Elsevier

Food Chemistry

Volume 406, 16 April 2023, 135070
Food Chemistry

Lysozyme–phenolics bioconjugates with antioxidant and antibacterial bifunctionalities: Structural basis underlying the dual-function

https://doi.org/10.1016/j.foodchem.2022.135070Get rights and content

Highlights

Abstract

This work aims at adopting an Electron Paramagnetic Resonance (EPR) spectroscopic technique to help understanding protein–phenolic conjugation and final functionalities relationship as well as the underlying structural basis of antioxidant and antibacterial dual functionalities. Specifically, lysozyme (Lys) was conjugated with two natural phenolic acids, i.e. rosmarinic acid (RA) and gentisic acid (GA, our previous work) with obviously different molecular features. Lys-RA displayed 8.6- and 4.0-times enhanced antioxidant stoichiometry compared to the native Lys and ones with GA, respectively, due to the stronger antioxidant activity of RA. However, RA conjugation mitigated both enzymatic and antibacterial activities of Lys-RA conjugates. Such inhibition effect is attributed to the greater structural and surface property changes of Lys upon conjugating with RA. Furthermore, the polyphenol conjugation related structural basis of disturbance, reactivity and selectivity were explored via site-directed spin labeling (SDSL)–EPR. A dynamic picture of reactivity and selectivity of phenolics conjugation on Lys was proposed.

Introduction

Protein-based conjugates, including polymer- and small compound-protein conjugates, are the new frontiers of materials science benefiting drug/protein delivery, biocatalysts, sustainable materials, and nutrition (Milczek, 2018, Quan et al., 2019b). Conjugating a functional synthetic or natural “partner” to the host protein can endorse the product multiple (at least dual) functionalities, namely the biological function of the protein and the function/property of the “partner” (Boutureira and Bernardes, 2015, Ekladious et al., 2019). In addition, the “partner” can induce changes in the chemical/physical properties and the dynamics/structure of the host protein, complexing the outcome of the conjugation and the resultant biomaterials function (Hou et al., 2016, Krall et al., 2016). As compared to the polymer counterpart, small functional compounds have the special advantage of minimal perturbation to the host protein while endorsing new functions. Simultaneously, conjugation with protein could solve biocompatibility problem of small compounds. Thus, small compound-protein conjugates become an attractive research area (Schuler and Eaton, 2008, Xu et al., 2021). Some promising examples include the conjugation of polyphenols to food proteins to improve their antioxidant capability or the attachment of anticancer drugs on protein surface for cell targeted cancer therapy (Abd El-Maksoud et al., 2018, Kuan et al., 2018, Li et al., 2019).

In the past decades, food proteins were modified by conjugation with distinct bioactive/functional molecules to improve biological/physicochemical functionalities or yield new characteristics for particular applications. Accordingly, research in protein conjugates especially food proteins were mainly focused on the methodological development of conjugation and improving their functionalities (Nakamura et al., 1991, Zha et al., 2021). However, a systematic understanding and control of protein conjugation and final functionality is the “black box” in food science, especially for future food design. This is the main reason that the desired functionality modification currently has to be carried out by trial and error. The concern is especially serious when adverse effects are developed, such as structural deformation, instability and aggregation. In principle, site-directed mutagenesis approaches can help better understanding the structure–function relationship of food proteins in order to achieve the desired functionality (Watanabe et al., 2018, Zhang et al., 2019). However, practically it is difficult to genetically alter food proteins in commercial scale (Hoyt et al., 2019). The only alternative becomes wisely selecting the conjugation partners, for example, polyphenols. However, there is a lack of knowledge on how different partners influence the function of food proteins upon conjugation, making it not straightforward to select the best partner for desired function. Protein conjugation complexities caused by uncontrollable conjugation site/abundancy, uniform composition and structure and crosslinking contribute to the functionality uncertainty of result protein conjugates (Rohn, 2014, Xu et al., 2021). Although site-selective modification of protein has emerged as a promising strategy, only certain amino acids with specific functional group on the host protein could be constructed, which greatly limits broad applications in protein conjugation (Boutureira and Bernardes, 2015, Reddy et al., 2020). In addition, conjugation may induce host protein changes in surface charge, hydrophobicity, and structure/dynamics (DeSantis and Jones, 1999, Quan et al., 2019b). While the surface charge and hydrophobicity of a protein upon conjugation can be measured experimentally, the resultant data often cannot directly explain the final function change (Poklar Ulrih, 2017). The structural and dynamic changes are, therefore, believed more important and thus, required to understand the functions of the conjugated protein, and eventually help guide the partner selection.

Revealing the structural basis of protein conjugation requires knowledge on the number and positions of conjugate molecules on the host protein as well as the perturbation to the structure and dynamics of the host protein at a sufficiently high resolution. The complexity, dynamics, and heterogeneity of the resultant conjugates challenge a number of experimental tools for protein structure probing. A potential solution is site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy (Klare and Steinhoff, 2009, Pan et al., 2018, 2021). SDSL allows site-specific attachment of a small nitroxide radical to a residue of interest of the host protein, while EPR detects the backbone dynamics of the labeled side chain regardless of the complexity of the system (Li et al., 2021, Pan et al., 2021). Such information reveals not only the local motional changes of the protein due to the conjugation but also the possible attachment site of the conjugating “partner”. Scanning through multiple regions of the host protein will lead to an overall picture of the possible attachment sites of the “partner” as well as the alteration in protein surface hydrophobicity (Sun et al., 2019), and relative reactivity of the “partner” toward different protein regions, serving as the structural basis of the protein conjugate design (Borbat & Freed, 2007).

Polyphenols are especially attractive for food protein modification due to their rich natural resources, high structural diversity and strong antioxidant functionality (Quan et al., 2019a, Reed and De Freitas, 2020). Previously, we have conjugated a model protein, lysozyme (Lys) with antimicrobial function, and a phenolic compound, gentisic acid (GA), to prepare and optimize dual functional (antioxidant-antimicrobial) conjugates (Li et al., 2022). However, there was no structural basis probed in that work. In fact, the structure and properties of polyphenol may determine the structure–function relationship of the generated protein conjugates. To prove it, we select rosmarinic acid (RA) as a comparison to probe how the structure of polyphenol influences the structure–function relationship of Lys conjugates. As compared to GA, RA (Fig. 1) was chosen since it has higher potential to improve antioxidant capacity of the conjugates duo to the double number of phenolic hydroxyl groups and o-diphenol structure (Lucarini and Pedulli, 2010, Moazzen et al., 2022). Also, two phenolic rings structure makes RA more hydrophobic, which may impact surface properties and interaction sites of the protein. Furthermore, the relatively large molecular size of RA could induce larger structural disturbance to the host protein and result in obvious functionality difference. Lastly, the electronic environment difference around carboxyl group may change reactivity selection and order when polyphenols interact with protein. We did observe potential antioxidant capacity improvement but antibacterial activity drops of the Lys-RA conjugates in comparison with Lys-GA ones, which is likely caused by the structural and dynamic disturbance of Lys caused by chemically grafting RA to Lys surfaces. Therefore, we employed SDSL-EPR spectroscopy to probe the structural basis of such functional difference between Lys-RA and Lys-GA conjugates, which revealed the relative reactivity and selectivity of GA and RA to different Lys surface residues/regions. Our work facilitates the rational design of protein conjugates based on the structure and function of the product as well as the possibility of conjugating different partners to tune the functionalities of bioactive product. This work serves as the beginning of efforts to demonstrate the possibility of filling in the “black box” in future protein conjugate design in order to guide the partner selection for food protein functionalities modification.

Section snippets

Materials

Lys from chicken egg white (lyophilized powder, protein content ≥ 90 %), GA, RA, 4-morpholinepropanesulfonic acid (MOPS), phosphate buffered saline (PBS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 1-ethyl-3-(3-dimethyl amino-propyl) carbodiimide (EDC), N-hydroxysulfosuccinimide sodium salt (sNHS), 2-(N-morpholino) ethanesulfonic acid (MES) hydrate, glycerol for molecular biology (≥99.0 %), potassium phosphate monobasic, and potassium phosphate dibasic were purchased from MilliporeSigma (St. Louis,

Research design

As introduced above, the main goals of this work are to i) compare the antioxidant and antibacterial capacity of the Lys-polyphenol conjugates and ii) elucidate the structural/dynamic changes underlying the functional alterations of Lys upon conjugation and the associated polyphenol selectivity. To achieve the first goal, we employed RA which has doubled hydroxyl groups per molecule as compared to GA (Fig. 1) while still possessing a carboxyl group for conjugation with lysyl residues in

Conclusions

In summary, we have successfully demonstrated that RA, a phenolic compound with stronger antioxidant activity as compared to GA, can be used to conjugate with an antibacterial enzyme to enable potentially enhanced antioxidant capability while, to a certain extent, retaining acceptable antibacterial functionality. Structure and properties differences of GA and RA contributed to the changes of antioxidant and antibacterial dual-functionalities of the corresponding Lys conjugates. By adopting

CRediT authorship contribution statement

Hui Li: Investigation, Methodology, Formal analysis, Data curation, Writing – original draft, Writing – review & editing. Yanxiong Pan: Methodology, Writing – review & editing. Chun Li: Methodology, Writing – review & editing. Zhongyu Yang: Conceptualization, Supervision, Project administration, Writing – review & editing. Jiajia Rao: Resources, Supervision, Writing – review & editing. Bingcan Chen: Conceptualization, Supervision, Project administration, Funding acquisition, Writing – review &

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work is supported by the National Institute of Food and Agriculture, United States Department of Agriculture Grant 2019-67018-29186. We gratefully acknowledge Prof. Teresa M. Bergholz (Department of Food Science and Human Nutrition, Michigan State University) for providing strains for antimicrobial study. We acknowledge the NDSU Electron Microscopy Center core facility and the corresponding supported National Science Foundation (Grant No. 0923354).

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