitive to puckering of the ribose ring or for rotation around the sugar-base bond. Interpretation of these markers requires careful analysis of complex vibrational modes. On the other hand, different conformations can be observed much more directly by vibronic spectroscopy when they produce multiple origins. As we will show below, we observe two origins in our spectra, which we can associate with the syn and the anti orientations of the base relative to the ribose moiety. We have published details of our setup for laser desorption jet cooling REMPI spectrometry elsewhere.10 Sample preparation consisted of depositing neat material in powder form on graphite substrates. We moved the substrate slowly while acquiring spectra, gradually exposing fresh material. For desorption we used pulses from a Nd:YAG laser at 1064 nm with fluences on the order of 1 mJ/cm2. Desorbed neutral molecules were entrained in a supersonic expansion with Ar drive gas, injected by a pulsed solenoid valve. Downstream, the entrained molecules were onecolor two-photon photoionized, and the ions were detected in a reflectron time-of-flight mass spectrometer. The first photon resonantly excites the molecule, while a second photon from the same laser ionizes the excited molecule. By varying the wavelength while monitoring specific mass peaks we obtained mass selected excitation spectra. The typical ionization laser fluence was on the order of 0.1 mJ/cm2. Figure 1 shows the REMPI spectra of (a) Gs, (b) 3′deoxyGs, and (c) 2′deoxyGs. We assign the lowest-energy peak in each of the spectra as a 0-0 transition to the S1 excited state. Careful scans to lower energy by 1000 cm-1 do not reveal any additional peaks. The same was true when performing two-color ionization with a second photon at 193 nm. Therefore, we do not believe that we are observing a cutoff in the spectrum related to the ionization potential of Gs. Furthermore we have measured the ionization potential of guanine as 8.1 eV by two-color ionization.11,12 That is 1 eV less than the two-photon energy at the Gs origin. † Heinrich Heine Universität. (1) Nir, E.; Grace, L.; Brauer, B.; de Vries, M. S. J. Am. Chem. Soc. 1999, 121, 4896. (2) Faurskov-Nielsen, O.; Lund, P.; Petersen, S. J. Raman Spectrosc. 1981, 11, 493. (3) Nishimura, Y.; Tsuboi, M.; Sato, T.; Aoki, K. J. Mol. Struct. 1986, 146, 123. (4) Chinsky, L.; Jolles, B.; Laigle, A.; Turpin, P. J. Raman Spectrosc. 1987, 18, 195. (5) Carmona, P.; Molina, M. J. Mol. Struct. 1990, 219, 323. (6) Toyama, A.; Takino, Y.; Takeuchi, H.; Harada, I. J. Am. Chem. Soc. 1993, 115, 11092. (7) Urabe, H.; Sugawara, Y.; Kasuya, T. Phys. ReV. B 1995, 51, 5666. (8) Toyama, A.; Hamuara, M.; Takeuchi, H. J. Mol. Struct. 1996, 15, 99. (9) Toyama, A.; Hanada, N.; Ono, J.; Yoshimitsu, E.; Takeuchi, H. J. Raman Spectrosc. 1999, 30, 623. (10) Meijer, G.; de Vries, M. S.; Hunziker, H. E.; Wendt, H. R. Appl. Phys. B 1990, 51, 395. (11) Hopkins, J. B.; Powers, D. R.; Smalley, R. E. J. Phys. Chem. 1981, 85, 3739. (12) Nir, E.; Grace, L.; de Vries, M. S. To be published. Figure 1. REMPI spectra of (a) guanosine, (b) 3′deoxyGs, and (c) 2′deoxyGs. In this energy range guanine itself does not exhibit any vibronic activity since its lowest energy peak is at 238 cm-1 above the origin. The syn and anti labels indicate origins of two possible conformers, and the numbers indicate vibrational modes and their combinations and overtones, for example 122 indicates one quantum of mode 1 and two quanta of mode 2; f indicates fundamental vibration. 8091 J. Am. Chem. Soc. 2000, 122, 8091-8092

Reassignment of ground and first excited state vibrations in phenol

Abstract The phenol molecule has been chosen to demonstrate how dispersed fluorescence spectroscopy can be used to assign hot bands, i.e. vibronic transitions starting from the vibrationally excited electronic ground state. The procedure outlined in this paper provides important information on fundamental out-of-plane vibrations in the electronic excited S1 state that are not observed in jet-cooled spectra. The results obtained lead to a new interpretation of the most intense hot bands in the UV absorption spectrum of phenol. We derive vibrational frequencies for the modes 10b1, 10b1 and 41 (Varsanyis nomenclature) which are different from those given in the literature.

The S1 state geometry of phenol determined by simultaneous Franck–Condon and rotational constants fits

We describe a program for Franck–Condon simulations of dispersed fluorescence spectra obtained from excitation of single vibronic fundamental, overtone and combination levels. The S1 state geometry of phenol has been determined by a simultaneous fit of the geometry to the vibronic intensities and effective rotational constants in the harmonic limit based on ab initio force constants.

Catalytic Mechanism of DNA Backbone Cleavage by the Restriction Enzyme EcoRV: A Quantum Mechanical/Molecular Mechanical Analysis

Endonucleases, such as the restriction enzyme EcoRV, cleave the DNA backbone at a specific recognition sequence. We have investigated the catalytic mechanism of backbone phosphodiester hydrolysis by the restriction enzyme EcoRV by means of hybrid quantum mechanical/molecular mechanical calculations. An exhaustive computation of different reaction pathways is performed, thus generating a network of pathways. Comparison of the computed (AM1d/MM) enzymatic reaction pathways with an analogous mechanism for small-molecule model systems [AM1/d and B3LYP/6-31++G(d,p)] reveals that the transition barriers for associative hydrolysis, which is more probable in the model systems, are not lowered by the enzyme. Instead, a reaction mechanism which has mostly dissociative character is more likely. The protein environment is tuned to significantly electrostatically stabilize the transition state structures. The direct catalytic impact of essential residues is determined: The magnesium metal ion activates a water molecule, thus facilitating protonation of the leaving group. A reduction of the coordination number of the magnesium metal ion from six to four upon the positioning of the attacking water molecule explains why larger metal ions, such as calcium, are not catalytically active. The nucleophile is generated by the transfer of a proton from the attacking water molecule to a carboxylic oxygen atom of aspartate 90. The catalytic effect of lysine 92 involves proper positioning of the scissile phosphate group and, more importantly, stabilization of the metaphosphate intermediate in an orientation optimal for attack of the nucleophile.

Geometry change of simple aromatics upon electronic excitation obtained from Franck-Condon fits of dispersed fluorescence spectra.

The S(1) state geometries of benzonitrile, p-cyanophenol, o-cyanophenol, chlorobenzene, and p-chlorophenol were determined by Franck-Condon simulations and a fit of the geometry to the vibronic intensities and effective rotational constants in the harmonic limit based on ab initio force constants.

Mechanism of DNA recognition by the restriction enzyme EcoRV.

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV-cognate DNA crystallographic complex, a sharp kink of 50 degrees has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA(CH(3))TATC (TA-CH(3)). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.

Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

Magnesium-dependent active-site conformational selection in the Diels-Alderase ribozyme.

The Diels-Alderase ribozyme, an in vitro-evolved ribonucleic acid enzyme, accelerates the formation of carbon-carbon bonds between an anthracene diene and a maleimide dienophile in a [4 + 2] cycloaddition, a reaction with broad application in organic chemistry. Here, the Diels-Alderase ribozyme is examined via molecular dynamics (MD) simulations in both crystalline and aqueous solution environments. The simulations indicate that the catalytic pocket is highly dynamic. At low Mg(2+) ion concentrations, inactive states with the catalytic pocket closed dominate. Stabilization of the enzymatically active, open state of the catalytic pocket requires a high concentration of Mg(2+) ions (e.g., 54 mM), with cations binding to specific phosphate sites on the backbone of the residues bridging the opposite strands of the pocket. The free energy profile for pocket opening at high Mg(2+) cation concentration exhibits a double minimum, with a barrier to opening of approximately 5.5 kJ/mol and the closed state approximately 3 kJ/mol lower than the open state. Selection of the open state on substrate binding leads to the catalytic activity of the ribozyme. The simulation results explain structurally the experimental observation that full catalytic activity depends on the Mg(2+) ion concentration.