Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red

Biochemistry
Eugenia PolveriniThomas Gensch

Abstract

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye ...Continue Reading

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Citations

Nov 22, 2011·The Journal of Physical Chemistry. B·Mily Bhattacharya, Samrat Mukhopadhyay
Aug 6, 2015·Biophysical Journal·Eugene G MaksimovAndrew B Rubin
Oct 24, 2012·The International Journal of Biochemistry & Cell Biology·Mehrnaz Azami-MovahedMohsen Nemat-Gorgani

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