Lipopolysaccharides inhibit REG3A self-aggregation on gold

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Lipopolysaccharides inhibit REG3A self-aggregation on gold nanoparticles: A combined study of multivariate analysis on time-resolved localsurface-plasmon-resonance spectra and molecular modeling Zhenxin Han, Xi Ren, Qiang Huang, Ting Shi, Yuping Lai, and Yi-Lei Zhao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00087 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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Langmuir

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Lipopolysaccharides inhibit REG3A self-aggregation on gold nanoparticles: A

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combined study of multivariate analysis on time-resolved local-surface-plasmon-

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resonance spectra and molecular modeling

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Zhenxin Han1⸆, Xi Ren1⸆, Qiang Huang1, Ting Shi1, Yuping Lai2, and Yi-Lei Zhao1*

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State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic

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& Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University,

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Shanghai, 200240, China

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Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal

University, Shanghai, 200241, China

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⸆ The

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* To whom correspondence should be addressed. Tel: 86-21-34207190; Fax: 86-21-34207347; Email:

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[email protected]

two authors equally contribute to this work.

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ACS Paragon Plus Environment

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Abstract

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Aggregation behavior of proteins on the surface of gold nanoparticles (AuNPs) has been

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extensively studied for its promising applications in biosensing, bioimaging, photodynamic

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therapy, drug delivery, etc. In this work, we studied adsorption kinetics of an antimicrobial

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protein, REG3A (regenerating islet-derived protein 3-alpha), on the surface of as-

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synthesized citrate-capped AuNPs under the influence of lipopolysaccharides (LPSs), with

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a combined method of UV-vis spectroscopy, multivariate analysis, and molecular dockings.

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In the AuNPs-REG3A binary system a component with the “up-and-down” signal was

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detected by the in-depth data analysis on the time-resolved spectroscopic data,

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corresponding with the protein agglomeration and exfoliation observed in the TEM

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(Transmission Electron Microscopy) and AFM (Atomic Force Microscopy) experiments.

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Intriguingly, LPSs can rescue the spectral oddity - the adsorption pattern in the AuNPs-

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REG3A-LPS ternary system becomes normal and similar to a typical single-layer mode as

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in our previous study of the serum albumin – AuNP system (Spectroscopy letters, 2016,

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49, 434-443). The following-up molecular modeling suggests that LPS molecules mainly

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interact with three segments of REG3A amino acid sequence, i.e. P109-T110-Q111-G112,

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P115-N116, and P137-S138-T139. The latter two protein-ligand interactions impair the

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REG3A-REG3A protein-protein interaction (PPI) between the two subunits (E114-P115-

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N116-G117-E118 and N136-P137-S138-T139-I140). Thus, our results elucidate the LPS

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inhibitory effect on fibrous protein self-aggregation at the AuNP surface and the molecular

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dockings give a plausible mechanism to rationalize the competition among protein-protein

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and protein-ligand interactions.

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Keywords: REG3A; AuNPs; lipopolysaccharides; adsorption kinetics; molecular modeling

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Introduction

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REG3A, also known as HIP/PAP, is a secreted intestinal bactericidal protein that

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contains a C-type lectin domain. REG3A can also regulate keratinocyte proliferation to

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promote wound healing and induce a negative regulator SHP-1 to control wound

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inflammation.1,2 Particularly, REG3A is highly polarized by a variety of positively and

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negatively charged amino acid residues and feasibly forms a fibril-like structure.3,4 The

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formation of REG3A hexamer unit is in the initiator of antibiosis – the hexamer units stack

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together to construct fibril-like transmembrane channel within the outer membrane of

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bacteria and then kill bacteria. Interestingly, this antimicrobial activity is only effective for

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gram-positive bacteria but invalid to gram-negative bacteria; it is believed that the

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exclusive lipopolysaccharide molecules (LPSs) of gram-negative bacteria might play

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essential roles in their escaping from REG3A antibiosis.5 LPSs are secreted by gut

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microbiota and function as endotoxin in human body. LPSs can elicit severe immune

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responses in human body and also cause many metabolic diseases such as diabetes and

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obesity.6-8 However, little is known about the interplay of LPSs and REG3A particularly

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regarding their intermolecular interactions.

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AuNPs have long been employed as a useful material in bionanotechnology because

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of their unique properties(i.e. size-dependent optical property,9 moderate biotoxicity,10

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stability and high extinction coefficients).11 Our previous studies have successfully

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characterized adsorption patterns between AuNPs and serum albumin (both human and

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bovine) by using the UV-vis spectroscopic data,12 and

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widely reported in literatures .13-16 Multiple experimental approaches about thermodynamic

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and kinetic measurements have been developed to investigate interactions on the interface

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of protein and AuNPs.14,17-18 Nevertheless, kinetic features of a fiber-forming protein

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REG3A on the surface of AuNPs and the influence of LPSs are never elucidated yet.

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Therefore, it is of great interest to study their adsorptive features in the REG3A-AuNPs

parallel bioconjugations have been

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binary system and the LPSs effect in the REG3A-AuNPs-LPS ternary system.

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In this work, aggregative characteristics of REG3A on AuNPs were first compared

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with bovine serum albumin (BSA) with the transmission electron microscopy (TEM) and

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atomic force microscopy (AFM). Then local-surface-plasmon-resonance kinetic

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measurements of the binary and ternary systems with the UV-vis spectroscopy were

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presented. Adsorption features were distinguished from kinetic UV-vis data of the two

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systems with the combined multivariate analyses of principal component analysis (PCA),19

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non-negative matrix factorization (NMF),20-21 and multivariate curve resolution alternating

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least squares (MCR-ALS).22-24 Moreover, molecular modeling methods, including

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ZDOCK25 and Autodock Vina26 , were employed to generate docking poses and

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intermolecular interactions between REG3A and LPSs and within REG3A self-aggregation

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were analyzed.

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Materials and methods

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1. Chemicals and Materials

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All chemicals were of analytical grade and used without further purification. Milli-Q

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purified double distilled water was used throughout experiments. Trisodium citrate

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dehydrate was purchased from Aladdin (Shanghai, China). Chloroauric acid tetrahydrate

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and BSA were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). LPSs

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was obtained from Sigma-Aldrich (Germany). REG3A was expressed and purified

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according to published methods, provided by Dr. Yuping Lai from East China Normal

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University.4,27

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2. Synthesis of AuNPs

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All glassware used in the experiment was cleaned in a bath of freshly prepared 3:1

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HCl/HNO3, rinsed thoroughly in Milli-Q purified water, and oven dried prior to use. 4


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AuNPs were prepared following Frens’s method.28 Typically, a 51.0 mL aqueous solution

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of chloroauric acid tetrahydrate (2.06 mL, 1%) was heated to boiling, then 1.90 mL of

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trisodium citrate (1%) was added. The boiling solution was stirred for another 30 min, then

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the AuNPs solution was diluted until the spectral absorption peak reached about 0.8 at the

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wavelength of 520 nm for further conjugation experiments, then stored at 4 °C in the dark.

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The concentration of AuNPs solution was determined to be 3.9 nM by dividing the

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absorbance at 450 nm by the extinction coefficient of 9.57 × 107 M-1·cm-1 (c = A450 /

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ε450).29 The size and morphology of AuNPs were characterized with TEM (JEM-2100).

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3. AFM Characterization and Dynamic Light Scattering (DLS) Analysis

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AFM experiments were performed with the Multimode Nanoscope III (Digital

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Instruments, Santa Barbara, CA) operating in the tapping mode at ambient conditions. The

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sample was a mixture with a ratio of 80 nM protein solution to 3.9 nM AuNPs solution.

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DLS Analysis and zeta potential measurement were conducted using a Zetasizer Nano

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ZS90 instrument from Malvern Instruments Ltd. (Westborough, MA). The analyses were

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performed with He-Ne Laser (633 nm) at a scattering angle of 175 ̊ at 25 °C. For dynamic

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light scattering detection, samples were measured every 30 seconds for 8 minutes. The

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particle sizes were reported as averages of three measurements. High salt condition was

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achieved by adding 5 × PBS buffer.

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4. UV-vis Kinetic Measurements

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UV-visible measurements were conducted with an Agilent 8453 UV-vis spectrometer

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(Agilent, USA), using a quartz cuvette with 1 cm path length. AuNPs solutions were diluted

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to specified concentrations in advance and LPSs were diluted to 10 μg/mL. For UV-vis

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measurement of the REG3A-AuNPs system, REG3A solution was diluted to different

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concentrations (10 to 100 nM). AuNPs solution (1 mL) and protein solution (1 mL) were

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micropipetted into a 1 cm × 1 cm × 4 cm cuvette and mixed thoroughly. For small molecule 5


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concentration screening experiment, 1 mL AuNPs solution, 0.5 mL small molecule solution

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and 0.5 mL of 30 nM protein solution were micropipetted into the cuvette and mixed

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thoroughly. UV-vis kinetic measurements were taken immediately. The cuvette and all

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solutions remained at 25 °C before and during the UV-vis measurements by being

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connected with a thermostat (BILON-T-3001S, Shanghai, China). Every single

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measurement was collected automatically from 190 nm to 1100 nm with intervals of 1.5 s

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for 480 s. After measurements, the protein-AuNPs mixture in the cuvette was collected and

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its final pH was determined to be around 5.95. The binary systems were tested in the

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triplicated experiments and the ternary systems in the duplicated experiments, to confirm

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the adsorptive features of the two systems. After the initial assessment on all the data (See

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Fig. S1 in the Supporting Information), the following sections were exhibited with a

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representative dataset for each system.

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5. Data processing and analysis

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Pretreatment of the measured UV-vis datasets was achieved by calculating the

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absorbance shifts. Therefore, the Delta-Absorbance profiles (DetA) were used for

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preliminary judgement of time-resolved stability for each system. Secondly, PCA was

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applied to each system to determine the total principal component number and extract the

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featured signals. The scores submatrices represented the concentration while the loadings

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submatrices represented the spectral information for each principal component. The

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number of principal components for each system was then re-evaluated by calculating a

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series of parameters such as cumulative contribution rate, correlation coefficients, and the

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residual sum of squares. Furthermore, the PCA-resolved spectral profiles and time-

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resolved concentration profiles were refined using the NMF and MCR-ALS approaches

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for a final verification.

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6. Molecular modeling 6


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Molecular modeling studies were carried out to understand the structural details of

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molecular interactions. A rigid-body docking program, ZDOCK, was used to generate

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multiple docking poses of REG3A-REG3A and REG3A-LPS in a time-saving and efficient

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manner. The shape complementarity, desolvation, and electrostatics were optimized for the

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two molecules. After thoroughly searching for possible rotational and translational poses

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of ligand, the ZDOCK energy-based scoring function gave a ZSCORE value for each pose.

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Specifically speaking, a typical and fundamental structure of LPS molecule (Kdo2-

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lipid A) was built up in silicon with the GaussView 5 software, then the LPS structure was

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optimized by Discovery Studio 3.5 (DS) software. Structure of active form REG3A was

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acquired from the PDB database (PDB code:4MTH, http://www.rcsb.org). Molecular

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docking between REG3A and LPS, and REG3A-REG3A were performed with both the

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ZDOCK and Autodock Vina methods. The Dipole moment of REG3A molecule was

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calculated with Protein Dipole Moments Server (http://dipole.weizmann.ac.il/).30

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Schematic 2D diagrams of protein-ligand interactions were illustrated with the LigPlot+

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program.31

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Results and discussion

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REG3A aggregates in the presence of AuNPs

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Characteristic parameters of the synthesized AuNPs such as its average diameter and

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Zeta potential were calculated from TEM, DLS and Zeta potential experiments (Fig. S2,

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Table S1). The average diameter of AuNPs was determined to be 12.6 nm by calculating

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more than 100 particles. The hydrodynamic diameter of AuNPs was measured as 28.9 ±

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0.8 nm, and the electrostatic potential was − 42.9 ± 1.6 mV. After incubated with BSA and

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REG3A, the Zeta potential of the protein-AnNPs system increased by 13.8 mV and 23.2

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mV respectively, indicating that REG3A could increase more surface electrostatic potential

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of AuNPs. 7


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To study mechanical properties of REG3A with the presence of AuNPs, AFM

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experiments were performed, and the fibrous network structure of REG3A molecules

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around AuNPs was clearly observed (Fig. 1). As indicated by the red arrows, some of the

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REG3A fibrils were not directly attached to the surface of AuNPs, suggesting that these

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fibrils might fall off the surface of AuNPs. In contrast, BSA exhibits little self-aggregation

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tendencies in the presence of AuNPs.32,33 Consequently, given the fact that REG3A

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dissolves in aqueous solution in the form of monomer in the absence of AuNPs, it is

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reasonable to conclude that AuNPs act as a nucleation center in the aggregation of REG3A.

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Moreover, REG3A fibrils would break down from AuNPs when it grew up to a certain

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degree. Thus, a variety of protein-nanoparticle complexes and protein self-aggregates

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coexisted in the system.

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Stability experiments revealed the formation of protein corona and its protection on

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AuNPs under high ionic strength. Without coating proteins, the hydrodynamic diameter of

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AuNPs increased significantly at higher ionic strengths, indicating the swift aggregation of

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destabilized AuNPs into larger particles in the case of lacking the protection of protein

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corona. However, after incubating for only an hour, REG3A and BSA both showed a

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protective effect on AuNPs, and this phenomenon was rather significant after the

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incubation duration was prolonged to 48 hours (Fig. S3). Compared with the single layer

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formation of the protein corona in BSA-AuNPs system, the REG3A-AuNPs system

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seemed much less stable with the increase of ionic strength, possibly due to the relative

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loose protein-fiber structure on the surface of AuNPs.

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Figure 1. AFM images of (a) pure REG3A, (b) and (c) AuNPs incubated with REG3A, and

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(d) AuNPs incubated with BSA.

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REG3A-AuNPs absorption characteristics in the absence and presence of LPSs

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In the UV-vis spectra of the REG3A-AuNPs binary system, the absorbance peak at

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the wavelength of 521 nm reduced the intensity slightly with a 1 nm redshift within 480

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seconds. Compared with the REG3A-AuNPs-LPSs ternary system, the differences between

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the initial and final spectra of the binary system were relatively more significant than that

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of the ternary system, particularly in the long-wavelength range (Fig. 2a and b). The subtle

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differences suggest a relative instability of the binary system compared with the ternary

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system. In the time-resolved viewpoint, the behavior of absorbance at the wavelength of

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520 nm changed quite differently between the binary and ternary systems: the absorbance

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signal decreased steadily, with several “up-and-down” stages in the binary system; by

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contrast, in the ternary system the absorbance changed much smaller in quantity with a 9


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moderate declining trend even with a higher noise (Fig. S4). In agreement with the AFM

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results, these differences appeared in the two systems suggested a complexity in dynamic

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pattern of the binary system, possibly due to repetitive binding and falling of REG3A on

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the surface of AuNPs.

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The concentration-dependent DetA profile was calculated by subtracting the

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minimum UV-vis absorbance intensity from the maximum, using the protein concentration

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in a range of 10 to 100 nM. With the increasing protein concentration, the overall

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magnitude of DetA spectra increased, accompanied with a considerable redshift (12 nm for

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the binary system and 15 nm for the ternary system). After the addition of LPSs, the

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maximum value in the DetA profile decreased from 0.05 to 0.04, and the band broadened

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to over 50 nm (Fig. 2c and d). Insets showed the integrals of the peak area in the highlighted

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region, which exhibited a dose-dependent relationship with the REG3A concentration. In

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the case of the binary system, the peak area changed much significantly once the

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concentration of REG3A reached 80 nM while with the introduction of LPSs, the peak area

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increased smoothly. In the controlled experiment, the variation of absorbance of pure

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AuNPs solution was only about 0.01 under the same UV-vis irradiation condition (Fig. S5).

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Figure 2. The initial (t = 0 s) and final (t = 480 s) UV-vis absorption spectra of (a) the

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AuNPs-REG3A binary system and (b) the AuNPs-REG3A-LPSs ternary system,

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respectively, and the UV-vis Delta spectra with different REG3A concentrations of (c) the

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binary system and (d) the ternary system (insets are the integrals of the highlighted peak

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areas in the two systems).

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Determination of the number of components

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In order to distinguish the dynamic features from the time-resolved spectra in the two

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systems, principal component analysis (PCA) was performed to determine the total number

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of the components for each system, and a score plot was produced. The first four principal

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components were then extracted from the PCA-resolved spectral profiles. (Fig. S6). The

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fourth component signals of all cases presented random and noisy signals, but the third

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component showed significant difference between the two systems. The PCA analysis

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suggests that, with p-value < 0.0001, the component number of REG3A-AuNPs binary 11


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system is three, while the REG3A-AuNPs-LPSs ternary system consists of two principal

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components. To further validate the two component numbers in the two systems, the NMF

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analysis was carried out according to the rank from 1 to 4, obtaining more reliable spectral

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profiles and kinetic concentration profiles (Fig. S7 and Fig. S8). Consistently, the NMF

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estimation of the numbers of components gave the same results, which was three and two

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for the binary and ternary system, respectively.

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Figure 3. The PCA analysis of the two systems, including cumulative contribution rates of (a)

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the binary system and (b) the ternary system, and (c) the PCA scores plot of the first two

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principal components with the different concentrations of REG3A concentrations, and (d)

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the corresponding PCA loadings information.

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For further verification, standard parameters such as cumulative contribution rate

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(CCR) and cophenetic correlation coefficient as well as the residual sum of squares (RSS)

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were calculated for re-evaluation of the number of components in each system. In each 12


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system, the first two principal components contributed most of the signals in the UV-vis

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spectra. The third principal component is significant in the binary system but neglectable

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in the ternary system (Fig. 3a and b). Because a high quality resolution result of a given

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dataset is usually characterized by relatively larger cophenetic coefficients and little RSS,

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the number of components were determined to be three for the binary system and two for

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the ternary system (Fig. S9), once again in agreement with the previous conclusion.

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Feature extraction: pure spectra and time-resolved kinetic profiles

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The UV-vis data were then decomposed for feature extraction of pure spectra profiles

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and time-resolved concentration profiles, according to the best-determined number of

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components for each system. Two resolved submatrices from the NMF analysis were

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selected as the initial estimation for MCR-ALS optimization. According to the total number

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of principal component, spectral curves and the time-resolved kinetic evolution curves of

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both systems were refined by the NMF based MCR-ALS optimization (Fig. 4a and b). The

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resolved absorption peak of the component one (the black curve) was located around 519

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nm, which is the characteristic absorption peak of AuNPs; therefore, this component

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represented signals of AuNPs both in the binary and ternary systems. Component two and

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three in the binary system were postulated as different species of REG3A-AuNPs complex.

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the absorption peaks of these complexes were at 564 nm and the overall signal intensity of

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the blue curve was stronger than the red species. With the addition of LPSs, the second

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component disappeared from the binary system, resulting in the existence of only two

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principal components in the ternary system and the complex redshift of the absorption peak

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to 596 nm.

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More intriguingly, the kinetic signals of the third component in the binary system were

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highly disordered, but with the presence of LPSs, this odd accumulating trend was

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eliminated and replaced with a distinctive steadily accumulating pattern (Fig. 4 c and d).

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When the concentration of REG3A reached to 70 nM, the up-and-down signals of the 13


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binary system were significant and random; scores were only reliable with values higher

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than 0.01 and the trend was neither predictable nor correlated with the increased

3

concentration of REG3A. However, the kinetic evolution curves of the ternary system

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exhibited an accumulation trend with time after the addition of 10 μg/mL LPSs and

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continued to exist even the concentration of REG3A was increased from 10 nM to 100 nM.

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The extracted kinetic information from both systems are in agreement with the results from

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DetA analysis demonstrated in Fig. 2.

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Figure 4. The PCA/NMF/MCR-ALS optimized spectral profiles (a and b) and time-

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resolved concentration profiles (c and d), in which (a) and (c) for the binary system and (b)

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and (d) for the ternary system.

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Molecular Modeling

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Although REG3A did not show self-aggregation behavior in a nM-level dilute

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solution, the self-aggregation tendency of REG3A molecule has already been characterized

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in biological systems. the hexamer complex of REG3A was proved to be initialized as the 14


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functional unit related with bactericidal activity.5 More importantly, the introduction of

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negatively charged AuNPs elicited the complex-forming phenomenon. With highly

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polarized surface charge distribution on REG3A, electrostatic interactions could play an

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essential role in both of the REG3A self-aggregation and the interplay of REG3A and

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AuNPs. Polar amino acid residues of REG3A briefly separate to two sides in the 3D

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structure, resulting in a relatively large dipole moment (PDB code: 4MTH, 753 Debye)

7

compared to BSA (PDB code: 3V03, 579 Debye- the average dipole moments of 14960

8

protein is 542.66±417.88 Debye according to the Protein Dipole Moments Server).30 In

9

previous studies, electrostatic interactions have been considered to dominate in the BSA-

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AuNPs interaction.14

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To illustrate the details of specific intermolecular interactions within REG3A complex,

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we carried out ZDOCK modeling with two REG3A molecules to mimic the REG3A self-

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aggregation. As shown in Fig. 5a, most docking sites were clustering at the top and side

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positions of REG3A, representing the two dominant patterns of top-to-top interactions and

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side-by-side stacking in the REG3A hexamer structure. In particular, the protein docking

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result indicates that the top-to-top poses were in higher ranking and the majority part,

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superior to the side-by-side pattern. The top-ranking pose (Fig. 5b) was selected from a

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total of 2000 poses as a representative structure for the binding analysis. It is found that

19

one coiling structure (Glu114-Pro115-Asn116-Gly117-Glu118) and an alpha helix

20

(Asn136-Pro137-Ser138-Thr139-Ile140) were the most responsible moieties during

21

protein-protein conjugation, located at the center of the REG3A hexamer. In the DIMPLOT

22

(Fig. 5c), key residues in the REG3A (A)-REG3A (B) interface were clearly shown.

23

Intermolecular interactions between coiling structures were mainly hydrophobic

24

interactions (formed by Glu114, Pro115, Asn116 and Gly117). the REG3A dimer was

25

stabilized by hydrogen bonds formed by polar residues (Asn136 and Ser138) of the alpha

26

helix.

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1 2

Figure 5. The molecular docking results of REG3A self-binding study, including (a) two

3

dominant binding modes (“top” and “side”) and the high-ranked 10 docking clusters, (b)

4

The highest-ranked pose of “top-top” interaction of two REG3A molecules, and (c)

5

schematic DIMPLOT diagram of the interface.

6 7

A similar ZDOCK modeling method was applied to the REG3A and LPS interaction

8

system to understand how LPSs competed with the self-binding interaction. The 2000

9

docking sites from 3600 poses were clustered in 100 groups and the top 3 clusters showed

10

two coils (Pro109-Thr110-Gln111-Gly112 and Pro115-Asn116). an alpha helix (Pro137-

11

Ser138-Thr139) of REG3A was critical during binding with LPSs, as shown in Fig. 6 (and

12

Fig. S10). The two coils are the “carbohydrate-binding loop” of REG3A, which has been

13

previously reported to be specific in recognizing and binding carbohydrate,27 and the “EPN”

14

motif (Glu-Pro-Asn) of REG3A, which can conjugate with the carbohydrate chain of

15

peptidoglycan.34 These results are in good agreement with the previous literatures and

16

Autodock Vina docking gave similar results (Fig. S11). Moreover, the average ZDOCK 16


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scores for 3600 poses of REG3A-LPS were calculated to be7.11±0.91--larger than that

2

for REG3A-REG3A interaction (6.49±0.87). Since the scoring function of the ZDOCK

3

program is energy based and a higher ZDOCK score represents a more favorable docking

4

pose, it is reasonable to conclude that the binding interaction between LPS and REG3A is

5

stronger, because the formation of REG3A hexamer was inhibited by LPS. Indeed, LPS

6

was tightly bound to REG3A primarily by hydrogen bonds and hydrophobic interactions.

7

As illustrated in Fig. 6, common amino acid residues such as Pro115, Asn116, Ser141, and

8

Ser142 of REG3A contributed positively to intermolecular binding. When compared, the

9

REG3A-LPS complex is more stable than REG3A self-interaction due to more parts of

10

LPS molecule are interacting with receptor REG3A, and the structure of LPS is more

11

flexible compared with another REG3A ligand.

12

In the binary system, REG3A aggregated via hexamer to form a fibrous structure on

13

the negatively-charged surface of AuNPs in REG3A-AuNPs binary system. With the

14

stacking of hexamers, increased electrostatic energy led to the fibrils falling off (Fig. S10),

15

which gave the observed fluctuations in the spectrum. In the ternary system, REG3A

16

conjugated to the surface of AuNPs in the presence of LPSs. The introduction of LPSs

17

blocked the intermolecular interactions between REG3A molecules, especially the self-

18

binding interactions responsible for the formation of the central pore in the REG3A

19

hexamer. As a result, the self-aggregation pattern of REG3A disappeared in the ternary

20

system, leaving only REG3A mono-layer and REG3A-LPSs complexes adsorbed on the

21

surface of AuNPs.

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1 2

Figure 6. The molecular docking results of LPS onto REG3A, including (a-c)

3

representative poses from the highest-ranked 3 clusters, and. (d-f) the Ligplot 2D-diagrams

4

of the corresponding binding pose. (Hydrogen bonds are shown in green dotted lines,

5

hydrophobic interactions are presented with the red spoked arcs, and key residues are

6

highlighted with red circles).

7 8

Conclusions

9 10

LPSs present an inhibitory effect on the bactericidal function of human intestinal

11

antimicrobial protein REG3A. In this work, the AFM, TEM, UV-vis spectral kinetic

12

measurements, and multivariate analysis methods, as well as molecular modeling, were

13

combined to characterize the REG3A adsorption on the surface of AuNPs and the LPS

14

influence. The conjugation of REG3A on AuNPs was featured by three components of UV18


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vis spectroscopic absorbance, different from serum protein. REG3A showed a strong

2

tendency of self-aggregation on the surface of AuNPs. When growing up to a certain degree,

3

the REG3A fibrous complex would fall off from the surface of AuNPs, resulting in the

4

random fluctuations in the UV-vis spectra. The molecular docking results indicates that

5

LPS may impede the hydrophobic interaction and hydrogen bonding between the coil

6

structure (E114-P115-N116-G117-E118) and helical structure (N136-P137-S138-T139-

7

I140), which were entangled with each other in the protein-protein interaction. LPS can

8

bind REG3A protein very tightly on the multiple motifs such as P109-T110-Q111-G112,

9

P115-N116, and P137-S138-T139. In the presence of LPSs, the strong interaction of

10

REG3A-LPS blocked the REG3A-REG3A self-interaction, the number of components in

11

the ternary system decreased to two, and the adsorption kinetic curves became similar to

12

serum protein.

13 14

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1

ASSOCIATED CONTENT

2 3

Supporting Information

4

The Supporting Information is available free of charge on the ACS Publications website at

5

DOI: 10.1021/acs.langmuir.?????. PCA and NMF analyses of the UV-vis absorbance data,

6

TEM image/DLS/zeta potential/stability of AuNPs with/without protein, and the calculated

7

binding affinity in the molecular docking.

8 9

AUTHOR INFORMATION

10

Corresponding Author

11

*State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory

12

of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology,

13

Shanghai Jiao Tong University, Shanghai, 200240, China. Tel: 86-21-34207190; Fax: 86-

14

21-34207347; Email: [email protected]

15 16

Notes

17

The authors declare no competing financial interests.

18 19

Author Contributions

20

YPL and YLZ conceived and designed the investigation. XR and QH conducted the

21

experiments, and ZXH and XR performed the calculations and analyses. ZXH, XR, TS,

22

YPL, and YLZ wrote up the paper.

23 24

ACKNOWLEDGMENT

25

The authors thank the National Science Foundation of China (21377085 and 31770070), 20


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SJTU-YG2016MS33, and the SJTU-HPC computing facility award for financial support

2

and computational hours. ZXH gives his thanks to Mr. Yuanqi Wang and Mr. Ashfaqur

3

Rehman for making language revisions to this manuscript.

4

References

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nanoparticle-based colorimetric probe for pyrophosphate using a competition assay approach. Chemical communications 2013, 49, 152-154. (12) Ren, X.; Li, M.; Chen, M.; Dai, Y.; Shi, T.; Zhao, Y. L. Characterization of Proteinconjugating Kinetics Based on Localized Surface Plasmon Resonance of the Gold Nanoparticle. Spectroscopy Letters 2016, 49: 434-443 (13) Cañaveras, F.; Madueño, R.; Sevilla, J. M.; Blázquez, M.; Pineda, T. Role of the Functionalization of the Gold Nanoparticle Surface on the Formation of Bioconjugates with Human Serum Albumin. The Journal of Physical Chemistry C 2012, 116, 1043010437. (14) Boulos, S. P.; Davis, T. A.; Yang, J. A.; Lohse, S. E.; Alkilany, A. M.; Holland, L. A.; Murphy, C. J. Nanoparticle-protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces. Langmuir 2013, 29, 14984-14996. (15) Gao, G.; Zhang, M.; Gong, D.; Chen, R.; Hu, X.; Sun, T. The size-effect of gold nanoparticles and nanoclusters in the inhibition of amyloid-beta fibrillation. Nanoscale 2017, 9, 4107-4113. (16) Mirsadeghi, S.; Dinarvand, R.; Ghahremani, M. H.; Hormozi-Nezhad, M. R.; Mahmoudi, Z.; Hajipour, M. J.; Atyabi, F.; Ghavami, M.; Mahmoudi, M. Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process. Nanoscale 2015, 7, 5004-5013. (17) Wang, A.; Perera, Y. R.; Davidson, M. B.; Fitzkee, N. C. Electrostatic Interactions and Protein Competition Reveal a Dynamic Surface in Gold Nanoparticle-Protein Adsorption. The journal of physical chemistry. C, Nanomaterials and interfaces 2016, 120, 2423124239. (18) Wang, Y.; Ni, Y. Combination of UV-vis spectroscopy and chemometrics to understand protein-nanomaterial conjugate: a case study on human serum albumin and gold nanoparticles. Talanta 2014, 119, 320-330. (19) Jolliffe, I. T.; Cadima, J. Principal component analysis: a review and recent developments. Philosophical transactions. Series A, Mathematical, physical, and engineering sciences 2016, 374, 20150202. (20) Lee, D. D.; Seung, H. S. Learning the parts of objects by non-negative matrix factorization. Nature 1999, 401, 788-791. (21) Brunet, J. P.; Tamayo, P.; Golub, T. R.; Mesirov, J. P. Metagenes and molecular pattern discovery using matrix factorization. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, 4164-4169. (22) Jaumot, J.; Gargallo, R.; Juan, A. D.; Tauler, R. A graphical user-friendly interface for MCR-ALS: a new tool for multivariate curve resolution in MATLAB. Chemometrics & Intelligent Laboratory Systems 2005, 76, 101-110. (23) Jaumot, J.; de Juan, A.; Tauler, R. MCR-ALS GUI 2.0: New features and applications. Chemometrics and Intelligent Laboratory Systems 2015, 140, 1-12. (24) Ruckebusch, C.; Blanchet, L. Multivariate curve resolution: a review of advanced and 22


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tailored applications and challenges. Analytica chimica acta 2013, 765, 28-36. (25) Chen, R.; Li, L.; Weng, Z. ZDOCK: an initial-stage protein-docking algorithm. Proteins-structure Function & Bioinformatics 2003, 52, 80-87. (26) Trott, O.; Olson, A. J., AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry 2010, 31, 455-461. (27) Cash, H. L.; Whitham, C. V.; Behrendt, C. L.; Hooper, L. V. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 2006, 313, 1126-1130. (28) Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nature Physical Science 1973, 241, 20-22. (29) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G.; Chem, A. Determination of size and concentration of gold nanoparticles from UV-vis spectra. Analytical chemistry 2007, 79, 4215-4221. (30) Felder, C. E.; Prilusky, J.; Silman, I.; Sussman, J. L. A server and database for dipole moments of proteins. Nucleic acids research 2007, 35 (Web Server issue), 512-521. (31) Laskowski, R. A.; Swindells, M. B.; LigPlot+: Multiple Ligand–Protein Interaction Diagrams for Drug Discovery. Journal of Chemical Information & Modeling, 2011, 51, 2778-2786. (32) Chaudhary, A.; Gupta, A.; Khan, S.; Nandi, C. K. Morphological effect of gold nanoparticles on the adsorption of bovine serum albumin. Physical Chemistry Chemical Physics 2014, 16, 20471-20482. (33) Naveenraj, S.; Anandan, S.; Kathiravan, A.; Renganathan, R.; Ashokkumar, M. The interaction of sonochemically synthesized gold nanoparticles with serum albumins. Journal of Pharmaceutical & Biomedical Analysis 2010, 53, 804. (34) Lehotzky, R. E.; Partch, C. L.; Mukherjee, S.; Cash, H. L.; Goldman, W. E.; Gardner, K. H.; Hooper, L. V. Molecular basis for peptidoglycan recognition by a bactericidal lectin. Proceedings of the National Academy of Sciences of the United States of America 2010, 107, 7722-7727.

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Table of Content

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Figure 1. AFM images of (a) pure REG3A, (b) and (c) AuNPs incubated with REG3A, and (d) AuNPs incubated with BSA. 130x99mm (300 x 300 DPI)


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Figure 2. The initial (t = 0 s) and final (t = 480 s) UV-vis absorption spectra of (a) the AuNPs-REG3A binary system and (b) the AuNPs-REG3A-LPSs ternary system, respectively, and the UV-vis Delta spectra with different REG3A concentrations of (c) the binary system and (d) the ternary system (insets are the integrals of the highlighted peak areas in the two systems). 130x99mm (300 x 300 DPI)


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Figure 3. The PCA analysis of the two systems, including cumulative contribution rates of (a) the binary system and (b) the ternary system, and (c) the PCA scores plot of the first two principal components with the different concentrations of REG3A concentrations, and (d) the corresponding PCA loadings information. 130x99mm (300 x 300 DPI)


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Figure 4. The PCA/NMF/MCR-ALS optimized spectral profiles (a and b) and time-resolved concentration profiles (c and d), in which (a) and (c) for the binary system and (b) and (d) for the ternary system. 130x99mm (300 x 300 DPI)


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Figure 5. The molecular docking results of REG3A self-binding study, including (a) two dominant binding modes (“top” and “side”) and the high-ranked 10 docking clusters, (b) The highest-ranked pose of “top-top” interaction of two REG3A molecules, and (c) schematic DIMPLOT diagram of the interface. 130x99mm (300 x 300 DPI)


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Figure 6. The molecular docking results of LPS onto REG3A, including (a-c) representative poses from the highest-ranked 3 clusters, and. (d-f) the Ligplot 2D-diagrams of the corresponding binding pose. (Hydrogen bonds are shown in green dotted lines, hydrophobic interactions are presented with the red spoked arcs, and key residues are highlighted with red circles). 130x99mm (300 x 300 DPI)


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TOC 44x23mm (300 x 300 DPI)


Lipopolysaccharides inhibit REG3A self-aggregation on gold

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