Craig Don Paul | Georg-August-Universität Göttingen (original) (raw)
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Papers by Craig Don Paul
The steady improvement in the imaging of cellular processes in living tissue over the last 10-15 ... more The steady improvement in the imaging of cellular processes in living tissue over the last 10-15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).
In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (T... more In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed.
We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call P... more We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.
Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluores... more Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74. 63, b = 75.38, c = 84.51 Å , = 90.96, = 89.92, = 104.03 . The Matthews coefficient (V M = 2.26 Å 3 Da À1 ) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.
The steady improvement in the imaging of cellular processes in living tissue over the last 10-15 ... more The steady improvement in the imaging of cellular processes in living tissue over the last 10-15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).
In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (T... more In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed.
We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call P... more We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.
Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluores... more Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74. 63, b = 75.38, c = 84.51 Å , = 90.96, = 89.92, = 104.03 . The Matthews coefficient (V M = 2.26 Å 3 Da À1 ) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.