Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies

doi:10.1016/j.saa.2016.04.008

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Volume 164, 5 July 2016, Pages 103-109

https://doi.org/10.1016/j.saa.2016.04.008 Get rights and content

Highlights

•
This work studied the interacting mechanism of copper with lysozyme.
•
Copper could spontaneously interact with lysozyme through hydrophobic forces.
•
Binding of copper to lysozyme was a static quenching process.
•
Copper induced the conformational changes of lysozyme.
•
Copper could affect the function of lysozyme by inhibiting its catalytic activity.

Abstract

Although copper is essential to all living organisms, its potential toxicity to human health have aroused wide concerns. Previous studies have reported copper could alter physical properties of lysozyme. The direct binding of copper with lysozyme might induce the conformational and functional changes of lysozyme and then influence the body's resistance to bacterial attack. To better understand the potential toxicity and toxic mechanisms of copper, the interaction of copper with lysozyme was investigated by biophysical methods including multi-spectroscopic measurements, isothermal titration calorimetry (ITC), molecular docking study and enzyme activity assay. Multi-spectroscopic measurements proved that copper quenched the intrinsic fluorescence of lysozyme in a static process accompanied by complex formation and conformational changes. The ITC results indicated that the binding interaction was a spontaneous process with approximately three thermodynamical binding sites at 298 K and the hydrophobic force is the predominant driven force. The enzyme activity was obviously inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites. This study helps to elucidate the molecular mechanism of the interaction between copper and lysozyme and provides reference for toxicological studies of copper.

Graphical abstract

Copper could spontaneously bind lysozyme through hydrophobic forces with a positive ΔH, a favorable ΔS, and a negative ΔG. Binding of copper to lysozyme caused static quenching of the fluorescence, the change of the microenvironment of tryptophan residues and conformational changes of lysozyme. Besides, the lysozyme activity was also inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites. Thus, the direct interaction between copper and lysozyme might induce the conformational and functional alterations of lysozyme.

Introduction

Copper is an essential micronutrient that is vital to a wide range of metabolic processes [1]. It is widely used in military weapons, machinery manufacturing, dental products, intrauterine devices and cosmetics [2]. Although copper is a required element, exposure to elevated concentrations of copper becomes toxic. Copper poisoning may result in hepatocellular necrosis, acute tubular necrosis lethargy and anorexia in the early stages [2], [3]. The massive utilizations of copper have aroused considerable concerns regarding its biological roles and potential health impact [4], [5].

Lysozyme is abundant in various biological fluids and tissues, including skin, saliva, tears, liver blood and lymphatic tissues of humans and other animals [6], [7].It is a glycoside hydrolase consisting of 129 amino acid residues, which contains four disulfide bond, six tryptophan (Trp), three tyrosine (Tyr) and three phenylalanine (Phe) residues [6]. Previous research has shown that lysozyme is associated with innate immune system and can protect the cells from death by lysing the cytoderm of bacteria [8], [9]. Lysozyme also plays a vital role in physiological functions such as anti-tumor effects, anti-inflammatory, anti-viral and detumescence [10], [11], [12].

Studies have reported that excessive copper could lead to irreversible inactivation of lysozyme and extensive changes in the physical properties in weakly alkaline solution [13].Studies also have investigated the interaction between copper and hen egg-white lysozyme at different copper concentrations by electrospray ionization mass spectrometry to determine the binding capacity [14]. Thus, the direct interaction between copper and lysozyme might induce the conformational and functional alterations of lysozyme. The potential toxicity and toxic mechanisms of copper on lysozyme is significant for understanding heavy metal toxicity at molecular level. Although the interaction between copper and lysozyme has been explored by enzymatic, spectroscopy microscopy and spectroscopic techniques [15], [16], no research data were available on detailed thermodynamic descriptions of the interaction. Isothermal titration calorimetry (ITC) technique was used in this study to determine binding and thermodynamic parameters. Molecular docking studies were carried out with MOE software to illustrate the binding mode between lysozyme and copper and figure out the underlying mechanism. What's more, the fluorescence quenching mechanism, the microenvironment changes of Trp and Tyr residues and the binding sites in lysozyme have not been systematically determined in previous studies. Time-resolved fluorescence measurements was used to explore the fluorescence quenching mechanism (static or dynamic quenching).

In this work, we investigated the interacting mechanism of copper with lysozyme by multiple fluorescence measurements, UV–visible absorption, circular dichroism (CD) spectroscopy, time-resolved fluorescence measurements, ITC, molecular docking study and enzyme activity assay. Fluorescence quenching mechanism, conformational and functional changes of lysozyme, thermodynamic parameters, binding constants and modes were discussed in details. This study helps to understand comprehensively the effects of copper on lysozyme activity and toxic mechanism of copper on lysozyme.

Section snippets

Materials

Lysozyme (from chicken egg white) was purchased from Amersco (USA) and was dissolved in ultrapure water to form a 1.0 × 10^− 4 M solution (preserved at 4 °C). A1 × 10^− 2 M CuCl₂ solution was prepared by dissolving 0.175 g CuCl₂·2H₂O (Tianjin Kermel Chemical Reagent Research Institute) in 100 mL using ultrapure water. 0.2 M acetic acid (HAc) and 0.2 M sodium acetate (NaAc) were used to prepare 0.2 M HAc-NaAc buffer solutions (pH = 5.5).

All the reagents were analytical-reagent grade unless otherwise noted and

Influence of copper on the fluorescence intensity of lysozyme

Fluorescence measurements were utilized to explore the alteration of lysozyme fluorescence with the copper addition. The absorption of proteins during excitation and emission processes resulted in the inner filter effect of fluorescence value [19]. For the sake of eliminating the possible inner filter effect (IFE), preliminary experiments were conducted. In this work, the sum of the absorption at 278 nm (excitation wavelength) and 338 nm (emission wavelength) could cause < 5% error, so IFE could

Conclusions

In this study, we investigated the interacting mechanism of copper with lysozyme by multiple spectroscopic measurements, ITC techniques, molecular docking study and enzyme activity assay. The experimental results showed that static quenching was the predominant quenching mechanism of the interaction between lysozyme and copper. In addition, copper induced the conformational changes of lysozyme and the microenvironment changes of Trp residues in lysozyme. ITC results revealed that copper could

Acknowledgments

This work is supported by NSFC (20875055, 21277081, 21477067), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China (708058, 20130131110016), Science and Technology Development Plan of Shandong Province (2014GSF117027) are also acknowledged.

References (43)

M.C. Linder
Copper and genomic stability in mammals
Mutat. Res. Fundam. Mol. Mech. Mutagen.
(2001)
L.M. Gaetke et al.
Copper toxicity, oxidative stress, and antioxidant nutrients
Toxicology
(2003)
G.J. Brewer
Copper toxicity in Alzheimer's disease: cognitive loss from ingestion of inorganic copper
J. Trace Elem. Med. Biol.
(2012)
J. Chongqiu et al.
Lysozyme enhanced europium–metacycline complex fluorescence: a new spectrofluorimetric method for the determination of lysozyme
Anal. Chim. Acta
(2004)
G. Paramaguru et al.
Interaction of anthraquinone dyes with lysozyme: evidences from spectroscopic and docking studies
J. Hazard. Mater.
(2010)
M. Bayarri et al.
Properties of lysozyme/low methoxyl (LM) pectin complexes for antimicrobial edible food packaging
J. Food Eng.
(2014)
T. Croguennec et al.
Iron and citrate interactions with hen egg white lysozyme
Food Chem.
(2000)
R.E. Feeney et al.
Irreversible inactivation of lysozyme by copper
Arch. Biochem. Biophys.
(1956)
S. Ghosh et al.
Fibrillation of hen egg white lysozyme triggers reduction of copper (II)
Int. J. Biol. Macromol.
(2012)
Z. Cao et al.
The effect of sarafloxacin on Cu/ZnSOD structure and activity
Spectrochim. Acta A Mol. Biomol. Spectrosc.
(2015)

X. Zhao et al.

Recent progress and perspectives on the toxicity of carbon nanotubes at organism, organ, cell, and biomacromolecule levels

Environ. Int.

(2012)

H. Zhang et al.

Interactions of lead (II) acetate with the enzyme lysozyme: a spectroscopic investigation

J. Lumin.

(2013)

Z. Gu et al.

Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation

Int. J. Biochem. Cell Biol.

(2004)

H. Sun et al.

Spectroscopic investigations on the effect of N-acetyl-L-cysteine-capped CdTe quantum dots on catalase

Spectrochim. Acta A Mol. Biomol. Spectrosc.

(2014)

M.S. Ali et al.

Spectroscopic studies on the interaction between novel polyvinylthiol-functionalized silver nanoparticles with lysozyme

Spectrochim. Acta A Mol. Biomol. Spectrosc.

(2015)

J. Wang et al.

Molecular mechanism on cadmium-induced activity changes of catalase and superoxide dismutase

Int. J. Biol. Macromol.

(2015)

X. Li et al.

Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques

Chem. Biol. Interact.

(2015)

Y. Liu et al.

Investigation on the interaction of the toxicant, gentian violet, with bovine hemoglobin

Food Chem. Toxicol.

(2013)

T. Chen et al.

Binding of dihydromyricetin to human hemoglobin: fluorescence and circular dichroism studies

Spectrochim. Acta A Mol. Biomol. Spectrosc.

(2012)

V.I. Teichberg et al.

A spectrofluorometric study of tryptophan 108 in hen egg-white lysozyme

FEBS Lett.

(1970)

V.I. Teichberg et al.

Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme

J. Mol. Biol.

(1974)

Cited by (73)

Molecular mechanisms of polystyrene nanoplastics and alpha-amylase interactions and their binding model: A multidimensional analysis
2024, Science of the Total Environment
Plastic fragments are widely distributed in different environmental media and has recently drawn special attention due to its difficulty in degradation and serious health and environmental problems. Among, nanoplastics (NPs) are smaller in size, larger in surface/volume ratio, and more likely to easily adsorb ambient pollutants than macro plastic particles. Moreover, NPs can be easily absorbed by wide variety of organisms and accumulate in multiple tissues/organs and cells, thus posing a more serious threat to living organisms. Alpha-amylase (α-amylase) is a hydrolase, which can be derived from various sources such as animals, plants, and microorganisms. Currently, no studies have concentrated on the binding of NPs with α-amylase and their interaction mechanisms by employing a multidimensional strategy. Hence, we explored the interaction mechanisms of polystyrene nanoplastics (PS-NPs) with α-amylase by means of multispectral analysis, in vitro enzymatic activity analysis, and molecular simulation techniques under in vitro conditions. The findings showed that PS-NPs had the capability to bind with the intrinsic fluorescence chromophores, leading to fluorescence changes of these specific amino acids. This interaction also caused the alterations in the micro-environment of the fluorophore residues mainly tryptophan (TRP) and tyrosine (TYR) residues of α-amylase. PS-NPs interaction promoted the unfolding and partial expansion of polypeptide chains and the loosening of protein skeletons, and destroyed the secondary structure (increased random coil contents and decreased α-helical contents) of this protein, forming a larger particle size of the PS-NPs-α-amylase complex. Moreover, the enzymatic activity of α-amylase in vitro was found to be inhibited in a concentration dependent manner, thereby impairing its physiological functions. Further molecular simulation found that PS-NPs had a higher tendency to bind to the active site of α-amylase, which is the cause for its structural and functional changes. Additionally, the hydrophobic force played a major role in mediating the binding interactions between PS-NPs and α-amylase. Taken together, our study indicated that PS-NPs interaction can initiate the abnormal physiological functions of α-amylase through PS-NPs-induced structural and conformational alternations.
Molecular mechanisms of nano-sized polystyrene plastics induced cytotoxicity and immunotoxicity in Eisenia fetida
2024, Journal of Hazardous Materials
Nanoplastics (NPs) are currently everywhere and environmental pollution by NPs is a pressing global problem. Nevertheless, until now, few studies have concentrated on the mechanisms and pathways of cytotoxic effects and immune dysfunction of NPs on soil organisms employing a multidimensional strategy. Hence, earthworm immune cells and immunity protein lysozyme (LZM) were selected as specific receptors to uncover the underlying mechanisms of cytotoxicity, genotoxicity, and immunotoxicity resulting from exposure to polystyrene nanoplastics (PS-NPs), and the binding mechanisms of PS-NPs-LZM interaction. Results on cells indicated that when earthworm immune cells were exposed to high-dose PS-NPs, it caused a notable rise in the release of reactive oxygen species (ROS), resulting in oxidative stress. PS-NPs exposure significantly decreased the cell viability of earthworm immune cells, inducing cytotoxicity through ROS-mediated oxidative stress pathway, and oxidative injury effects, including reduced antioxidant defenses, lipid peroxidation, DNA damage, and protein oxidation. Moreover, PS-NPs stress inhibited the intracellular LZM activity in immune cells, resulting in impaired immune function and immunotoxicity by activating the oxidative stress pathway mediated by ROS. The results from molecular studies revealed that PS-NPs binding destroyed the LZM structure and conformation, including secondary structure changes, protein skeleton unfolding/loosening, fluorescence sensitization, microenvironment changes, and particle size changes. Molecular docking suggested that PS-NPs combined with active center of LZM easier and inhibited the protein function more, and formed a hydrophobic interaction with TRP 62, a crucial amino acid residue closely associated with the function and conformation of LZM. This is also responsible for LZM conformational changes and functional inhibition /inactivation. These results of this research offer a fresh outlook on evaluating the detriment of NPs to the immune function of soil organisms using cellular and molecular strategies.
pH-dependent interactions of biologically important metal ions with hen egg white lysozyme based on its hydration properties: Thermodynamic and mechanistic insights
2024, International Journal of Biological Macromolecules
Importance of metal ion selectivity in biomolecules and their key role in proteins are widely explored. However, understanding the thermodynamics of how hydrated metal ions alter the protein hydration and their conformation is also important. In this study, the interaction of some biologically important Ca²⁺, Mn²⁺, Co²⁺, Cu²⁺, and Zn²⁺ ions with hen egg white lysozyme at pH 2.1, 3.0, 4.5 and 7.4 has been investigated. Intrinsic fluorescence studies have been employed for metal ion-induced protein conformational changes analysis. Thermostability based on protein hydration has been investigated using differential scanning calorimetry (DSC). Thermodynamic parameters emphasizing on metal ion-protein binding mechanistic insights have been well discussed using isothermal titration calorimetry (ITC). Overall, these experiments have reported that their interactions are pH-dependent and entropically driven. This research also reports the strongly hydrated metal ions as water structure breaker unlike osmolytes based on DSC studies. These experimental results have highlighted higher concentrations of different metal ions effect on the protein hydration and thermostability which might be helpful in understanding their interactions in aqueous solutions.
Insights into the potential toxicity of Zn(II) to catalase and their binding mechanisms
2024, Journal of Molecular Liquids
As a common metal, Zinc was widely used in the in the galvanising industry. However, the overuse had contributed to the environmental overexposure of zinc, which had caused a number of health issues in environmental organisms. Catalase (CAT) plays a critical part in regulating oxidative stress in the body of humans. Interactions between Zn(II) and CAT were examined in this paper using a variety of spectroscopic techniques, molecular docking, isothermal titration calorimetry (ITC) and enzyme activity assay. According to the results, Zn(II) can interact with CAT fluorophore residues like Trp and Tyr and demonstrate fluorescence sensitization to these amino acids. Direct contact between CAT and Zn(II) led to the disruption of protein backbone structure and peptide chain tightening. When exposed to Zn(II), the secondary structure of CAT was altered, including the decreased α-helix of CAT, increased β-sheet content, and the increased grain size of the zinc-CAT complex. Measurement of thermodynamic parameters of the binding reaction by ITC showed that the force between Zn(II) and CAT was electrostatic force. In addition, the binding site of Zn(II) is close to the active center of CAT which boosted enzyme activity. In conclusion, our research demonstrates that Zinc has an impact on both the structure and function of CAT.
Lysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking
2024, Archives of Biochemistry and Biophysics
Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer. Also, tensiometric measuring of the interfacial tension was used for calculation of binding parameters accordingly to Fainerman model. The treatment of complexes with atomic tritium followed by trypsinolysis and analysis of tritium distribution in the lysozyme peptides reveals the binding sites, binding energies in which were analyzed using molecular docking. Formation of complexes with amikacin and levofloxacin preserves secondar structure of protein. However, the formation of complex with amikacin leads to the almost total loss of the enzymatic activity of lysozyme and the redshift of the maximum on the lysozyme fluorescence band. A slight decrease in the distribution coefficient of lysozyme in the presence of amikacin assumes that the complex has higher hydrophilicity in comparison to lysozyme without additives. The most favorable for binding were the positions of the active centers that included amino acids Asp52 and Glu35, as well as in the vicinity of peptide His15-Arg21, with the participation of amino acids Tyr20, Arg14. In the case of levofloxacin, the formation of lysozyme-ligand complex in aqueous solution is possible without changing the microenvironment of the active center of the protein. Binding of levofloxacin to the active center of the enzyme was the most favorable, but Asp52 and Glu35 that are responsible for the enzymatic activity of lysozyme, were not affected.
Elucidating binding mechanisms of naringenin by alpha-chymotrypsin: Insights into non-binding interactions and complex formation
2023, International Journal of Biological Macromolecules
As an inevitable parameter in the description of enzyme properties, the investigation of enzyme-ligand interactions has attracted a lot of attention. Alpha-Chymotrypsin (α-Chy) is essential for protein digestion and plays an important role in human health. Naringenin (NAG) as a potent antioxidant has recently been applied in the pharmaceutical industry. Using multispectral methods and computational simulation techniques, the binding strength of NAG to α-Chy was investigated in this research. UV–vis and fluorescence quenching data showed significant spectral changes upon binding of NAG to α-Chy. As demonstrated by fluorescence techniques, NAG could employ a static quenching process to decrease the intrinsic fluorescence of α-Chy. Both circular dichroism (CD) and FTIR spectroscopic analyses revealed that binding of NAG to α-Chy caused more flexible conformation. The slight increases in RMSD (0.06 nm) were observed for the NAG-(α-Chy) compound was supported by the results of thermal stability data. Docking computation confirmed that hydrogen and Van der Waals interactions are the important forces, which is in exact agreement with thermodynamics studies. Kinetic analysis of the enzyme showed an increase in activity, which was consistent, with the MD simulation results. The findings from the in-silico studies were in complete agreement with the experimental results.

View all citing articles on Scopus

View full text

Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Influence of copper on the fluorescence intensity of lysozyme

Conclusions

Acknowledgments

Mutat. Res. Fundam. Mol. Mech. Mutagen.

Toxicology

J. Trace Elem. Med. Biol.

Anal. Chim. Acta

J. Hazard. Mater.

J. Food Eng.

Food Chem.

Arch. Biochem. Biophys.

Int. J. Biol. Macromol.

Spectrochim. Acta A Mol. Biomol. Spectrosc.

Environ. Int.

J. Lumin.

Int. J. Biochem. Cell Biol.

Spectrochim. Acta A Mol. Biomol. Spectrosc.

Spectrochim. Acta A Mol. Biomol. Spectrosc.

Int. J. Biol. Macromol.

Chem. Biol. Interact.

Food Chem. Toxicol.

Spectrochim. Acta A Mol. Biomol. Spectrosc.

FEBS Lett.

J. Mol. Biol.