Publications by authors named "Prajna Paramita Pal"

6 Publications

  • Page 1 of 1

Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins.

Cytometry A 2014 Jul 19;85(7):621-7. Epub 2014 Feb 19.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, 55131, Mainz, Germany.

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.
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http://dx.doi.org/10.1002/cyto.a.22451DOI Listing
July 2014

Gold fluorescent annexin A5 as a novel apoptosis detection tool.

Cytometry A 2009 Jul;75(7):626-33

Max Planck Institute of Neurobiology, Department of Neuroimmunology, Martinsried, Germany.

We describe a golden fluorescent apoptosis detection tool, which we generated by a fusion of golden fluorescent protein (GdFP) with human annexin A5 (anxA5). GdFP was obtained by replacement of tryptophan at position 66 with 4-aminotryptophan in the chromophore of enhanced cyan fluorescent protein. The GdFP-anxA5 construct combines highly desirable features originating from both fusion partners. These include (i) strong binding to membrane phosphatidylserine patches of apoptotic cells in the presence of Ca(2+) which is brought about by anxA5, (ii) the stable and homogeneous monomeric state, (iii) as well as the red-shifted fluorescence maximum at 574 nm originating from GdFP. We found that GdFP-anxA5 is equally well applicable for apoptosis studies as a routinely used fluorescein 5'-isothiocyanate-annexin A5 conjugate. Golden fluorescent annexin A5 represents a new, stable, and homogeneous red-shifted optical probe for the efficient detection of apoptosis by fluorescence microscopy or by flow cytometry.
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http://dx.doi.org/10.1002/cyto.a.20737DOI Listing
July 2009

Efforts towards the design of 'teflon' proteins: in vivo translation with trifluorinated leucine and methionine analogues.

Chem Biodivers 2004 Oct;1(10):1465-75

Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried.

In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.
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http://dx.doi.org/10.1002/cbdv.200490107DOI Listing
October 2004

Designing novel spectral classes of proteins with a tryptophan-expanded genetic code.

Biol Chem 2004 Oct;385(10):893-904

Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany.

Fluorescence methods are now well-established and powerful tools to study biological macromolecules. The canonical amino acid tryptophan (Trp), encoded by a single UGG triplet, is the main reporter of intrinsic fluorescence properties of most natural proteins and peptides and is thus an attractive target for tailoring their spectral properties. Recent advances in research have provided substantial evidence that the natural protein translational machinery can be genetically reprogrammed to introduce a large number of non-coded (i.e. noncanonical) Trp analogues and surrogates into various proteins. Especially attractive targets for such an engineering approach are fluorescent proteins in which the chromophore is formed post-translationally from an amino acid sequence, like the green fluorescent protein from Aequorea victoria. With the currently available translationally active fluoro-, hydroxy-, amino-, halogen-, and chalcogen-containing Trp analogues and surrogates, the traditional methods for protein engineering and design can be supplemented or even fully replaced by these novel approaches. Future research will provide a further increase in the number of Trp-like amino acids that are available for redesign (by engineering of the genetic code) of native Trp residues and enable novel strategies to generate proteins with tailored spectral properties.
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http://dx.doi.org/10.1515/BC.2004.117DOI Listing
October 2004

Crystallographic evidence for isomeric chromophores in 3-fluorotyrosyl-green fluorescent protein.

Chembiochem 2004 May;5(5):720-2

Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, 82152 Martinsried, Germany.

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http://dx.doi.org/10.1002/cbic.200300818DOI Listing
May 2004

Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code.

Biol Chem 2004 Feb;385(2):191-202

Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany.

The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphur-aromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.
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http://dx.doi.org/10.1515/BC.2004.038DOI Listing
February 2004