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

Bogumil Zelent, Chris Bialas, Ignacy Gryczynski, Pan Chen, Rahul Chib, Karina Lewerissa, Maria G. Corradini, Richard D. Ludescher, Jane M. Vanderkooi, Franz M. Matschinsky

Research output: Contribution to journalArticle

2 Scopus citations

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.

Original languageEnglish
Pages (from-to)1621-1631
Number of pages11
JournalJournal of Fluorescence
Volume27
Issue number5
DOIs
StatePublished - 1 Sep 2017

Keywords

  • Aromatic groups
  • Dark complexes
  • Glucokinase
  • Glucose
  • Glycerol
  • Osmolites
  • Tryptophan fluorescence

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    Zelent, B., Bialas, C., Gryczynski, I., Chen, P., Chib, R., Lewerissa, K., Corradini, M. G., Ludescher, R. D., Vanderkooi, J. M., & Matschinsky, F. M. (2017). Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase. Journal of Fluorescence, 27(5), 1621-1631. https://doi.org/10.1007/s10895-017-2099-x