PubMed Health⌕ Search

Biomedical subjects

T Kulinski

Publications and source records attributed to T Kulinski.

8 recordsLinked to original sources
PubMed2000

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.

Base Pairing↗
PubMed1998

A molecular dynamics computer simulation study of nucleotide analogues. Comparison of the hydration pattern of dithymidine phosphate with those of the dithymidine methylphosphonate diastereomers.

The hydration pattern of thymidyl(3'->5') thymidine 1 and those of Rp and Sp diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between Rp and Sp diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5'-yl units.

Computer Simulation↗
PubMed1997

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

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, and are consistent with the extended and folded backbone conformations of two alpha-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.

Chemical Phenomena↗
PubMed1995

Dynamics of the peptide hormone motilin studied by time resolved fluorescence spectroscopy.

Time resolved fluorescence was used to study the dynamics on the nanosecond and subnanosecond time scale of the peptide hormone motilin. The peptide is composed of 22 amino acid residues and has one tyrosine residue in position 7, which was used as an intrinsic fluorescence probe. The measurements show that two rotational correlation times, decreasing with increasing temperature, are needed to account for the fluorescence polarization anisotropy decay data. Viscosity measurements combined with the fluorescence measurements show that the rotational correlation times vary approximately as viscosity with temperature. The shorter rotational correlation time (0.08 ns in an aqueous solution with 30% hexafluoropropanol, HFP at 20 degrees C) should be related to internal movement of the tyrosine side chain in the peptide while the longer rotational correlation time (2.2 ns in 30% HFP at 20 degrees C) describes the motion of the whole peptide. In addition, the interaction of motilin or the derivative motilin (Y7F) -23W (with tyrosine substituted by phenylalanine and with a tryptophan fluorophore added to the C-terminal) with negatively charged phospholipid vesicles (DOPG) was studied. The results show the development of a long anisotropy decay time which reflects partial immobilization of the peptide by interaction with the vesicles.

Animals↗
PubMed1993

Site specific point mutation changes specificity: a molecular modeling study by free energy simulations and enzyme kinetics of the thermodynamics in ribonuclease T1 substrate interactions.

We have theoretically and experimentally studied the binding of two different ligands to wild-type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrate 3'-GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3'-monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3'GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i,e., the perturbation from Glu to Gln, than in the perturbation from 3'-GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength.

2-Aminopurine↗
PubMed1993

The interaction of pyrene labeled diacylglycerol with protein kinase C in mixed micelles.

The binding of protein kinase C (PKC) to pyrene-labeled diacylglycerol (pDG) has been studied in a mixed micellar system by monitoring resonance energy transfer from excited tryptophans to pyrene with time-correlated single photon counting. The average lifetime of the excited state of the tryptophans in PKC showed a clear dependence on the mole percentage pDG in micelles in contrast with pyrene-labeled phosphatidylcholine (pPC). The binding data has been analyzed to a simple model which encompasses the size of the micelles and the binding constant of the pDG-PKC complex. From our data, though, these quantities cannot be determined independently. If we have no size information on the micelles we can determine a lower boundary of this quantity compatible with the data. When the micellar size is known, a binding constant for the DG-PKC complex can be extracted. The presented analytical approach can be applied to other systems in which lipid-protein interactions must be quantified.

Animals↗
PubMed1987

Spectroscopic investigations of the single tryptophan residue and of riboflavin and 7-oxolumazine bound to lumazine apoprotein from Photobacterium leiognathi.

Spectroscopic techniques have been applied to investigate the conformation, local structure, and dynamic properties of the apoprotein of the lumazine protein from Photobacterium leiognathi and the holoprotein reconstituted with either the natural ligand 6,7-dimethyl-8-ribityllumazine or the closely related analogues riboflavin and 6-methyl-7-oxo-8-ribityllumazine (7-oxolumazine). The analogues are bound similarly to the natural prosthetic group. They exhibit similar shifts on binding in their absorption and fluorescence spectra, single-exponential fluorescence decays, and no independent motion from the protein as evident from a long-lived anisotropy decay (single-exponential phi = 10 ns, 20 degrees C) and high initial anisotropy. Steady-state anisotropy measurements result in similar KD's (40 nM, 20 degrees C, 50 mM inorganic phosphate) for all ligands. Circular dichroism in the far-UV region (190-250 nm) indicates no change in secondary structure on binding to the apoprotein. In the spectral region of 250-310 nm relatively large changes occur, indicating changes in the environment of the tyrosine and tryptophan residues. The single tryptophan residue shows a three-exponential decay of its fluorescence in both the apoprotein and the holoprotein. Radiationless energy transfer also occurs from the tryptophan to the bound ligand, especially evident with 7-oxolumazine. We have designed a new method for evaluation of the rate constant of energy transfer by measuring the (picosecond) rise time of the acceptor fluorescence. The anisotropy decay of the tryptophan residue shows two correlation times, a short one (phi approximately equal to 0.4 ns) representing rapid but restriced oscillation of this residue and a longer one (phi 2 = 5-7 ns, 20 degrees C) representing the motion of a larger segment of the protein.

Apoproteins↗