Biomedical subjects

Jeffrey A Stuart

Publications and source records attributed to Jeffrey A Stuart.

7 recordsLinked to original sources

PubMed2006

Photoinduced surface potential change of bacteriorhodopsin mutant D96N measured by scanning surface potential microscopy.

We report the direct measurement of photoinduced surface potential differences of wild-type (WT) and mutant D96N bacteriorhodopsin (BR) membranes at pH 7 and 10.5. Atomic force microscopy (AFM) and scanning surface potential microscopy (SSPM) were used to measure the BR membrane with the extracellular side facing up. We present AFM and SSPM images of WT and mutant D96N in which the light-dark transition occurred in the mid-scan of a single BR membrane. Photosteady-state populations of the M state were generated to facilitate measurement in each sample. The photoinduced surface potential of D96N is 63 mV (peak to valley) at pH 10.5 and is 48 mV at pH 7. The photoinduced surface potential of WT is 37 mV at pH 10.5 and approximately 0 at pH 7. Signal magnitudes are proportional to the amount of M produced at each pH. The results indicated that the surface potentials were generated by photoformation of surface charges on the extracellular side of the membrane. Higher surface potential correlated with a longer lifetime of the charges. A mechanistic basis for these signals is proposed, and it is concluded that they represent a steady-state measurement of the B2 photovoltage.

Bacteriorhodopsins↗

PubMed2006

Structure, function, and wavelength selection in blue-absorbing proteorhodopsin.

The absorption maximum of blue proteorhodopsin (BPR) is the most blue-shifted of all retinal proteins found in archaea or bacteria, with the exception of sensory rhodopsin II (SRII). The absorption spectrum also exhibits a pH dependence larger than any other retinal protein. We examine the structural origins of these two properties of BPR by using optical spectroscopy, homology modeling, and molecular orbital theory. Bacteriorhodopsin (BR) and SRII are used as homology parents for comparative purposes. We find that the tertiary structure of BPR based on SRII is more realistic with respect to free energy, dynamic stability, and spectroscopic properties. Molecular orbital calculations including full single- and double-configuration interaction within the chromophore pi-electron system provide perspectives on the wavelength regulation in this protein and indicate that Arg-95, Gln-106, Glu-143, and Asp-229 play important, and in some cases pH-dependent roles. A possible model for the 0.22 eV red shift of BPR at low pH is examined, in which Glu-143 becomes protonated and releases Arg-95 to rotate up into the binding site, altering the electrostatic environment of the chromophore. At high pH, BPR has spectroscopic properties similar to SRII, but at low pH, BPR has spectroscopic properties more similar to BR. Nevertheless, SRII is a significantly better homology model for BPR and opens up the question of whether this protein serves as a proton pump, as commonly believed, or is a light sensor with structure-function properties more comparable to those of SRII. The function of BPR in the native organism is discussed with reference to the results of the homology model.

Amino Acid Sequence↗

PubMed2006

Mitochondrial DNA maintenance and bioenergetics.

Oxidative phosphorylation requires assembly of the protein products of both mitochondrial and of nuclear genomes into functional respiratory complexes. Cellular respiration can be compromised when mitochondrial DNA (mtDNA) sequences are corrupted. Oxidative damage resulting from reactive oxygen species (ROS) produced during respiration is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction. Here, we review mechanisms of mitochondrial ROS production, mtDNA damage and its relationship to mitochondrial dysfunction. We focus particular attention on the roles of mtDNA repair enzymes and processes by which the integrity of the mitochondrial genome is maintained and dysfunction prevented.

Animals↗

PubMed2005

Energy, quiescence and the cellular basis of animal life spans.

Animals are routinely faced with harsh environmental conditions in which insufficient energy is available to grow and reproduce. Many animals adapt to this challenge by entering a dormant, or quiescent state. In some animals, such as the nematode Caenorhabditis elegans, quiescence is coincident with increased stress resistance and longevity. Here we review evidence that the rules of life span extension established in C. elegans may be generally true of most animals. That is, that the rate of animal aging correlates inversely with cellular resistance to physiological stress, particularly oxidative stress, and that stress resistance is co-regulated with the quiescence adaptation (where the latter occurs). We discuss evidence for highly conserved intracellular signalling pathways involved in energy sensing that are sensitive to aspects of mitochondrial energy transduction and can be modulated in response to energetic flux. We provide a broad overview of the current knowledge of the relationships between energy, metabolism and life span.

Adaptation, Physiological↗

PubMed2005

Characterization and photochemistry of 13-desmethyl bacteriorhodopsin.

The photochemistry of the 13-desmethyl (DM) analogue of bacteriorhodopsin (BR) is examined by using spectroscopy, molecular orbital theory, and chromophore extraction followed by conformational analysis. The removal of the 13-methyl group permits the direct photochemical formation of a thermally stable, photochemically reversible state, P1(DM) (lambda(max) = 525 nm), which can be generated efficiently by exciting the resting state, bR(DM) with yellow or red light (lambda > 590 nm). Chromophore extraction analysis reveals that the retinal configuration in P1(DM) is 9-cis, identical to that of the retinal configuration in the native BR P1 state. Fourier transform infrared and Raman experiments on P1(DM) indicate an anti configuration around the C15=N bond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient, time-resolved studies indicate that the P1(DM) state forms primarily via thermal relaxation from the L(D)(DM) state. Theoretical studies on the BR binding site show that 13-dm retinal is capable of isomerizing into a 9-cis configuration with minimal steric hindrance from surrounding residues, in contrast to the native chromophore in which surrounding residues significantly obstruct the corresponding motion. Analysis of the photokinetic experiments indicates that the Arrhenius activation energy of the bR(DM) --> P1(DM) transition in 13-dm-BR is less than 0.6 kcal/mol (vs 22 +/-5 kcal/mol measured for the bR --> P (P1 and P2) reaction in 85:15 glycerol:water suspensions of wild type). Consequently, the P1(DM) state in 13-dm-BR can form directly from all-trans, 15-anti intermediates (bR(DM) and O(DM)) or all-trans, 15-syn (K(D)(DM)/L(D)(DM)) intermediates. This study demonstrates that the 13-methyl group, and its interactions with nearby binding site residues, is primarily responsible for channeling one-photon photochemical and thermal reactions and is limited to the all-trans and 13-cis species interconversions in the native protein.

Bacteriorhodopsins↗

PubMed2004

Photoelectric response of polarization sensitive bacteriorhodopsin films.

Polarization sensitivity is introduced into oriented bacteriorhodopsin (BR) films through a photochemical bleaching process, which chemically modifies the structure of the purple membrane by breaking the intrinsic symmetry of the membrane-bound BR trimers. The resulting photovoltage generated in an indium-tin oxide (ITO)/BR/ITO detector is found to be anisotropic with respect to cross-polarized probe beams. A model, based on the polarization dependent photoselection of the BR molecules qualitatively explains the photochemical bleaching process and the observed anisotropic response. The effect reported here can be used to construct a polarization sensitive BR-based bio-photoreceiver.

Anisotropy↗

PubMed2002

Optimization of bacteriorhodopsin for bioelectronic devices.

Bacteriorhodopsin (BR) is the photoactive proton pump found in the purple membrane of the salt marsh archaeon Halobacterium salinarum. Evolution has optimized this protein for high photochemical efficiency, thermal stability and cyclicity, as the organism must be able to function in a hot, stagnant and resource-limited environment. Photonic materials generated via organic chemistry have yet to surpass the native protein in terms of quantum efficiency or cyclicity. However, the native protein still lacks the overall efficiency necessary for commercial viability and virtually all successful photonic devices using bacteriorhodopsin are based on chemical or genetic variants of the native protein. We show that genetic engineering can provide significant improvement in the device capabilities of proteins and, in the case of bacteriorhodopsin, a 700-fold improvement has been realized in volumetric data storage. We conclude that semi-random mutagenesis and directed evolution will play a prominent role in future efforts in bioelectronic optimization.

Bacteriorhodopsins↗