Shaw Ajay Kumar, Pal Samir Kumar
Unit for Nano Science and Technology, Department of Chemical, Biological and Macromolecular Sciences, SN Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India.
J Photochem Photobiol B. 2008 Mar 28;90(3):187-97. doi: 10.1016/j.jphotobiol.2008.01.001. Epub 2008 Jan 9.
Human serum albumin (HSA) is a very important transporter protein in the circulatory system. It is a multi-domain binding protein, which binds a wide variety of ligands in its multiple binding sites and aids in transport, distribution and metabolism of many endogenous and exogenous ligands. With change in pH, HSA is known to undergo conformational transformation, which is very essential for picking up and releasing them at sites of differing pH inside physiological system. Hence, the characterization of ligand binding to these pH-induced conformers is extremely important. We have explored binding interaction of a ligand protoporphyrin IX (PPIX), which is demonstrated (X-ray crystallography) to reside in domain-IB at the various pH-induced folded states of HSA. The ligand PPIX is found to remain attached to all the HSA conformers which offers an opportunity to use Förster's resonance energy transfer (FRET) between an intrinsic donor fluorophore (Trp214) located in domain-IIA to the acceptor ligand PPIX to characterize the inter-domain separation between IB and IIA. Additionally FRET between an extrinsic fluorophore 2-p-toluidinylnaphthalene-6-sulfonate (TNS) located in domain-IIIA and PPIX is also undertaken to quantify the inter-domain separation between IB and IIIA. Circular dichroism (CD) and dynamic light scattering (DLS) studies have been done in conjunction with picosecond time resolved fluorescence and polarization-gated spectroscopy to determine, respectively, the secondary and tertiary structures of various pH-induced folded states of the protein. Severe structural perturbation including swelling of the protein is observed in the low pH-induced conformer of HSA as evidenced from all the techniques used.
人血清白蛋白（HSA）是循环系统中一种非常重要的转运蛋白。它是一种多结构域结合蛋白，在其多个结合位点结合多种配体，并有助于许多内源性和外源性配体的运输、分布和代谢。已知随着pH值的变化，HSA会发生构象转变，这对于在生理系统内不同pH值的位点摄取和释放配体至关重要。因此，表征配体与这些pH诱导构象体的结合极其重要。我们研究了配体原卟啉IX（PPIX）的结合相互作用，X射线晶体学证明该配体在HSA的各种pH诱导折叠状态下位于结构域IB中。发现配体PPIX与所有HSA构象体保持结合，这为利用位于结构域IIA的内在供体荧光团（Trp214）与受体配体PPIX之间的福斯特共振能量转移（FRET）来表征IB和IIA之间的结构域间距离提供了机会。此外，还进行了位于结构域IIIA的外在荧光团2-对甲苯胺基萘-6-磺酸盐（TNS）与PPIX之间的FRET，以量化IB和IIIA之间的结构域间距离。结合皮秒时间分辨荧光和偏振门控光谱分别进行了圆二色性（CD）和动态光散射（DLS）研究，以确定蛋白质各种pH诱导折叠状态的二级和三级结构。所有使用的技术均证明，在低pH诱导的HSA构象体中观察到了包括蛋白质肿胀在内的严重结构扰动。
