Tadeusz Kulinski

Single Stranded Loops of Quadruplex DNA As Key Benchmark for Testing Nucleic Acids Force Fields.

We have carried out a set of explicit solvent molecular dynamics (MD) simulations on two DNA quadruplex (G-DNA) molecules, namely the antiparallel d(G4T4G4)2 dimeric quadruplex with diagonal loops and the parallel-stranded human telomeric monomolecular quadruplex d[AGGG(TTAGGG)3] with three propeller loops. The main purpose of the paper was testing of the capability of the MD simulation technique to describe single-stranded topologies of G-DNA loops, which represent a very challenging task for computational methods. The total amount of conventional and locally enhanced sampling (LES) simulations analyzed in this study exceeds 1.5 μs, while we tested several versions of the AMBER force field (parm99, parmbsc0, and a version with modified glycosidic χ torsion profile) and the CHARMM27 force field. Further, we compared minimal salt and excess salt simulations. Postprocessing MM-PBSA (Molecular Mechanics, Poisson-Boltzmann, Surface Area) free energy calculations are also reported. None of the presently available force fields is accurate enough in describing the G-DNA loops. The imbalance is best seen for the propeller loops, as their experimental structure is lost within a few ns of standard simulations with all force fields. Among them, parmbsc0 provides results that are clearly closest to the experimental target values but still not in full agreement. This confirms that the improvement of the γ torsional profile penalizing the γ trans substates in the parmbsc0 parametrization was a step in the right direction, albeit not sufficient to treat all imbalances. The modified χ parametrization appears to rigidify the studied systems but does not change the ultimate outcome of the present simulations. The structures obtained in simulations with the modified χ profile are predetermined by its combination with either parm99 or parmbsc0. Experimental geometries of diagonal loops of d(G4T4G4)2 are stable in standard simulations on the ∼10 ns time scale but are becoming progressively lost in longer and LES simulations. In addition, the d(G4T4G4)2 quadruplex contains, besides the three genuine binding sites for cations in the channel of its stem, also an ion binding site at each stem-loop junction. This arrangement of five cations in the quadruplex core region is entirely unstable in all 24 simulations that we attempted. Overall, our results confirm that G-DNA loops represent one of the most difficult targets for molecular modeling approaches and should be considered as reference structures in any future studies aiming to develop or tune nucleic acids force fields.

Conformations of Flanking Bases in HIV-1 RNA DIS Kissing Complexes Studied by Molecular Dynamics

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.

Effects of Base Substitutions in an RNA Hairpin from Molecular Dynamics and Free Energy Simulations

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.

The Apical Loop of the HIV-1 TAR RNA Hairpin Is Stabilized by a Cross-loop Base Pair

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.

Conformational Dynamics of a 5S rRNA Hairpin Domain Containing Loop D and a Single Nucleotide Bulge

Molecular modeling and molecular dynamics have been employed to study the conformation and flexibility of a 15-nucleotide fragment of the plant 5S rRNA containing loop D and a single uridine bulge. Two different model built initial structures were used: one with the bulge localized inside the helical stem and another with the bulge pointing out from the helix. Several independent 700-ps-long trajectories in aqueous solution with Na(+) conterions were produced for each starting structure. The bulge nucleotide inside the helix stayed in two main conformations, both of which affected the geometry of the stem part opposite the bulge. When the bulge nucleotide was located outside the helix, we found high base mobility and local backbone flexibility. The dynamics of the hydrogen bond network and conformational changes from a direct to a water mediated hydrogen bond in the sheared G-A basepair in the tetraloop was described. Our results correlate with lead ion induced cleavage patterns in 5S rRNA. Sites resistant to nonspecific lead cleavage appeared in all our simulations as the most rigid fragments independent of the localization of the bulge nucleotide.

Conformational analysis of galanin using end to end distance distribution observed by Förster resonance energy transfer.

Abstract The structural dynamics of the flexible neuropeptide galanin in solution were studied by Förster resonance energy transfer measurements at different temperatures by time-resolved fluorescence spectroscopy to determine its conformational heterogeneity. Endogenous tryptophan at position 2 acted as the fluorescent donor and the non fluorescent acceptor dinitrophenyl or the fluorescent acceptor dansyl were selectively attached to lysine 25 in porcine galanin. The coexistence of different structures of the neuropeptide galanin in trifluoroethanol solution was revealed by the model independent analysis of the distribution of relaxation times from the time-resolved resonance energy transfer data. Multiple conformational states are reflected by distinct end-to-end distance populations. The conformations differ in mean donor-acceptor distance by about 15 Å, and are consistent with the extended and folded backbone conformations of two α-helical regions separated by a flexible hinge. The effect that the labelling of galanin has on binding to the receptor was also evaluated. DNP-galanin showed the same high affinity to galanin receptors as unlabelled galanin, whereas DNS-galanin had significantly reduced affinity.

FLUORESCENCE LIFETIME MEASUREMENTS OF PSEUDOAZULENES USING PICOSECOND-RESOLVED SINGLE PHOTON COUNTING

The very rapidly decaying fluorescence of three different pseudoazulenes was measured, enabling to check simultaneously the time resolution and accuracy of the set-up with mode-locked and synchronously pumped lasers and time correlated single photon counting. Instrumental details are given to reach optimum time resolution and accuracy. The fluorescence decay of a widely used fluorescent standard was used in the deconvolution procedure.

Antivirally active ribavirin analogues--4,5-disubstituted 1,2,3-triazole nucleosides: biological evaluation against certain respiratory viruses and computational modelling.

Background: Ribavirin is a broad-spectrum antiviral agent that derives some of its activity from inhibition of cellular inosine monophosphate dehydrogenase (IMPDH), resulting in lower guanosine triphosphate (GTP) levels. Here we report the biological activities of three ribavirin analogues. Methods: Antiviral activities of test compounds were performed by in vitro cytopathic effect inhibition assays against influenza A (H1N1, H3N2 and H5N1), influenza B, measles, parainfluenza type 3 (PIV-3) and respiratory syncytial viruses. Compounds were modelled into the ribavirin 5‘-monophosphate binding site of the crystallographic structure of the human type II IMPDH (hIMPDH2) ternary complex. Effects of compounds on intracellular GTP levels were performed by strong anion exchange HPLC analysis. Results: Of the three compounds evaluated, the 5-ethynyl nucleoside (ETCAR) exhibited virus-inhibitory activities (at 1.2–20 μM, depending upon the virus) against most of the viruses, except for weak activity against PIV-3 (62 μM). Antiviral activity of ETCAR was similar to ribavirin; however, cytotoxicity of ETCAR was greater than ribavirin. Replacing the 5-ethynyl group with a 5-propynyl or bromo substituent (BrCAR) considerably reduced antiviral activity. Computational studies of ternary complexes of hIMPDH2 enzyme with 5‘-monophosphates of the compounds helped rationalize the observed differences in biological activity. All compounds suppressed GTP levels in cells; additionally, BrCAR suppressed adenosine triphosphate and elevated uridine triphosphate levels. Conclusions: Three compounds related to ribavirin inhibited IMPDH and had weak to moderate antiviral activity. Cytotoxicity adversely affected the antiviral selectivity of ETCAR. As with ribavirin, reduction in intracellular GTP may play a role in virus inhibition.

Time‐resolved fluorescence studies of flavodoxin Demonstration of picosecond fluorescence lifetimes of FMN in Desulfovibrio flavodoxins

Picosecond‐resolved fluorescence spectroscopy on FMN bound in flavodoxin from Desulfovibrio vulgaris and Desulfovibrio gigas revealed fluorescence lifetimes of about 30 ps. The lifetime shortening can be explained by charge transfer interaction in the flavin excited state with aromatic amino acid residues. In addition to the short lifetime component, nanosecond lifetimes were also observed in both flavodoxins. The latter fluorescence lifetime in D. vulgaris flavodoxin (5.6 ns) is even longer than that of free FMN (4.7 ns A fixed lifetime analysis of the results from a diluted D. vulgaris flavodoxin solution yielded the dissociation constant of the FMN‐apoflavodoxin complex: K d = 9.4 ∓ 1.4 nM at 20 °C and pH 7.0.

Conformational transitions of flanking purines in HIV-1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

Dimerization of HIV‐1 genomic RNA is initiated by kissing loop interactions at the Dimerization Initiation Site (DIS). Dynamics of purines that flank the 5′ ends of the loop–loop helix in HIV‐1 DIS kissing complex were explored using explicit solvent molecular dynamics (MD) simulations with the CHARMM force field. Multiple MD simulations (200 ns in total) of X‐ray structures for HIV‐1 DIS Subtypes A, B, and F revealed conformational variability of flanking purines. In particular, the flanking purines, which in the starting X‐ray structures are bulged‐out and stack in pairs, formed a consecutive stack of four bulged‐out adenines at the beginning of several simulations. This conformation is seen in the crystal structure of DIS Subtype F with no interference from crystal packing, and was frequently reported in our preceding MD studies performed with the AMBER force field. However, as CHARMM simulations progressed, the four continuously stacked adenines showed conformational transitions from the bulged‐out into the bulged‐in geometries. Although such an arrangement has not been seen in any X‐ray structure, it has been suggested by a recent NMR investigation. In CHARMM simulations, in the longer time scale, the flanking purines display the tendency to move to bulged‐in conformations. This is in contrast with the AMBER simulations, which indicate a modest prevalence for bulged‐out flanking base positions in line with the X‐ray data. The simulations also suggest that the intermolecular stacking between purines from the opposite hairpins can additionally stabilize the kissing complex.