Description of the relationship between protein structure and function remains a primary focus in molecular biology, biochemistry, protein engineering and bioelectronics. Moreover, the investigation ...of the protein conformational changes after adhesion and dehydration is of importance to tackle problems related to the interaction of proteins with solid surfaces. In this paper the conformational changes of wild-type Discosoma recombinant red fluorescent proteins (DsRed) adhered on silver nanoparticles (AgNPs)-based nanocomposites are explored via surface-enhanced Raman scattering (SERS). Originality in the present approach is to work on dehydrated DsRed thin protein layers in link with natural conditions during drying. To enable the SERS effect, plasmonic substrates consisting of a single layer of AgNPs encapsulated by an ultra-thin silica cover layer were elaborated by plasma process. The achieved enhancement of the electromagnetic field in the vicinity of the AgNPs is as high as 10
. This very strong enhancement factor allowed detecting Raman signals from discontinuous layers of DsRed issued from solution with protein concentration of only 80 nM. Three different conformations of the DsRed proteins after adhesion and dehydration on the plasmonic substrates were identified. It was found that the DsRed chromophore structure of the adsorbed proteins undergoes optically assisted chemical transformations when interacting with the optical beam, which leads to reversible transitions between the three different conformations. The proposed time-evolution scenario endorses the dynamical character of the relationship between protein structure and function. It also confirms that the conformational changes of proteins with strong internal coherence, like DsRed proteins, are reversible.
Protecting grapevine pruning wounds by inoculating them with Trichoderma spp. can prevent infection from trunk disease pathogens. The growth and interactions of both, the biological control agent ...Trichoderma spp. and the vine pathogens, are not well understood. Green fluorescent protein (GFP)-labelled Trichoderma harzianum and red fluorescent protein (DsRed)-labelled T. harzianum, were dual-inoculated with Phaeomoniella chlamydospora (DsRed) or Eutypa lata (GFP) on fresh pruning wounds of one-year-old Cabernet Sauvignon and Sauvignon blanc shoots. The inoculated fungi were recovered from varying depths within the shoots at 30-day-intervals for 90 days. Trichoderma harzianum suppressed the pathogens and grew deeper in the presence of the pathogens than when it was singly inoculated; possibly an indication of pathogen recognition and competitive response. Eutypa lata was completely eliminated from Sauvignon blanc in dual-inoculated canes after 90 days. The mycelium of P. chlamydospora (DsRed) grew extensively in the xylem vessels and possibly contributed to vessel occlusion. Phaeomoniella chlamydospora and E. lata caused blockage of the vessels and thickening of the vessel walls. Grapevine wood produced both tyloses and gums (gels) that blocked xylem vessels as a result of infection. A thickening of the cell walls of xylem fibres occurred only in E. lata-inoculated shoots, indicative of a different mode of pathogenesis from P. chlamydospora.
The Arabidopsis genome has two similar dynamin-like proteins, ADL2a and ADL2b (76.7% identity). ADL2a is reported to be localized in chloroplasts Kang et al. (1998) Plant Mol. Biol. 38: 437, while ...ADL2b functions in mitochondrial division Arimura and Tsutsumi (2002) Proc. Natl. Acad. Sci. USA 99: 5727. Using GFP fusion proteins, we observed both ADL2a and ADL2b in portions of mitochondria but not in chloroplasts. Furthermore, cells transformed with ADL2a and ADL2b with a defective GTPase domain had normal chloroplasts but elongated mitochondria. These results imply that both ADL2b and ADL2a are involved in the division of plant mitochondria.
To better engineer and analyze beneficial biofilms as well as to develop strategies to control detrimental biofilms (e.g., biomedical device-based infections), it is critical to quantify bacterial ...species compositions within biofilms. A non-invasive method is described here that determines local and overall bacterial concentrations within a biofilm, using optical microscopy and digital image analysis techniques. The method is based upon a calibration of cell fluorescence to known cell number concentrations and is verified by direct cell counts of destructive samples of cultivated biofilms. Two GFP mutants, each with unique emission colors were used with both epi-fluorescent microscopy and one-photon confocal microscopy to determine local spatial biofilm cell concentrations in pure and mixed-strain biofilms. Our microbial system comprises Pseudomonas putida containing either green fluorescent protein (GFP) or containing the red fluorescent protein (DsRed). Strains expressing a green or red fluorescent protein were detected by two different microscopy methods: epi-fluorescence and single-photon confocal laser scanning microscopy. Overall biofilm cell concentrations determined directly from destructive samples were in good agreement with non-invasive measurements of adherent cell concentrations calculated from the measured “integrated fluorescent density” minus any background fluorescence. Results show the areal cell concentration (cell number/area) determined from non-destructive direct counts in a pure culture or binary-strain biofilm varied with the biofilm depth. Use of this method to estimate local dynamic plasmid segregational loss and plasmid conjugation transfer kinetics will be reported in a subsequent manuscript.
We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and ...protease‐cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)‐coding sequences were amplified by PCR and cloned into pMAL (MBP‐EGFP) or pT7His (His10‐mDsRed) prokaryotic expression vectors. Then the fluorescent proteins were expressed in Rosetta (DE3) pLysS by IPTG induction or autoinduction. We purified the fluorescent proteins by affinity chromatography (Amylose and metal ion‐chelating column), anion‐exchange chromatography (High Q column), size exclusive chromatography (Sephacryl S‐200 column), and hydrophobic interaction chromatography (Methyl HIC column) to exhibit the protein‐purification techniques. After purification, the fusion protein MBP‐EGFP was cleaved by TEV protease. The recombinant mDsRed protein was crystallized by hanging drop vapor diffusion technique to show students the basic operation of crystallization. The whole procedure can be monitored real time by naked eyes when using fluorescent proteins. The demonstration of expression, purification, crystallization, and protease cleavage became much more vivid and interesting, which greatly deepened the students' understanding of modern protein‐science techniques.
In the present study, a red fluorescent protein (DsRed) from the coral Discosoma was fused to the C‐terminus of protein ZZ, a synthetic artificial IgG‐Fc‐fragment‐binding protein derived from the ...B‐domain of staphylococcal Protein A. The chimaeric protein, tagged with six histidine residues at the N‐terminus, was expressed in Escherichia coli and easily purified by one‐step Ni2+‐chelating affinity chromatography. Its fluorescence and IgG‐binding activities were validated using fluorescence‐spectrum analysis, ELISA and dot‐blot analysis. Furthermore, in subsequent dot‐blotting immunoanalysis of glutathione S‐transferase and tumour necrosis factor‐α, and immunofluorescent microscopy assay of interferon regulatory factor 3, the chimaeric protein enabled effective detection of target molecules. Compared with fluorescence‐conjugated antibodies, ZZ–DsRed is less susceptible to photobleaching and easy to produce. In addition, unlike HRP (horseradish peroxidase)‐conjugated antibodies, using ZZ‐DsRed needs no addition of a chromogenic reagent. Our results indicate that ZZ–DsRed shows a wide and promising application potential in immunological detection as a substitute for fluorescent or HRP‐conjugated anti‐IgGs.
We describe a novel homogeneous protein-protein interaction assay based on bioluminescence resonance energy transfer (BRET) from mutant firefly luciferase to red fluorescent protein (DsRed). The ...association of glutathione S-transferase (GST)-luciferase and protein G-DsRed, which was driven by the addition of anti-GST antibody, was successfully monitored by BRET.
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