Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix
TL;DR: The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers, and it is proposed that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.
Abstract: It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-micron spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, approximately 0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.
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01 Jan 2008
3 citations
Cites background or methods from "Rapid Diffusion of Green Fluorescen..."
...1a, dotted circle), the assumption of one-dimensional diffusion cannot be made (as in [218, 219, 220, 244, 245])....
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...In addition to the whole cell image analysis [218, 219, 220], conventional FRAP measurements were performed on organelles of similar size as a bacterial cell, most notably mitochondria [243, 244]....
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...The half recovery time t 1 2 was taken from the curve, which was then converted into a diffusion constant by using a complex mathematical model [244, 245, 246] to account for the orientation and geometry of the mitochondria....
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01 Jan 2006
TL;DR: In this paper, the cell biology of AMPAR synthesis, transport, targeting and surface expression is of crucial importance for correct neuronal function, which relies on spatially and temporally coordinated protein-protein interactions to facilitate and regulate receptor trafficking.
Abstract: The cell biology of AMPAR synthesis, transport, targeting and surface expression is of crucial importance for correct neuronal function. These processes rely on spatially and temporally coordinated protein-protein interactions to facilitate and regulate receptor trafficking. While considerable advances have been achieved in unravelling the complexity and roles of some of these interactions, the dynamic aspects of AMPAR trafficking are less well-characterized. We have been working to visualize AMPAR movement in living hippocampal neurons to define the properties of AMPAR trafficking under basal and activated conditions. Here, we review current techniques and the progress achieved.
3 citations
01 Jan 2010
TL;DR: This thesis is focused on the role of mitochondria in pancreatic β-cells and brown adipose tissue and the two main aspects of mitochondrial functional efficiency and the ...
Abstract: This thesis is focused on the role of mitochondria in pancreatic β-cells and brown adipose tissue (BAT). Two main aspects of mitochondria were explored; mitochondrial functional efficiency and the ...
3 citations
01 Jan 2013
TL;DR: Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state.
Abstract: Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state. The process has been character ...
2 citations
01 Jan 2013
TL;DR: Interestingly, urate increased superoxide production and oxygen consumption by neutrophil-like cells (differentiated HL-60 cells).
Abstract: PATRICIO, E. S. Evaluation of the interaction of urate hydroperoxide with protein disulfide isomerase (PDI) in inflammatory processes. 2014. 99 p. M. Sc. Dissertation – Graduate Program in Biochemistry, Chemistry Institute, University of Sao Paulo, Sao Paulo. The oxidation of the uric acid (7,9-dihidro-1H-purine-2,6,8(3H)-trione) by myeloperoxidase (MPO) generates the urate radical. In inflammatory conditions the superoxide reacts with urate radical to form the urate hydroperoxide (HOOU). Taking into account the high amount of MPO, urate and superoxide in the atheroma plaque, it is likely that HOOU is being formed in this inflammatory environment. The HOOU is a strong oxidizing agent and can react with thiol groups from proteins, like the protein disulfide isomerase (PDI). As a consequence of its oxidation, PDI positively modulates NADPH oxidase (Nox) and increases superoxide production by neutrophils. Therefore, the formation of HOOU in the vascular sheet would contribute to tissue damage and would explain the positive correlation between hyperuricemia and the risk for cardiovascular disease. To investigate the interaction of HOOU with PDI, we performed the chemical synthesis of the compound by the Type I photooxidation, using UVA irradiation and riboflavin as a photosensitizer. Initially, we standardized the irradiation light and the time of the reaction that produced the highest income. The HOOU and its reduced product 5hydroxiisourate were separate, identified and characterized by liquid chromatography coupled to mass spectrometry (LC/MS). We also determine the molar extinction coefficient of HOOU at 308 nm (ɛ308 = 6537 ± 377 M -1 .cm -1 ). The half-life of the compound was 41 min at 22 o C. The HOOU selectively oxidized methionine and glutathione. The reaction of HOOU with glutathione did not form any stable adducts. Thus, all consumed glutathione generated glutathione disulfide. When incubated with PDI (10 μM), 70 and 100% of the total amount of HOOU (3 μM) was consumed within 30 and 120 seconds, respectively. Besides, the PDI (23 μM) had its thiol groups oxidized after incubation with 140 μM HOOU for 30 min at 22oC. The HOOU oxidized the cysteine residues from the catalytical sites of PDI with a second order rate constant of 1.5 ± 0.04 x 10 3 M 1 .s -1 . This result suggests a favorable interaction of HOOU with this protein in the biological system, as well as a possible modulatory role of HOOU on the PDI-Nox pathway. Interestingly, urate increased superoxide production and oxygen consumption by neutrophil-like cells (differentiated HL-60 cells). This effect could be mediated by the formation of HOOU during the neutrophil oxidative burst, followed by the oxidation of PDI, a positive regulation of Nox and an increase in superoxide production.
2 citations
References
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TL;DR: The theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments are presented and some model experiments on aqueous solutions of rhodamine 6G are described.
2,594 citations
"Rapid Diffusion of Green Fluorescen..." refers background in this paper
...As discussed by Axelrod et al. (1976) for conventional two-dimensional spot photobleaching, this approximation is reasonably valid for practical laser/lens systems; the same considerations would apply for bleaching of long thin mitochondria where bleach profile is nearly constant across the thin…...
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TL;DR: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products.
Abstract: The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.
2,232 citations
TL;DR: The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 Å by multiwavelength anomalous dispersion phasing methods and the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.
Abstract: The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of s-sheet with an α-helix inside and short helical segments on the ends of the cylinder. This motif, with s-structure on the outside and α-helix on the inside, represents a new protein fold, which we have named the s-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr86 with reduction of its Cα-Cs bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.
1,502 citations
"Rapid Diffusion of Green Fluorescen..." refers background in this paper
...The three–amino acid chromophore in GFP is fixed rigidly within a barrel structure (Yang et al., 1996; Örmo et al., 1996)....
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1,070 citations
TL;DR: This minireview has attempted to provide some overall perspective on the question of how various forms of diffusion in reduced dimensions, or diffusion within a nonspecifically bound state, can speed biological interactions beyond the limits normally set by three-dimensional diffusion processes.
1,017 citations