The development of innovative and performing analytical techniques is crucial in order to better understand the role of xenobiotics and biotics in biological systems. The study of interesting biomolecules is nowadays a key issue that connects different scientific fields, proceeding through medicine, physics, chemistry, engineering and even mathematics and represents the most powerful mean to find and design effective, pathogen-specific therapeutics. In recent years, bacteria and fungi have developed alarming drug resistance attitudes, becoming extremely dangerous for the patients’ subsistence. In particular, the dramatic increase of invasive mycoses, due to opportunistic fungal pathogens [1], represents both a serious threat and a challenging scientific issue. One highly strict dominating factor appoints the biggest obstacle to find efficient pathogen-specific drugs that will not produce unwanted side-effects in patients: both fungi and humans share essential metabolic pathways, as they belong to Eukaryota domain. In order to develop a highly specific antifungal drug, it is crucial to focus on differences between human and pathogen metabolism, such as the transport system of zinc. In our work, we focus on the zinc uptake and vacuole storage controlled by COT1, a transmembrane protein composed of 199 amino acid residues, located in the vacuolar, mitochondrial and cell membrane. Its main function is the transport of cobalt and zinc ions and its structure remains unsolved [2]. We used Phyre2 [3] to simulate and analyze the predicted, highly probable structure. It occurs that the most probable Zn(II) binding sites of COT1 are located at the Cterminal region, in the extracellular, or in the case of vacuolar transporter, in cytoplasmic part of the protein. We also decided to investigate Cu(II) binding, since Cu(II) is a good reference metal for Zn(II) as well as a necessary nutrient for C. Albicans, a fungal pathogen containing COT1. In the present work, the binding affinity of Zn(II) and Cu(II) towards three peptides (Ac-FHEHGHSHSHGSGGGGGGNH2, Ac-SHSHSHSHS-NH2 and Ac-FHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS -NH2), models for the COT1 metal binding sites, were characterized exploiting different analytical techniques. High-resolution mass spectra were obtained using a time-of-flight mass analyzer (TOF) with an electrospray ionization (ESI) source spectrometer. ESI-TOFs are popular in bioanalytical chemistry, since these kinds of instruments can be routinely used to get accurate mass measurements of small molecules, peptides or even intact proteins. Furthermore, mass spectra provide useful information about stoichiometry of complexes at a given pH. Stability constants for proton, Zn(II) and Cu(II) complexes were calculated from pH/metric titrations. Finally, spectroscopic techniques, such as UV-Vis, Circular Dichroism and Electron Paramagnetic Resonance, provided information about structure and coordination modes of the investigated peptides. REFERENCES [1] P. Carver et al., Ann. Pharmacother. 2015, 49825-49837. [2] D. Conklin, J. McMaster, M. Culbertson, C. Kung, Mol. Cell Biol., 1992, 12(9), 3678- 3688. [3] L. A. Kelley et al., Nature Protocols, 2015, 10, 845-858.

Application of bioanalytical techniques in the investigation of binding sites in COT1, a zinc transporter in Candida albicans

Denise Bellotti
;
Maurizio Remelli
2017

Abstract

The development of innovative and performing analytical techniques is crucial in order to better understand the role of xenobiotics and biotics in biological systems. The study of interesting biomolecules is nowadays a key issue that connects different scientific fields, proceeding through medicine, physics, chemistry, engineering and even mathematics and represents the most powerful mean to find and design effective, pathogen-specific therapeutics. In recent years, bacteria and fungi have developed alarming drug resistance attitudes, becoming extremely dangerous for the patients’ subsistence. In particular, the dramatic increase of invasive mycoses, due to opportunistic fungal pathogens [1], represents both a serious threat and a challenging scientific issue. One highly strict dominating factor appoints the biggest obstacle to find efficient pathogen-specific drugs that will not produce unwanted side-effects in patients: both fungi and humans share essential metabolic pathways, as they belong to Eukaryota domain. In order to develop a highly specific antifungal drug, it is crucial to focus on differences between human and pathogen metabolism, such as the transport system of zinc. In our work, we focus on the zinc uptake and vacuole storage controlled by COT1, a transmembrane protein composed of 199 amino acid residues, located in the vacuolar, mitochondrial and cell membrane. Its main function is the transport of cobalt and zinc ions and its structure remains unsolved [2]. We used Phyre2 [3] to simulate and analyze the predicted, highly probable structure. It occurs that the most probable Zn(II) binding sites of COT1 are located at the Cterminal region, in the extracellular, or in the case of vacuolar transporter, in cytoplasmic part of the protein. We also decided to investigate Cu(II) binding, since Cu(II) is a good reference metal for Zn(II) as well as a necessary nutrient for C. Albicans, a fungal pathogen containing COT1. In the present work, the binding affinity of Zn(II) and Cu(II) towards three peptides (Ac-FHEHGHSHSHGSGGGGGGNH2, Ac-SHSHSHSHS-NH2 and Ac-FHEHGHSHSHGSGGGGGGSDHSGDSKSHSHSHSHS -NH2), models for the COT1 metal binding sites, were characterized exploiting different analytical techniques. High-resolution mass spectra were obtained using a time-of-flight mass analyzer (TOF) with an electrospray ionization (ESI) source spectrometer. ESI-TOFs are popular in bioanalytical chemistry, since these kinds of instruments can be routinely used to get accurate mass measurements of small molecules, peptides or even intact proteins. Furthermore, mass spectra provide useful information about stoichiometry of complexes at a given pH. Stability constants for proton, Zn(II) and Cu(II) complexes were calculated from pH/metric titrations. Finally, spectroscopic techniques, such as UV-Vis, Circular Dichroism and Electron Paramagnetic Resonance, provided information about structure and coordination modes of the investigated peptides. REFERENCES [1] P. Carver et al., Ann. Pharmacother. 2015, 49825-49837. [2] D. Conklin, J. McMaster, M. Culbertson, C. Kung, Mol. Cell Biol., 1992, 12(9), 3678- 3688. [3] L. A. Kelley et al., Nature Protocols, 2015, 10, 845-858.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2480173
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