ZnO nanoparticles assist the refolding of denatured green fluorescent protein

Proteins are essential for cellular and biological processes. Proteins are synthesized and fold into the native structure to become active. The inability of a protein molecule to remain in its native conformation is called as protein misfolding, and this is due to several environmental factors. Protein misfolding and aggregation handle several human diseases. Protein misfolding is believed to be one of the causes of several disorders such as cancer, degenerative diseases, and metabolic pathologies. The zinc oxide (ZnO) nanoparticle was significantly promoted refolding of thermally denatured green fluorescent protein (GFP). In the present study, ZnO nanoparticles interaction with GFP was investigated by ultraviolet ‐ visible spectrophotometer, fluorescence spectrophotometer, and dynamic light scattering. Results suggest that the ZnO nanoparticles significantly assist the refolding of denatured GFP. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of SpyTag/SpyCatcher cyclization on stability and refolding of green fluorescent protein

Biotechnology Letters - To study the effect of SpyTag/SpyCatcher cyclization on stability and refolding of protein, we constructed a cyclized green fluorescent protein (SRGFP) and its derivative to...     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.     

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.     

Structure and dynamics of green fluorescent protein

Many marine organisms are luminescent. The proteins that produce the light include a primary light producer (aequorin or luciferase) and often a secondary photoprotein that red shifts the light for better penetration in the ocean. Green fluorescent protein is one such secondary protein. It is remarkable in that it autocatalyzes the formation of its own fluorophore and thus can be expressed in variety of organisms in its fluorescent form. The recent determination of its 3D structure and other physical characterizations are revealing its molecular mechanism of action     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

Acid denaturation of mustard 12S protein

The effect of low pH on the molecular properties of mustard 12S protein has been studied by the techniques of ultracentrifugation, viscometry, electrophoresis, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism. Ultracentrifugation and electrophoresis experiments indicated dissociation of the protein in the pH range 5.0 to 3.0 and below this pH reaggregation was indicated. Viscosity, turbidimetry, u.v. difference spectroscopy, fluorescence spectroscopy and circular dichroism studies showed that denaturation of the protein occurred between pH 5.0 and 3.0 and refolding at pH values below 3.0.     

Vibrations-determined properties of green fluorescent protein

The physicochemical characteristics of the green fluorescent protein (GFP), including the thermodynamic properties (entropy, enthalpy, Gibbs' free energy, heat capacity), normal mode vibrations, and atomic fluctuations, were investigated. The Gaussian 03 computational chemistry program was employed for normal mode analysis using the AMBER force field. The thermodynamic parameters and atomic fluctuations were then calculated from the vibrational eigenvalues (frequencies) and eigenvectors. The regions of highest rigidity were shown to be the beta-sheet barrel with the central alpha-helix, which bears the chromophore. The most flexible parts of the GFP molecule were the outlying loops that cover the top and bottom of the beta-barrel. This way, the balance between rigidity and flexibility is maintained, which is the optimal relationship for protein stability in terms of Gibbs' free energy. This dual-schemed structure satisfies the requirements for GFP function. In this sense, the structure of GFP resembles a nanoscale drum: a stiff cylinder with flexible vibrating end(s).     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.     

The dark side of green fluorescent protein

Here, severe interference of chlorophyll with green fluorescent protein (GFP) fluorescence is described for medicago (Medicago truncatula), rice (Oryza sativa) and arabidopsis (Arabidopsis thaliana). This interference disrupts the proportional relationship between GFP content and fluorescence that is intrinsic to its use as a quantitative reporter. The involvement of chlorophyll in the loss of GFP fluorescence with leaf age was shown in vivo, by the removal of chlorophyll through etiolation or by ethanol extraction, and in vitro, by titration of a GFP solution with chlorophyll solutions of various concentrations. A substantial decrease in fluorescence in early development of medicago and rice leaves correlated with chlorophyll accumulation. In all three species tested, removal of chlorophyll yielded up to a 10-fold increase in fluorescence. Loss of GFP fluorescence in vitro was 4-fold greater for chlorophyll b than for chlorophyll a. Differences exist between plant species for the discrepancy between apparent GFP fluorescence and its actual level in green tissues. Substantial errors in estimating promoter activity from GFP fluorescence can occur if pigment interference is not considered.     

Imaging the environment of green fluorescent protein

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.     

Quantitative measurement of green fluorescent protein expression

Summary Data presented here shows a time course analysis of E. coli shake flask cultures expressing the reporter gene green fluorescent protein (GFP) with simultaneous comparison of microbial fluorescence intensity measurements and GFP concentration measured by Western blot. There is an apparent lag between the presence of GFP and its fluorescence due to the time required for formation of the chromophore. We demonstrate that GFP fluorescence can be used as a quantifiable reporter gene, provided the cyclization time for chromophore formation is considered.     

A photostable monomeric superfolder green fluorescent protein

The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N‐ and C‐terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general‐purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.     

Charge transfer in green fluorescent protein.

Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene-imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the ramifications of the observation of strong conformational perturbations below the protein dynamical transition at approximately 200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.     

Engineering and characterization of a superfolder green fluorescent protein

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.