Employment of Iron-Binding Protein from Haemophilus influenzae in

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Employment of Iron-Binding Protein from Haemophilus influenzae in Functional Nanopipettes for Iron Monitoring Gonca Bulbul, Goksin Liu, Namrata Rao Vithalapur, Canan Atilgan, Zehra Sayers, and Nader Pourmand ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00263 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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ACS Chemical Neuroscience

Employment of Iron-Binding Protein from Haemophilus influenzae in Functional Nanopipettes for Iron Monitoring Gonca Bulbul1, Goksin Liu2, Namrata Rao Vithalapur1, Canan Atilgan2, Zehra Sayers2, Nader Pourmand*1 1 2

Department of Biomolecular Engineering, UC Santa Cruz, Santa Cruz, California, USA Faculty of Engineering and Natural Sciences, Sabanci University, Tuzla 34956, Istanbul,

Turkey

Abstract

Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, the interest in the iron status of central nervous system has increased significantly in the last decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington’s, Parkinson’s and Alzheimer’s. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the ironbinding protein (FBP) from Haemophillus influenzae, specific for the ferrous ions. Subsequently, we employed this protein in our nanopipette platform, and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 µM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

Keywords: Haemophilus influenza, iron, nanopipette, sensing, iron characterization

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1. Introduction Iron is one of the most essential transition metals in the body and is involved in a number of biological processes1. It is a crucial component of proteins that operate in many important cellular processes including oxygen transport, mitochondrial respiration, and DNA synthesis and repair2. In living cells, iron can be found as ferrous (Fe2+) or ferric (Fe3+) ions3. Although iron is an essential element for the body, free ferrous ions are toxic due to their propensity to react with oxygen, which generates reactive oxygen species (ROS) via Fenton and Haber-Weiss reactions4. Since ROS can damage DNA, proteins, lipids, and the cell membrane5; the mechanisms involving iron storage and incorporation into the proteins by promoting their solubility and controlling its redox state must be managed carefully. Because iron concentration contributes to oxidative stress, it is tightly regulated in living organisms. Damage due to iron concentration may lead to aging and a number of diseases, such as stroke, cancer, and neurological diseases, e.g., Parkinson’s disease, Alzheimer’s disease and atherosclerosis6. Iron is present in the body bound to hemoglobin, in the oxygen storage protein myoglobin in the muscles, and in iron-containing enzymes, the cytochromes7. The amount of free iron in body fluids is controlled by the proteins of the transferrin family8. They play a crucial role in sequestering and solubilizing iron9, which is then taken up in the cells by the transferrin receptors10. FbpA is a periplasmic iron-binding protein, a bacterial transferrin found in Neisseria gonorrhoeae and Haemophilus influenzae11. It has been shown that a relatively high affinity is required for the removal and transport of iron from the host transferrin to the bacterial periplasm12. Bacterial FbpA and human Tf are similar in both structure and function11, 13. ApohFbpA from H. influenzae is a homologue with 71% amino acid sequence similarity. FbpA (from Neisseria species and H. influenzae) binds iron with four amino acid side-chains: glutamate, histidine, and two tyrosines and the metal coordination is further stabilized by the presence of a synergistic PO4 anion present in the binding site. Quantification of iron in cells and in extracellular fluid is of great interest, as both iron deficiency and overload impair cellular functions14. Iron functions as a cofactor in various metabolic reactions and is integral to the formation of heme and iron-sulfur clusters, thus indispensable to mammalian metabolism. Iron-sulfur clusters in the mitochondria along with


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hemes participate in oxidative phosphorylation. Abnormalities in brain iron metabolism is linked to the pathogenesis of a number of neurodegenerative brain diseases15. Determination of free iron in cells may improve our understanding of its functions in transport pathways, including in pathological conditions. Several methods such as spectrophotometry16-17, flow injection analysis18, fluorescence19, chemiluminescence20, radiometric21, and high-performance liquid chromatography22 have been suggested for quantitative analysis of iron. Although quantification of iron in cells and extracellular fluid can be achieved with atomic absorption spectroscopy (AAS), paramagnetic resonance spectroscopy, and colorimetric analysis, owing to technological barriers, detection of iron in single cells continues to be limited to use of fluorescence probes. Quantification of total iron in cultured cells can be done using the ferrozine method, which distinguishes between ferrous and ferric ion and distinctly measures ferrous ions at 550 nm.14 Although this method is simple and reliable compared to other radioactive detection methods and AAS, it cannot be used for the detection of ferrous ions in a single live cell. Considering that intracellular iron concentrations vary depending on environmental conditions and also on the cell type23 it is crucial to develop a tool that can be adapted to single-cell iron monitoring. Recently, the development of SERS active nanopipettes for quantitative detection of hemeproteins and Fe+3 in single cells was reported.24 Moreover, there has been limited success in designing of Fe2+selective sensors that can be adapted for real-time single cell analysis. Recently, nanopipettes have gained importance as novel sensing tools and have been utilized as sensitive, minimally invasive, non-destructive and selective biosensors for detection and quantification of proteins, metal cations, glucose, pH

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. As iron tightly binds to hFbpA,

the protein from Haemophilus influenzae was purified and characterized in this work. Holo- and apo-FBP were cloned and efficiently expressed in E. coli. and the recombinant protein was purified using the His6-tag. The isolated protein was biotinylated and used to functionalize quartz nanopipettes. Functionalized nanopipettes used in this work can further allow for continuous monitoring and site-specific detection of intracellular iron levels in the future. In this paper, we illustrate the synthesis, and characterization of holo- and apo-FBP and demonstrate its immobilization on the nanopipette platform for ferrous detection. Research is underway to employ these apo-hFBP- functionalized probes directly in a single cell to monitor and quantify concentrations of ferrous ions in real-time.


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2.Results and Discussion The FBP was purified by using Ni-affinity chromatography, and its purity was monitored by SDS-PAGE (Figure 1). The pink color of the purified FBP further indicated the presence of the holoform of the protein.

Figure 1. 12% SDS-PAGE analysis of purified recombinant ferric-binding protein for [2] apoand [3] holo FBP in comparison to [1] Molecular Weight Marker. 6 µg protein was loaded to each gel.

2.1. Characterization of Ferrous Binding Protein 2.1.1. Absorbance Spectroscopy Measurement of the biophysical properties of the holo- and apo-FBP as well as the metal bound form reconstituted from apo-FBP was carried out spectroscopically; the absorbance spectra are presented in Figure 2(a) and 2(b). A comparison of holo-, reconstituted and apo-FBP absorption spectra shows that they display the expected 280 nm protein peak. However, the 257 nm minimum observed in holo- and reconstituted FBP is shifted to 252 nm in the apo-protein.


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Figure 2. Absorption spectra of (a) Apo- and holo-FBP at 1.2 mg/ml in 50 mM Tris.HCl pH 8.0, 100 mM NaCl (b) Reconstituted holo-FBP in 10 mM Tris.HCl pH 8.0.

2.1.2. Dynamic Light Scattering (DLS) Dynamic light scattering (DLS) was used to determine monodispersity, stability and conformational differences between apo- and holo-FBP. Light-scattering data were collected from samples at 1mg/ml in 50 mM Tris.HCl pH 8.0, 100 mM NaCl buffer using Nano-Zetasizer (Malvern Instruments, USA) equipment. The hydrodynamic radius (Rh) of holo-FBP changes from 3.08 nm to 3.22 nm in the apo form, yielding a 4-5% increase upon depletion of iron. This change indicates a more open conformation of the FBP structure when the metal is removed.


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Figure 3. Scattered intensity plotted against d=2xRh for (a) Apo-FBP (1.2 mg/ml) in 10 mM Tris.HCl pH 8.0 and (b) Holo-FBP (3.4 mg/ml) in 50 mM Tris.HCl pH 7.4

2.1.3. Small-Angle X-Ray Scattering (SAXS) Measurements SAXS measurements are used to determine the low-resolution structural parameters and shape of the proteins in solution and can also be used to follow structural changes under the influence of varying environmental conditions30. SAXS measurements were conducted on apo- and holo-FBP at the p12 beamline of the EMBL outstation on the PETRA III synchrotron radiation source at the DESY site in Hamburg, Germany. Data were collected in the momentum transfer range 0.08 < S < 6 nm-1 (S = 4πsinθ/λ, where 2θ is the scattering angle and λ= 0.15 nm is the wavelength) using a photon-counting PILATUS 1M pixel detector (67 x 420 mm2) positioned at a distance of 2.7 m away from the sample. Typical analyses from the SAXS data are given in Figure 4. The scattering profiles of normalized apo-FBP (4 mg/ml) (black) and holo-FBP (1.5 mg/ml) (red) are shown in figure 4(a), where the scattering intensity is plotted against the momentum transfer (S).


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(a)

(b)

(c)

(d)

Figure 4. (a) SAXS profiles; (b) Guinier plots; (c) Pair distribution functions for apo-FBP (black) and holo-FBP (red); (d) Kratky analysis for apo-FBP.

Although the signal-to-noise ratio is low at large values of S at these concentrations, differences in the scattering patterns for the two proteins can be detected at lower angles. The Guinier approximation, in which the intensity is represented as I(S)= I(0)exp(-(SRg)2/3) at very low angles (0.3

Employment of Iron-Binding Protein from Haemophilus influenzae in

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