Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein 1 1Edited by D. C. Rees

Wachter, Rebekka M.; Yarbrough, Daniel K.; Kallio, Karen; Remington, S.J.

doi:10.1006/jmbi.2000.3905

Cited by 161 publications

(226 citation statements)

References 45 publications

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“…However, in addition to the maltose-induced hinge-twist motion, the overall protein structure of the nanosensor may be affected by other factors, such as ionic conditions. Furthermore, EYFP is highly sensitive to halides and acidic pH, because both parameters favor protonation of the chromophore anion (25). Although introduction of a Q69M mutation in EYFP reduces sensitivity (26), nonspecific conformational changes still cannot be overcome.…”

Section: Fig 2 In Vitro Substrate Titrations Of Purified Nanosensormentioning

confidence: 99%

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Fehr

Frommer

Lalonde

2002

Proc. Natl. Acad. Sci. U.S.A.

315

279

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Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing proteinbased nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications. Limited information is derived from methods requiring fixation or fractionation of tissue (1, 2). Static analysis of metabolite composition in organs, tissues, and cellular compartments involves cell disruption. Most techniques neither measure metabolite changes in real-time nor account for likely variations in local metabolite concentration at the cellular level. Current methods have low resolution and are prone to artifacts, e.g., contamination by other cell types or subcellular compartments. Thus, little is known about the dynamic changes in concentration of metabolites such as sugars and amino acids͞neurotransmit-ters at the site of transport, i.e., in the synaptic cleft relative to the cytosol of adjacent neurons and glia or at the loading site of the phloem in plants, but also in the distribution of different sugars within cellular compartments. To better understand metabolism and compartmentation, a noninvasive technique would be of significant advantage.To generate a set of multifunctional nanosensors with specificity to a large number of different compounds, suitable binding proteins fulfilling a number of criteria are required. First, the binding proteins must undergo a conformational change on substrate binding. Ideally, they should belong to a family covering a wide spectrum of substrates. Furthermore, high-affinity binding would be advantageous, because it would provide a comparatively fast way to generate mutants with lower affinities suited for an optimal physiological detection range. Finally, for measurements in eukaryotes, ...

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Section: Fig 2 In Vitro Substrate Titrations Of Purified Nanosensormentioning

confidence: 99%

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Fehr

Frommer

Lalonde

2002

Proc. Natl. Acad. Sci. U.S.A.

315

279

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“…To this end, HeLa cells were cotransfected with cDNAs encoding N-terminal 81 aa residues of the Golgi-resident integral membrane protein GT fused to EYFP, EGFP or ECFP [Llopis et al, 1998]. It is important to highlight that (i) both EGFP and EYFP fluorescence intensities are highly pH-sensitive, (ii) ECFP, which is much less sensitive to pH that either of the others, is used as a reference to correct putative changes in the cell focussing that could modify fluorescence emission only due to expected pH changes, and (iii) only EGFP but no EYFP has been found to be insensitive to chloride concentration [Wachter and Remington, 1999;Wachter et al, 2000]. GT-EGFP, GT-EYFP and GT-ECFP mostly localized to the Golgi complex (middle/ trans cisternae) of HeLa cells [Llopis et al, 1998].…”

Section: Depolymerization But Not Stabilization Of F-actin Raises Intmentioning

confidence: 99%

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

Lázaro‐Diéguez

Jiménez

Barth

et al. 2006

Cell Motil. Cytoskeleton

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Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H þ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis. Cell Motil. Cytoskeleton 63: 778-791, 2006. '

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“…37,38 Other YFPs are very sensitive to halides due to easy ion access via a solvent channel or cavity formed close to the dimer interface. 39,40 In citrine, this cavity is filled by a mutation Gln69Met preventing the access to the ion. 37 A similar effect is desired in mCherry.…”

Section: Introductionmentioning

confidence: 99%

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

Chapagain

Regmi

Castillo

2011

The Journal of Chemical Physics

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Fluorescent proteins (FPs) are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. In this paper, we use molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variant. We employ implicit ligand sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. We also show that these pathways can be blocked or altered and barrel fluctuations can be reduced by strategic amino acid substitutions.

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Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein 1 1Edited by D. C. Rees

Cited by 161 publications

References 45 publications

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

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