Abstract
Neuroglobin (Ngb), a recently discovered globin, is predominantly expressed in the brain, retina, and other nerve tissues of vertebrates. The unfolding processes of apo-neuroglobin (apoNgb) induced by guanidine hydrochloride (GdnHCl) and urea were investigated by spectroscopic methods. In the unfolding processes, apoNgb's tertiary structural transition was monitored by the changes of intrinsic fluorescence emission spectra, and its secondary structural transition was measured by the changes of far-ultraviolet circular dichroism (CD) spectra. In addition, 8-anilino-1-naphthalenesulfonic acid (ANS), a hydrophobic cluster binding dye, was also used to monitor the unfolding process of apoNgb and to explore its intermediates. Results showed that GdnHCl-induced unfolding of apoNgb was via a three-state pathway, that is, Native state (N) → Intermediate state (I) → Unfolded state (U), during which the intermediate was inferred by an increase in fluorescence intensity and the change of CD value. Gibbs free energy changes are 10.2 kJ·mol−1 for the first unfolding transition and 14.0 kJ·mol−1 for the second transition. However, urea-induced unfolding of apoNgb only underwent a two-state transition: Native state (N) → Partially unfolded state (P). The result showed that GdnHCl can efficiently affect the conformational states of apoNgb compared with those of urea. The work will benefit to have an understanding of the unfolding mechanism of apoNgb induced by GdnHCl and urea.
Highlights
For a long time, hemoglobin (Hb) and myoglobin (Mb) have been considered as the only types of globin in vertebrates
The guanidine hydrochloride (GdnHCl)-induced and Urea-induced structural changes of apoNgb have been investigated by using fluorescence, ANS fluorescence, and far-UV circular dichroism (CD) spectroscopic techniques
It is well known that the intrinsic fluorescence of proteins originated from the tryptophan (Trp) and tyrosine (Tyr) fluorophores is very sensitive to the microenvironments of the amino acid residues
Summary
Hemoglobin (Hb) and myoglobin (Mb) have been considered as the only types of globin in vertebrates. Ngb shares only 20–25% identity in amino acid sequence with vertebrate Hb and Mb, it displays the structural determinants of the globin fold [3]. Both Hb and Mb display a pentacoordinated heme, in which the iron ion is bound by four nitrogen atoms of the porphyrin ring and a proximal histidine in the F helix (His F8). The apo form of Mb (apoMb) has been investigated intensively and some studies showed that apoMb rendered different transition states under varying denaturing conditions [9,10,11,12]. Results revealed that apoNgb formed a folding intermediate known as molten globule, which possessed native-like secondary structure and lost its tertiary structure significantly at pH 2.0
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