Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms
Preliminary results of human PrPC protein studied by spectroscopic techniques
Introduction
Neurodegenerative diseases such as Alzheimer or Parkinson diseases are tightly related to protein misfolding problem. There are almost 90 proteins that are considered to be potential amyloidogenic factors in this matter [1]. When protein, due to environmental influence, starts to fold incorrectly it may create amyloidogenic deposits in Central Nervous System [2] and other peripheral tissues [3]. These deposits are resistant to any known proteinase enzymes. In nervous tissue, constant deposition of protein plaques leads to neuron damage and cell death. Molecular mechanisms recognition concerning protein alteration from native, soluble and functional to toxic, insoluble aggregates is crucial in order to develop efficient treatment for those, nowadays, incurable diseases. One of those proteins, the PrPC is considered to have a receptor function for amyloid-β oligomers [4], [5] which are associated with development of Alzheimer disease. Additionally, it was shown that overexpression of PrPC lead to defects in cognitive skills [6].
In spite of many studies, the functions of PrPC protein in cells still remains unclear in details. However it is currently postulated that due to Cu(II) ions binding ability, PrPC protein is involved in maintenance of physiological concentration of copper [3], [7], [8]. Some experiments have shown that animals, with genes responsible for PrPC encoding being knock-out, have the level of active superoxide dismutase significantly lowered [9]. As a result, these animals suffered from high cellular damage due to increased Reactive Oxygen Species (ROS) activity. Therefore, it was suggested that copper bonded to PrPC protein has extremely high ROS production potential, and also that PrPC protein is included in copper transport from cell exterior to interior. Additionally, it was proven by the experiments on animals [10] that in presence of Cu(II) the N-terminal part of amino acid chain becomes stable. The same effect was observed when Cu(II) level in brain was lowered. Moreover, deletion of the PHGGGWGQ sequence significantly slowed down progression of neurodegenerative disease. On the other hand, in case of inherited form of disease with mutated PrPC protein, the PHGGGWGQ sequence was repeated 9 times [10]. Nevertheless, it is still unknown how copper ions influences the transformation process of PrPC into PrPSc pathological form and evaluation of coordination of Cu in PrPC-Cu(II) complex at different stoichiometry and pH conditions is necessary. Up to now, it was revealed that PrPC protein is involved in development and progression of Transmissible Spongiform Encephalopathies (TSE) diseases. This conditions are characterized by β-sheet rich, insoluble and proteinase resistant amyloid depositions composed of PrPC isoform [11] present in nervous tissue. Development of diseases are connected with changes in patient behavior, memory loss and progressive movement difficulties (ataxia), which finally leads to death. On molecular level, more clinical symptoms reflects more tissue and cell affected by pathologic PrPC → PrPSc alteration. Additionally, very recent research suggests that PrPC protein acts as critical receptor during formation of amyloid-β plaques oligomers (Aβo) [12]. It is worth to mention that complexes are in form of Aβo-PrPC therefore, in this case the prion protein remains in its native structure. PrPC protein has already been studied using various methods including Synchrotron Radiation Small Angle X-ray Scattering (SR-SAXS) spectroscopy on purified amyloid plaques [13], as well as Nuclear Magnetic Resonance (NMR) [14], [15], Circular Dichroism (CD) and biochemical methods [16]. Structure of Cu(II)-binding sites remains unknown due to high mobility of unstructured domain which provide to ambiguous results both in case of crystallographic and NMR techniques. X-ray Absorption Spectroscopy (XAS) experiments on prion proteins have been already carried out by: Shearer and Soh [17] and Morante et al. [18] on human PrPC (106–114) peptide and on synthetic αBoPrP (24–242) peptide, respectively. However, to the best of our knowledge, there is no XAS data on human PrPC-Cu(II) (23–231) model.
The PrPC protein is present in all mammals. In native form it is anchored on C-end in cellular membrane. It was proven that this particular protein might transform itself from native, harmless cellular form into pathologic so-called scrapie form (PrPSc) in process of posttranslational conversion. The cellular PrPC form has two main structural domains (Fig. 1). The N–terminal part of polypeptide chain, which includes amino acids from 23 to 124 is unstructured and very flexible. The second one, C-terminal domain (residues 128–231) has globular form containing three α-helixes and two short antiparallel β-sheets (Fig. 1) [19]. The scrapie form has much more tight β-sheets, which makes it very resistant to proteolysis. This transformation leads to large group of non-curable spongiform encephalopathies (TSE) diseases.
It was proven that PrPC protein is able to bind Cu(II) ions [8] as well as other divalent metal ions. N-terminal domain, comprised of 102 amino acid residues has ability to coordinate up to five Cu(II) ions mostly through a conservative fragment consisting of four tandem repeats of the PHGGGWGQ sequence [8]. Amount of bonded Cu(II) ions by PrPC protein varies with pH and copper concentration in solution [20]. In physiological pH and enough Cu(II) concentration each PHGGGWGQ fragment is able to bind one Cu(II) ion. It is coordinated in equatorial positions by N atom from imidazole group from histidine, another two N atoms from glycine and by O atom from CO group from glycine [20][21]. In this way, binding pocket is formed around the Cu(II) ion by of one 7- and two 5-member chelating rings [21]. Additionally, both crystallographic and EPR studies have shown that there is a double ring from tryptophan in Cu(II) coordination sphere, although it interacts indirectly with ion throughout hydrogen bond created with water molecule in axial position [22]. When PrPC is in large excess relatively to Cu(II), four PHGGGWGQ sequences are able to bind cooperatively one Cu(II) ion. This type of coordination includes four N atoms from histidine rings in equatorial positions. Moreover this type of coordination is around 104 times more stable than 1:1 coordination scheme. More types of coordination have been proposed up to now [7], [20], [21] including results obtained from ab initio calculations [23]. In first one O atom from CO group and 3 N atoms are included: from histidine residue and from deprotonated amide groups originating from two amino acids before histidine. In second model there are four N atoms and all are originating from deprotonated amide groups. In both cases there is no water in Cu(II) coordination shell and both models are equal [23].
Section snippets
Atomic Force Microscopy (AFM)
Atomic Force Microscopy (AFM) gives opportunity to study protein macroscopic structure on molecular level. AFM can be used in various modes e.g. force spectroscopy mode or topography imaging mode. In topography imaging mode sample is placed on piezoelectric table and tip is used to probe local surface of studied material. AFM tip is placed on cantilever that acts like a spring and is fixed to the rest of microscope. When the tip encounters a barrier, position of whole cantilever is interrupted.
Substrate preparation and AFM experiments
AFM topography in Fig. 2a reveals that prepared surface of mica was atomically flat, as expected, with root mean square (RMS) roughness of 0.054(2) nm. OZ scale of this AFM image was set in range of 0–7 nm to better compare it with further PrPC-Ni(II) and PrPC-Mg(II) AFM results. PrPC prion protein globular domain has ellipsoidal form with semi-major axis length around 4.5–5 nm. It can be seen, that expected size of the protein was two rows of magnitude bigger than roughness of prepared mica
Conclusions
Biochemical protocol of purifying PrPC protein was established and proved to be useful for both AFM and XAS experiments. PrPC proteins have been successfully attached to solid substrate and measured using AFM tapping mode. Average height of globular domain of human prion protein was determined with high accuracy. Developed experimental procedure will be useful for sample preparation for other spectroscopic experiments, including Nano-Infrared and TERS spectroscopy. Moreover, first attempt of
Acknowledgements
The research was performed using equipment purchased in the frame of a the project co-funded by the Małopolska Regional Operational Programme Measure 5.1 Krakow Metropolitan Area as an important hub of the European Research Area for 2007-2013, project No. MRPO.05.01.00-12-013/15.This work was supported by the Polish National Science Centre under grant No. 2014/15/B/ST4/04839. We acknowledge Swiss Light Source at Paul Scherrer Institute for granting the beamtime. The Authors would like to thank
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