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Vytas Reipa

Publications and source records attributed to Vytas Reipa.

7 recordsLinked to original sources
PubMed2006

Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy.

We have measured the temperature-dependent Raman spectra of two 30-mer ribonucleotides that represent the wild-type (WT) and dyskeratosis congenita (DKC) mutant (MT) GC (107-108) --> AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used these structures, previously characterized by UV-melting and NMR, as a model system for our Raman investigation. We observe that Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, reflect temperature-dependent alterations in the pentaloop and stem structures of these two oligonucleotides. We also observe that the intense nu(s)(O-P-O) band at 812 cm(-1) indicates the presence of A-form backbone structure at relatively low temperatures in both the WT and MT RNA sequences. The mutation induces a decrease in the intensity of the uridine (rU) band at 1244 cm(-1) associated with C2'-endo/anti ribose conformation in the pentaloop. Two transition temperatures (T(m) ) were determined from the analysis of Raman difference intensity-temperature profiles of the 1256 cm(-1) band, which is associated with vibrations of cytidine (rC) residues, in particular, the C2'-endo/anti ribose conformation (T(m) 1 = 23.6 +/- 1.6 degrees C for WT and 19.7 +/- 2.8 degrees C for MT; T(m) 2 = 68.9 +/- 1.8 degrees C for WT and 70.9 +/- 1.1 degrees C for MT). From these results we can conclude that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region and a slightly higher stability in the stem region than the WT 30-mer. This demonstrates that Raman bands, previously assigned to specific bases or conformational RNA markers, can be used to probe local structural features of the telomerase pseudoknot hairpin sequence.

Base Sequence↗
PubMed2006

Long-term monitoring of biofilm growth and disinfection using a quartz crystal microbalance and reflectance measurements.

A biofilm reactor was constructed to monitor the long-term growth and removal of biofilms as monitored by the use of a quartz crystal microbalance (QCM) and a novel optical method. The optical method measures the reflectance of white light off the surface of the quartz crystal microbalance electrode (gold) for determination of the biofilm thickness. Biofilm growth of Pseudomonas aeruginosa (PA) on the surface was used as a model system. Bioreactors were monitored for over 6 days. Expressing the QCM data as the ratio of changes in resistance to changes in frequency (DeltaR/Deltaf) facilitated the comparison of individual biofilm reactor runs. The various stages of biofilm growth and adaptation to low nutrients showed consistent characteristic changes in the DeltaR/Deltaf ratio, a parameter that reflects changes in the viscoelastic properties of the biofilm. The utility of white light reflectance for thickness measurements was shown for those stages of biofilm growth when the solution was not turbid due to high numbers of unattached cells. The thickness of the biofilms after 6 days ranged from 48 mum to 68 mum. Removal of the biofilm by a disinfectant (chlorine) was also measured in real time. The combination of QCM and reflectance allowed us to monitor in real time changes in the viscoelastic properties and thickness of biofilms over long periods of time.

Biofilms↗
PubMed2005

Optical properties of Alexa 488 and Cy5 immobilized on a glass surface.

The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret these red shifts in terms of a change in the polarizability and polarity of the effective solvent. A formula is given that can be used to estimate expected shifts in absorption and emission maxima for surface-immobilized fluorophores. Spectroscopic ellipsometry measurements provide identification of the fluorophores confined on a glass surface. These results suggest that the design of microarray detection systems should be based on the optical properties of fluorophores attached to the surface and not on the optical properties of fluorophores in solution.

Absorption↗
PubMed2004

Temperature-induced structural changes in putidaredoxin: a circular dichroism and UV-VIS absorption study.

Putidaredoxin (Pdx) is an 11,400-Da iron-sulfur protein that sequentially transfers two electrons to the cytochrome P450cam during the enzymatic cycle of the stereospecific camphor hydroxylation. We report two transitions in the Pdx UV-VIS absorption and circular dichroism (CD) temperature dependencies, occurring at 16.3+/-0.5 degrees C and 28.4+/-0.5 degrees C. The 16.3 degrees C transition is attributed to the disruption of the hydrogen bonding of the active center bridging sulfur atom with cysteine 45 and alanine 46. The transition at 28.4 degrees C occurs exclusively in the Pdx(ox) at very nearly the same temperature as the earlier reported biphasicity in the redox potential. The formal potential temperature slope constancy reflects the relative stability of the concentration ratio of both oxidation states. The lower temperature transition affects both Pdx(red) and Pdx(ox) to a comparable extent, and their concentration ratio remains constant. In contrast, the 28.4 degrees C transition preferentially destabilizes Pdx(ox) thereby accelerating the formal potential negative shift and lower redox reaction entropy. There is evidence to suggest that disrupting hydrogen bonding of the iron ligating cysteines 45, 39 with residues threonine 47, serine 44, glycine 41, and serine 42 causes the 28.4 degrees C transition. The sensitivity of the UV-VIS absorption and CD spectroscopy to subtle structural protein backbone transitions is demonstrated.

Alanine↗
PubMed2004

Direct spectroelectrochemical titration of glutathione.

Previous potentiometric attempts to determine the formal potential (E'(0)) of key intracellular redox buffer glutathione resulted in contradictory values. We have developed a spectroelectrochemical method using direct reduction on metal oxide electrodes. Disulfide absorbance at 258 nm was used to titrate glutathione in the thin layer cell reversibly. At conditions close to physiological ([GSH] = 0.001-0.005 mol/l, pH = 7.34; I = 0.1 mol/l; T = 298.15 K), we have measured glutathione E(0)' = -0.22 +/- 0.02 V (NHE), corroborating the results of equilibrium measurements.

Electrochemistry↗
PubMed2003

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy.

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.

Biochemical Phenomena↗