Zhao Qing, Zhang Yongqiang, Li Shu Jie
Department of Biophysics, School of Physics Science, Nankai University, 94 Weijin Road, Nankai District, Tianjin, 300071, People's Republic of China.
Biometals. 2014 Aug;27(4):793-802. doi: 10.1007/s10534-014-9751-6. Epub 2014 May 28.
The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His(244) or His(266) residue. The binding of Zn(2+) to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co(2+), Ca(2+), Ni(2+), Mg(2+), and Mn(2+). The strength of Ca(2+), Co(2+), Mg(2+), Mn(2+) and Ni(2+) binding to the site close to His(244) is stronger than that of these divalent metal ions binding to the site close to His(266).
电压门控质子通道Hv1以二聚体形式发挥作用，其中该蛋白质的细胞内C末端结构域负责二聚体结构并调节质子通透性。尽管众所周知二价金属离子会对质子通道活性产生影响，但二价金属离子与该通道的详细相互作用尚未得到充分阐明。在此，我们通过圆二色光谱（CD光谱）和荧光光谱研究了二价金属离子与人Hv1的C末端结构域的相互作用。二价金属离子的结合在pH 7时诱导了明显的构象变化，并使C末端结构域的热稳定性出现pH敏感的降低。通过荧光光谱实验进一步评估了这些相互作用。二价金属离子与Hv1的C末端结构域结合至少有两个结合位点，其中任何一个都靠近His(244)或His(266)残基。Zn(2+)与这两个位点的结合均增强了蛋白质在pH 7时的荧光，而其他二价金属离子与这两个位点的结合均导致荧光猝灭。二价金属离子与这两个位点结合强度从强到弱的顺序均为Co(2+)、Ca(2+)、Ni(2+)、Mg(2+)和Mn(2+)。Ca(2+)、Co(2+)、Mg(2+)、Mn(2+)和Ni(2+)与靠近His(244)位点的结合强度强于这些二价金属离子与靠近His(266)位点的结合强度。
