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7 Janelia Publications

Showing 1-7 of 7 results
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    12/01/09 | Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.
    Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL
    Nature Methods. 2009 Dec;6(12):875-81. doi: 10.1038/nmeth.1398

    Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

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    10/01/09 | Enzyme stabilization by domain insertion into a thermophilic protein.
    Kim C, Pierre B, Ostermeier M, Looger LL, Kim JR
    Protein Engineering, Design & Selection : PEDS. 2009 Oct;22(10):615-23. doi: 10.1093/protein/gzp044

    Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme’s desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37 degrees C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37 degrees C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.

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    07/26/09 | Three dimensional super resolution fluorescence imaging of single bacterial cells by stereo photoactivated localization microscopy.
    Tang J, Vaziri A, Akerboom JP, Looger L, Shank CV
    Microscopy and Microanalysis. 2009 July 26;15:564-5. doi: 10.1017/S1431927609095737
    03/06/09 | Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.
    Akerboom J, Rivera JD, Guilbe MM, Malavé EC, Hernandez HH, Tian L, Hires SA, Marvin JS, Looger LL, Schreiter ER
    The Journal of Biological Chemistry. 2009 Mar 6;284:6455-64. doi: 10.1074/jbc.M807657200

    The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

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    02/20/09 | Cofactor engineering of lactobacillus brevis alcohol dehydrogenase by computational design.
    Ronnie Machielsen , Loren L. Looger , John Raedts , Sjoerd Dijkhuizen , Werner Hummel , Hans‐Georg Hennemann , Thomas Daussmann , John van der Oost
    Engineering in Life Sciences. 2009 Feb 20;9(1):38-44. doi: 10.1002/elsc.200800046

    The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.

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    02/01/09 | A bright and photostable photoconvertible fluorescent protein.
    McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL
    Nature Methods. 2009 Feb;6(2):131-3. doi: 10.1038/nmeth.1296

    Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the approximately 10-nm localization precision of its parent.

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    01/01/09 | Modulating protein interactions by rational and computational design.
    Marvin JS, Looger LL
    Protein Engineering and Design. 2009:343-66