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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TL;DR: This work prepared a crowded environment in vitro consisting of hexaethylene glycol monododecyl ether and water and observed lysozyme diffusion between elongated micelles and fitted an anomalous diffusion model and a two-component normal diffusion model.
Abstract: Small proteins move in crowded cell compartments by anomalous diffusion. In many of them, e.g., the endoplasmic reticulum, the proteins move between lipid membranes in the aqueous lumen. Molecular crowding in vitro offers a systematic way to study anomalous and normal diffusion in a well controlled environment not accessible in vivo. We prepared a crowded environment in vitro consisting of hexaethylene glycol monododecyl ether (C12E6) nonionic surfactant and water and observed lysozyme diffusion between elongated micelles. We have fitted the data obtained in fluorescence correlation spectroscopy using an anomalous diffusion model and a two-component normal diffusion model. For a small concentration of surfactant (below 4 wt %) the data can be fitted by single-component normal diffusion. For larger concentrations the normal diffusion fit gave two components: one very slow and one fast. The amplitude of the slow component grows with C12E6 concentration. The ratio of diffusion coefficients (slow to fast) is...
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TL;DR: This chapter describes the implications for quantitative microscopy of the green fluorescent protein from the jellyfish Aequorea victoria and its mutants, which constitute a class of fluorophores that has revolutionized the evaluation of molecular interactions and visualization of biological systems.
Abstract: Publisher Summary This chapter describes the implications for quantitative microscopy. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its mutants constitute a class of fluorophores that has revolutionized the evaluation of molecular interactions and visualization of biological systems. When fused to proteins of interest and expressed in vivo , fluorescent proteins (FPs) act as versatile indicators of structure and function within cells and can be imaged with the full repertoire of fluorescence microscopy techniques. FPs and their constructs are finding increasing use in fluorescence lifetime imaging microscopy (FLIM) and fluorescence resonance energy transfer (FRET) modes of microspectroscopy for the elucidation of protein-protein interactions, signaling, and trafficking in cellular systems. The simple and the most common, application of FPs is as a passive marker fused to a target protein of interest for visualizing its spatiotemporal distribution. Mutations in and around the chromophore have systematic effects on the spectra. Green and red fluorescent proteins exhibit photophysical properties that are generally more complex than those of traditional fluorophores, for example, fluorescein and rhodamine.
39 citations
TL;DR: Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.
Abstract: Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chainreaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that meltingtemperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8 � C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.
38 citations
Cites background from "Rapid Diffusion of Green Fluorescen..."
...Rough estimates of MMC in terms of solids in solution range from 300– 400mg/ml in Escherichia coli (2), over 50–400mg/ml in eukaryotic cytoplasm (1), 270–560mg/ml in mitochondria (2) and 100–400mg/ml in nuclei (3)....
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TL;DR: Mge1p can act as a thermosensor for the mitochondrial Hsp70 system, regulating the nucleotide exchange rates under heat shock, as has been described for two bacterial GrpE proteins.
38 citations
TL;DR: The first families carrying recessive variants in the MSTO1 gene were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia, confirming the deleterious effect of the identified variants and the role of M STO1 in modulating mitochondrial dynamics.
Abstract: We report here the first families carrying recessive variants in the MSTO1 gene: compound heterozygous mutations were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia Human MSTO1 is a poorly studied protein, suggested to have mitochondrial localization and to regulate morphology and distribution of mitochondria As for other mutations affecting genes involved in mitochondrial dynamics, no biochemical defects typical of mitochondrial disorders were reported Studies in patients' fibroblasts revealed that MSTO1 protein levels were strongly reduced, the mitochondrial network was fragmented, and the fusion events among mitochondria were decreased, confirming the deleterious effect of the identified variants and the role of MSTO1 in modulating mitochondrial dynamics We also found that MSTO1 is mainly a cytosolic protein These findings indicate recessive mutations in MSTO1 as a new cause for inherited neuromuscular disorders with multisystem features
37 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