Biomedical subjects

R A Mathies

Publications and source records attributed to R A Mathies.

113 records · Page 7Linked to original sources

PubMed1985

Resonance Raman spectra of the acidified and deionized forms of bacteriorhodopsin.

The 568-nm absorption band of light-adapted bacteriorhodopsin (BR) shifts to 605 nm at pH 2, forming BR605A, and it shifts back to 565 nm at pH 0, forming BR565A. We have obtained resonance Raman spectra of BR605A and BR565A using purple membrane samples that have been suspended in a rotating Raman cell with a polyacrylamide gel. Raman spectra were also obtained of purple membrane in deionized solutions (BR605D). The spectra of BR605A and BR605D are very similar, and they correspond closely with the Raman spectrum of dark-adapted BR, which contains an approximately equal mixture of 13-cis and all-trans retinal protonated Schiff-base chromophores. This shows that BR605A and BR605D are not homogeneous molecular species but contain a mixture of pigment molecules with both 13-cis and all-trans retinal isomers. The Raman spectrum of BR565A is nearly identical to that of light-adapted BR, demonstrating that BR565A contains an all-trans protonated Schiff-base chromophore. These data provide constraints on the possible structural changes that can be invoked to explain the spectral shifts induced in the acid and deionized species.

Bacteriorhodopsins↗

PubMed1985

Vibrational analysis of the all-trans retinal protonated Schiff base.

We have obtained Raman spectra of a series of all-trans retinal protonated Schiff-base isotopic derivatives. 13C-substitutions were made at the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 positions while deuteration was performed at position 15. Based on the isotopic shifts, the observed C--C stretching vibrations in the 1,100-1,400 cm-1 fingerprint region are assigned. Normal mode calculations using a modified Urey-Bradley force field have been refined to reproduce the observed frequencies and isotopic shifts. Comparison with fingerprint assignments of all-trans retinal and its unprotonated Schiff base shows that the major effect of Schiff-base formation is a shift of the C14--C15 stretch from 1,111 cm-1 in the aldehyde to approximately 1,163 cm-1 in the Shiff base. This shift is attributed to the increased C14--C15 bond order that results from the reduced electronegativity of the Schiff-base nitrogen compared with the aldehyde oxygen. Protonation of the Schiff base increases pi-electron delocalization, causing a 6 to 16 cm-1 frequency increase of the normal modes involving the C8--C9, C10--C11, C12--C13, and C14--C15 stretches. Comparison of the protonated Schiff base Raman spectrum with that of light-adapted bacteriorhodopsin (BR568) shows that incorporation of the all-trans protonated Schiff base into bacterio-opsin produces an additional approximately 10 cm-1 increase of each C--C stretching frequency as a result of protein-induced pi-electron delocalization. Importantly, the frequency ordering and spacing of the C--C stretches in BR568 is the same as that found in the protonated Schiff base.

Retinaldehyde↗

PubMed1984

Structure of the retinal chromophore in the hR578 form of halorhodopsin.

Halorhodopsin is a retinal-containing pigment that is thought to function as a light-driven chloride ion pump in the cell membrane of Halobacterium halobium. To address the role of the retinal chromophore in chloride ion transport, resonance Raman spectra have been obtained of the hR578 form of chromatographically purified halorhodopsin (hR). The close similarity of the frequencies and intensities of the hR578 Raman bands with those of light-adapted bacteriorhodopsin (bR568) shows that the chromophore in hR578 has an all-trans configuration and that the protein environment around the chromophore in these two pigments is very similar. In addition, hR578 exhibits a Raman line at 1633 cm-1 which is assigned as the stretching vibration of a protonated Schiff base linkage to the protein based on its shift to 1627 cm-1 in D2O. The reduced frequency of the Schiff base stretching vibration compared with bR568 (1640 cm-1) is shown to result from a reduction of its coupling with the NH in-plane rock. This may be due to a reduction in hydrogen-bonding between the Schiff base proton and an electronegative counterion in halorhodopsin.

Bacteriorhodopsins↗

PubMedDate not supplied

On modeling the vibrational spectra of 14-s-cis retinal conformers in bacteriorhodopsin.

The vibrational properties of 13-cis, 14-s-trans and 13-cis, 14-s-cis protonated retinal Schiff base model compounds are explored with MNDO calculations. In particular, the effect of isomerization about the C14-C15 single bond on the vibrational properties of the deuterium in-plane rocking vibrations has been examined. Our MNDO calculations, using a variety of lysine models, lysine conformations and Schiff base charge environments, demonstrate that the C14-D and C15-D in-plane rocking vibrations in the 14,15-dideuterio retinal protonated Schiff base are strongly coupled in 13-cis, 14-s-cis molecules producing a splitting of ca. 80 cm-1 between the symmetric and antisymmetric rocking mode combinations but that these modes are only weakly coupled in 14-s-trans molecules. This analysis demonstrates that the 14,15-dideuterio labeling method developed earlier for determining C14-C15 conformation (S.P.A. Fodor, W. T. Pollard, R. Gebhard, E. M. M. van den Berg, J. Lugtenburg and R. A. Mathies, Proc. Natl. Acad. Sci. USA, 85, (1988) 2156-2160) is valid, and hence that the structure of the retinal chromophore in bacteriorhodopsin's L550 intermediate is 13-cis, 14-s-trans. The reasons for the misleading conclusions derived from MNDO calculations performed earlier by Schulten and Tavan are discussed.

Bacteriorhodopsins↗

PubMedDate not supplied

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.

DNA↗