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Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase

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Abstract

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme’s absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

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Acknowledgements

The work was supported by grants from NIDDK no. 22122 and 19,525 (F.M.M.), NSF grant CBET-1264608 (I.G.) and National Research Initiative or Agriculture and Food Research Initiative Grant no. 2014–67,017-2149 (R.D.L., B.Z.& M.G.C.) from USDA National Institute of Food and Agriculture, Improving Food Quality.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics and Diabetes Research Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Bogumil Zelent, Pan Chen & Franz M. Matschinsky

  2. Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA

    Bogumil Zelent, Rahul Chib, Karina Lewerissa, Maria G. Corradini & Richard D. Ludescher

  3. Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Chris Bialas & Jane M. Vanderkooi

  4. Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX, USA

    Ignacy Gryczynski

  5. Department of Food Science, University of Massachusetts Amherst, Massachusetts, MA, USA

    Maria G. Corradini

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  1. Bogumil Zelent
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  2. Chris Bialas
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  3. Ignacy Gryczynski
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  4. Pan Chen
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  5. Rahul Chib
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  6. Karina Lewerissa
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  7. Maria G. Corradini
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  8. Richard D. Ludescher
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  9. Jane M. Vanderkooi
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  10. Franz M. Matschinsky
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Correspondence to Bogumil Zelent or Franz M. Matschinsky.

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Zelent, B., Bialas, C., Gryczynski, I. et al. Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase. J Fluoresc 27, 1621–1631 (2017). https://doi.org/10.1007/s10895-017-2099-x

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  • DOI: https://doi.org/10.1007/s10895-017-2099-x

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