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Biomedical subjects

R A Mathies

Publications and source records attributed to R A Mathies.

At least 19 recordsLinked to original sources
PubMed1992

Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.

Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.

Bacteriorhodopsins↗
PubMed1992

DNA sequencing using capillary array electrophoresis.

A DNA sequencing method is presented that utilizes capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments are separated on an array of capillaries and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA sequencing fragments are separated in a single capillary and then distinguished by using a binary coding scheme where each fragment set is labeled with a characteristic ratio of two dye-labeled primers. Since only two dye-labeled primers are required, it is possible to select dyes that have identical mobility shifts. It is also shown that the ratio of the signal in the two detection channels provides a reliable identification of the sequencing fragment. DNA sequencing results on a 25-capillary array are presented.

Base Sequence↗
PubMed1992

Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.

Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.

Bacteriorhodopsins↗
PubMed1992

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.

Benzoxazoles↗
PubMed1992

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1991

Rapid-flow resonance Raman spectroscopy of bacterial photosynthetic reaction centers.

Rapid-flow resonance Raman vibrational spectra of bacterial photosynthetic reaction centers from the R-26 mutant of Rhodobacter sphaeroides have been obtained by using excitation wavelengths (810-910 nm) resonant with the lowest energy, photochemically active electronic absorption. The technique of shifted excitation Raman difference spectroscopy is used to identify genuine Raman scattering bands in the presence of a large fluorescence background. The comparison of spectra obtained from untreated reaction centers and from reaction centers treated with the oxidant K3Fe(CN)6 demonstrates that resonance enhancement is obtained from the special pair. Relatively strong Raman scattering is observed for special pair vibrations with frequencies of 36, 94, 127, 202, 730, and 898 cm-1; other modes are observed at 71, 337, and 685 cm-1. Qualitative Raman excitation profiles are reported for some of the strong modes, and resonance enhancement is observed to occur throughout the near-IR absorption band of the special pair. These Raman data determine which vibrations are coupled to the optical absorption in the special pair and, thus, probe the nuclear motion that occurs after electronic excitation. Implications for the interpretation of previous hole-burning experiments and for the excited-state dynamics and photochemistry of reaction centers are discussed.

Kinetics↗
PubMed1991

The first step in vision: femtosecond isomerization of rhodopsin.

The kinetics of the primary event in vision have been resolved with the use of femtosecond optical measurement techniques. The 11-cis retinal prosthetic group of rhodopsin is excited with a 35-femtosecond pump pulse at 500 nanometers, and the transient changes in absorption are measured between 450 and 580 nanometers with a 10-femtosecond probe pulse. Within 200 femtoseconds, an increased absorption is observed between 540 and 580 nanometers, indicating the formation of photoproduct on this time scale. These measurements demonstrate that the first step in vision, the 11-cis----11-trans torsional isomerization of the rhodopsin chromophore, is essentially complete in only 200 femtoseconds.

Animals↗
PubMed1991

High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.

Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes.

Benzothiazoles↗
PubMed1991

Resonance Raman spectra of bacteriorhodopsin mutants with substitutions at Asp-85, Asp-96, and Arg-82.

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.

Amino Acid Sequence↗
PubMed1991

High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner.

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Resonance Raman analysis of the Pr and Pfr forms of phytochrome.

Resonance Raman vibrational spectra of the Pr and Pfr forms of oat phytochrome have been obtained at room temperature. When Pr is converted to Pfr, new bands appear in the C = C and C = N stretching region at 1622, 1599, and 1552 cm-1, indicating that a major structural change of the chromophore has occurred. The Pr to Pfr conversion results in an 11 cm-1 lowering of the N-H rocking band from 1323 to 1312 cm-1. Normal mode calculations correlate this frequency drop with a Z----E isomerization about the C15 = C16 bond. A line at 803 cm-1 in Pr is replaced by an unusually intense mode at 814 cm-1 in Pfr. Calculations on model tetrapyrrole chromophores suggest that these low-wavenumber modes are hydrogen out-of-plane (HOOP) wagging vibrations of the bridging C15 methine hydrogen and that both the intensity and frequency of the C15 HOOP mode are sensitive to the geometry around the C14-C15 and C15 = C16 bonds. The large intensity of the 814-cm-1 mode in Pfr indicates that the chromophore is highly distorted from planarity around the C15 methine bridge. If the Pr----Pfr conversion does involve a C15 = C16 Z----E isomerization, then the intensity of the C15 HOOP mode in Pfr argues that the chromophore has an E,anti conformation. On the basis of a comparison with the vibrational calculations, the low frequency (803 cm-1) and the reduced intensity of the C15 HOOP mode in Pr suggest that the chromophore in Pr adopts the C15-Z,syn conformation.

Edible Grain↗
PubMed1990

Structure of the retinal chromophore in 7,9-dicis-rhodopsin.

Bovine rhodopsin was bleached and regenerated with 7,9-dicis-retinal to form 7,9-dicis-rhodopsin, which was purified on a concanavalin A affinity column. The absorption maximum of the 7,9-dicis pigment is 453 nm, giving an opsin shift of 1600 cm-1 compared to 2500 cm-1 for 11-cis-rhodopsin and 2400 cm-1 for 9-cis-rhodopsin. Rapid-flow resonance Raman spectra have been obtained of 7,9-dicis-rhodopsin in H2O and D2O at room temperature. The shift of the 1654-cm-1 C = N stretch to 1627 cm-1 in D2O demonstrates that the Schiff base nitrogen is protonated. The absence of any shift in the 1201-cm-1 mode, which is assigned as the C14-C15 stretch, or of any other C-C stretching modes in D2O indicates that the Schiff base C = N configuration is trans (anti). Assuming that the cyclohexenyl ring binds with the same orientation in 7,9-dicis-, 9-cis-, and 11-cis-rhodopsins, the presence of two cis bonds requires that the N-H bond of the 7,9-dicis chromophore points in the opposite direction from that in the 9-cis or 11-cis pigment. However, the Schiff base C = NH+ stretching frequency and its D2O shift in 7,9-dicis-rhodopsin are very similar to those in 11-cis- and 9-cis-rhodopsin, indicating that the Schiff base electrostatic/hydrogen-bonding environments are effectively the same. The C = N trans (anti) Schiff base geometry of 7,9-dicis-rhodopsin and the insensitivity of its Schiff base vibrational properties to orientation are rationalized by examining the binding site specificity with molecular modeling.

Animals↗
PubMed1990

Solid-state NMR studies of the mechanism of the opsin shift in the visual pigment rhodopsin.

Solid-state 13C NMR spectra have been obtained of bovine rhodopsin and isorhodopsin regenerated with retinal selectively 13C labeled along the polyene chain. In rhodopsin, the chemical shifts for 13C-5, 13C-6, 13C-7, 13C-14, and 13C-15 correspond closely to the chemical shifts observed in the 11-cis protonated Schiff base (PSB) model compound. Differences in chemical shift relative to the 11-cis PSB chloride salt are observed for positions 8 through 13, with the largest deshielding (6.2 ppm) localized at position 13. The localized deshielding at C-13 supports previous models of the opsin shift in rhodopsin that place a protein perturbation in the vicinity of position 13. Spectra obtained of isorhodopsin regenerated with 13C-labeled 9-cis-retinals reveal large perturbations at 13C-7 and 13C-13. The similar deshielding of the 13C-13 resonance in both pigments supports the presence of a protein perturbation near position 13. However, the chemical shifts at positions 7 and 12 in isorhodopsin are not analogous to those observed in rhodopsin and suggest that the binding site interactions near these positions are different for the two pigments. The implications of these results for the mechanism of the opsin shift in these proteins are discussed.

Animals↗
PubMed1990

Optimization of high-sensitivity fluorescence detection.

We present general expressions for the number of photons emitted by a fluorescent chromophore as a function of the intensity and the duration of illumination. The aim is to find optimal conditions for detecting fluorescent molecules in the presence of both ground-state depletion and photodestruction. The key molecular parameters are the absorption coefficient epsilon, the excited singlet-state lifetime tau f, the excited triplet-state decay rate kT, the intersystem crossing rate kI, and the intrinsic photodestruction time tau d. When only singlet saturation and photochemistry are important, the signal-to-noise ratio depends on two fundamental variables: k, the ratio of the absorption rate ka to the observed fluorescence decay rate kf, and tau, the ratio of the duration of illumination taut to the intrinsic photodestruction time tau d. Equations are also developed for the more complicated cases when triplet formation and photochemistry are important. This theory was tested by measuring the fluorescence from a solution of beta-phycoerythrin flowed through a focused argon ion laser beam. The dependence of the fluorescence on the incident light intensity and the illumination time agrees well with the theoretical prediction for singlet saturation and photochemistry. The signal-to-noise ratio is optimal when the light intensity and the flow rate are adjusted so that both K and tau are close to unity (5 X 10(22) photons cm-2 s-1 and a transit time tau t of 700 mus). This analysis should be useful for optimizing fluorescence detection in DNA sequencing, chromatography, fluorescence microscopy, and single-molecular fluorescence detection.

Fluorescence↗
PubMed1990

The role of back-reactions and proton uptake during the N----O transition in bacteriorhodopsin's photocycle: a kinetic resonance Raman study.

The kinetics of bacteriorhodopsin's photocycle have been analyzed at pH 5, 6, 7, 8, and 8.6 by using time-resolved resonance Raman spectroscopy. The concentrations of the various intermediates as a function of time were determined by following their resonance Raman intensities using 502-nm (L550, N550, BR568), 458-nm (M412), and 752-nm (O640) excitation. The spectral contributions to the pump + probe data from each intermediate were quantitatively separated by least-squares decomposition. These relative concentrations were then converted to absolute concentrations by using a conservation of molecules constraint. This enabled the unambiguous refinement of a variety of kinetic models to find the simplest one that accurately describes the data. The kinetic data, including the biphasic decay of L550 and M412, are best reproduced by a sequential scheme including back-reactions (BR----L----M----N----O----BR). In addition, the kinetics of the L----M and N----O steps are found to be pH-dependent. Both the forward and reverse rate constants connecting L550 and M412 increase with pH, confirming earlier proposals of catalyzed Schiff base deprotonation at alkaline pH. Below pH 7, the N550----O640 rate constant is independent of pH, but it decreases linearly with pH above 7. This indicates that the protein must pick up a proton during the N550----O640 transition and that this process becomes rate determining above pH 7. There must, therefore, be an intermediate between N550 and O640 which we denote as N+550. A molecular graphics model is presented which incorporates these observations into a mechanism for proton pumping.

Bacteriorhodopsins↗
PubMed1990

A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.

The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

DNA↗