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Biomedical subjects

Robert J Stanley

Publications and source records attributed to Robert J Stanley.

10 recordsLinked to original sources
PubMed2005

A cryogenic optical waveguide spectrometer for the measurement of low-temperature absorption spectra of dilute biological samples.

A cryogenic optical waveguide spectrometer that uses a Teflon-AF 2400 liquid core waveguide is described. In comparison to standard low-temperature absorption techniques, the liquid core waveguide approach not only affords the use of microliter samples but also provides significant improvements in sensitivity. Here we show low-temperature absorption spectra of various flavoproteins, including DNA photolyase, measured using this new technique. The technique has high reproducibility and can afford the detection of 15 ng of flavoprotein. In addition, the technique requires several hundredfold less protein than standard low-temperature techniques for the same sensitivity. The performance of the spectrometer in the ultraviolet (UV) region is investigated experimentally and compared with standard UV absorption techniques. Results indicate that, below 300 nm, the observed absorbances deviate from the Beer-Lambert law.

Cold Temperature↗
PubMed2003

Cis-syn thymidine dimer repair by DNA photolyase in real time.

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.

Bacteria↗
PubMed2003

Measurement of the electronic properties of the flavoprotein old yellow enzyme (OYE) and the OYE:p-Cl phenol charge-transfer complex using Stark spectroscopy.

Low-temperature absorption and Stark spectroscopy have been used to study the electronic properties of oxidized flavin mononucleotide (FMN) in old yellow enzyme (OYE) and OYE complexed with p-chlorophenol (p-Cl phenol). The low-temperature absorbance spectrum of OYE showed splittings of the blue and near-UV vibronic bands, which appears to be due to hydrogen bonding between the isoalloxazine moiety and the protein. A Stark spectroscopic analysis showed that the electronic structure of the FMN cofactor in OYE is not significantly perturbed relative to flavins in simple solvents. However, the charge-transfer band in the OYE:p-Cl phenol complex showed a large Stark effect indicative of substantial charge displacement. The magnitude and direction of this charge displacement are consistent with significant charge transfer along the charge-transfer transition dipole moment direction. In addition, the Stark spectrum of the CT band showed unexpected fine structure that could correlate with vibrational progressions in either the p-Cl phenol donor or the flavin acceptor.

Chlorophenols↗
PubMed2002

Cyclobutylpyrimidine dimer base flipping by DNA photolyase.

DNA Photolyase is a flavoprotein that uses light to repair cyclobutylpyrimidine dimers in DNA. From considerations of the crystal structure of the protein, it has been hypothesized that the dimer lesion is flipped out of the DNA double helix into the substrate binding pocket. We have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a probe of local double helical structure upon binding of the substrate to the protein. Our results show that the local structure around the thymidine lesion changes dramatically upon binding to Photolyase. This is consistent with base flipping of the lesion into the protein binding cavity with concomitant destacking of the opposing complementary 2-Ap nucleotide.

Base Pairing↗