Protein–Nanoparticle Interaction: Corona Formation and Conformational Changes in Proteins on Nanoparticles

Figures & data

Table 1 Representative Conformational Changes in Proteins Following Complexation with NPs

NPs	Surface Functional Group	Interacting Protein	Conformational Changes	Detection Method	Ref
SW CNT	None	Protein-G	The α-helicity diminished through hydrogen bond breakage.	Molecular dynamics (MD)	^Citation128
	None	Carbonic anhydrase (CA)	The CA-NP complex exhibited increased total α-helix content and decreased β-sheet content.	Circular dichroism (CD)	^Citation129
	None	Lysozyme	The α-helix to β-sheet transition was reported.	MD	^Citation130
	None, -COOH	Bovine serum albumin (BSA)	BSA interacted less strongly with pristine SWCNT than with carboxylated SWCNT. BSA lost more of its α-helix content upon binding to the carboxylated SWCNT.	CD	^Citation131
	None	Estrogen receptor α (ER)	ER binding to NPs triggered signal transduction by changing the structure of ER from the free form to the agonist-bound form.	Fluorescence MD	^Citation132
	-COOH	HRP, subtilisin, lysozyme	HRP maintained only 68% of the native α-helical structure after complexation. Subtilisin maintained only 76% of the native β-structure after complexation. Lysozyme retained 63% of the native structure.	CD	^Citation133
	-COOH	Human IgG, HSA, fibrinogen (FG)	The adsorption capacity to NPs was as follows: FG > HSA > IgG	Fluorescence MD	^Citation134
	None	Tau protein	The random coil structure → β-sheet transition	CD	^Citation53
MW CNT	None	Tau protein	No change in secondary structure was reported.	CD
	-COOH	Porcine trypsin (pTry)	The enzymatic activity of pTry reduced. The α-helical content reduced and unfolding started.	MD, UV, CD	^Citation135
	-OH	Amylase	The loss of the α-helical structure occurred, decreasing from 41.1% to 21.9%	CD	^Citation136
	None	BSA	The β-sheet content decreased from 33.3% to 29.8%; The β-turn content increased from 2% to 5%.	CD	^Citation137
	-COOH	HSA, FG, IgG, histone H1 (H1)	HAS, FG, and IgG showed a red shift in fluorescence, indicative of conformational changes in the hydrophobic core open, while H1 showed a blue shift.	TEM, CD, Fluorescence	^Citation138
	None, -COOH -PEG	BSA, IgG	The α-helicity diminished and the β-structure slightly increased for BSA. The effect was greater in COOH-NP and pristine-NP. The α-helicity greatly decreased and the β-structure was elevated for IgG. The overall folding moved to the unfolding state especially in COOH-NP and pristine-NP.	CD, TEM, DLS	^Citation76
AuNP	-MUA -TCOOH	Cytochrome-c	Reductions in the α-helical content and highly denatured form were observed.	CD	^Citation139
	-COOH	Cytochrome-c (CC), chymotrypsin (ChT)	CC: No change ChT: Complete denaturation upon binding	CD	^Citation140
	-COOH	Factor VIII IgG	Factor VIII, IgG: The extent of α-helical structure of both the proteins was reduced and structural transition occurred from α-helix to β-sheets.	Fluorescence CD	^Citation141
	-COOH	BSA	Reduction in the α-helical content	CD	^Citation142
	-COOH	BSA	Loss of α-helical content	CD	^Citation143
	-COOH	BSA	Loss of α-helical content and formation of more open structures.	ATR-FTIR fluorescence	^Citation144
	-Chloride -CTAB (GNR)	HSA	Reduction in α-helical content and an increase in random coil content were observed. The effects were greater in GNR.	CD, ITC	^Citation17
	-COOH, -CTAB TGNP GNR	BSA	Significant secondary structural changes were found in CTAB gold NP, TGNP, and GNR. The unfolding ability of citrate (-COOH) gold NP was weak.	CD	^Citation145
	-Chloride -CTAB (GNR)	BSA	The α-helical content was diminished.	FT-IR	^Citation146
	MHDA-GNR	Lysozyme, α-chymotrypsin (ChT)	Lysozyme: Ellipticity at 222 nm reduced by 15% for lysozyme. ChT: Ellipticity at 222 nm reduced by 10% for ChT.	CD	^Citation147
	MHDA-GNS	Lysozyme ChT	11% of ellipticity of lysozyme at 222 nm was reduced, while the structure of ChT was unchanged.	CD
AgNP	-BH4^−	α-A-Crystallin	The protein was partially unfolded with the exposure of two cysteine residues that could form coordinate bonds with AgNP.	FRET, FT-IR	^Citation148
	-COOH	BSA	The α-helix content decreased by up to 7% with the addition of AgNPs.	FT-IR, CD	^Citation149

Abbreviations: HRP, horseradish peroxidase; PEG, polyethylene glycol; MUA, mercaptoundecanoic acid; TCOOH, thioalkylated tetraethylene glycol; CTAB, cetyltrimethylammonium bromide; GNR, gold nanorods; TGNP, triangular gold nanoplates; MHDA, 16-mercaptohexadecanoic acid, anionic, nontoxic; GNS, gold nanosphere.

Table 2 Structural Changes in Model Proteins on Various Surfaces of Carbon Nanotubes

Protein	PI^a	Net Charge^a	Theoretical Effect^b	Secondary Structure Change^c	Melting Temperature (Tm) Change^c
Albumin	5.92	−11	NH2>OH>COOH	COOH>OH=NH2	Tm increase NH2>OH>COOH
α-1-Antitrypsin	5.37	−15	NH2>OH>COOH	OH>COOH>NH2	Tm decrease NH2>OH=COOH
Transferrin	6.81	−2	NH2>OH>COOH	NH2=OH=COOH	Tm decrease OH≥NH2≥COOH
Lysozyme	9.28	8	COOH>OH>NH2	COOH>OH>NH2	Tm decrease COOH>OH>NH2
Fibrinogen (α, β, r)	8.23, 8.54, 5.61	3, 6,−10	COOH=OH=NH2	COOH>NH2>OH	Tm increase COOH>NH2>OH

Notes: ^aThe expected charge properties were calculated using the amino acid sequences of each protein at a pH of 7.4. ^bThe expected amount of changes in the NP solution considering the net charge of proteins. ^cAll results were obtained using CD spectroscopy in phosphate buffer (pH 7.4).

Table 3 Secondary Structural Changes Depending on the Surface Characteristics

Proteins	Contents	Surfaces
		PBS	COOH	NH2	OH
Albumin	α-helix	40.7*	34.8	35.1	35.1
	β-sheet	13.7	16.2	16.0	16.0
	β-turn	15.6	16.4	16.4	16.4
	Random coil	30.0	32.6	32.5	32.5
α-1-Antitrypsin	α-helix	32.2	30.1	31.5	28.2
	β-sheet	17.5	18.6	17.9	19.8
	β-turn	16.8	17.2	16.9	17.5
	Random coil	33.5	34.1	33.7	34.5
Transferrin	α-helix	32.8	29.3	30.0	29.5
	β-sheet	17.3	19.2	18.8	19.1
	β-turn	16.9	17.4	17.3	17.4
	Random coil	33.0	34.1	33.9	34.0
Lysozyme	α-helix	59.9	48.1	52.8	51.3
	β-sheet	7.9	11.0	9.7	10.1
	β-turn	13.1	14.5	13.9	14.1
	Random coil	19.1	26.4	23.6	24.5
Fibrinogen (α, β, r)	α-helix	33.9	27.6	30.2	31.1
	β-sheet	16.6	20.2	18.6	18.1
	β-turn	16.6	17.7	17.2	17.0
	Random coil	32.9	34.5	34.0	33.8

Notes: *The calculated value is the percentage of each secondary structural element in the total structure. The percentages of structural elements obtained from the reference spectra (PBS) and those obtained from the most highly changed condition were represented by bold numbers.

Figure 1 Size comparison between proteins and NPs.

Notes: The sizes of NPs are compared to those of various proteins. The codes in brackets are those from the PDB database. Adapted with permission from Kopp M, Kollenda S, Epple M. Nanoparticle-Protein Interactions: Therapeutic Approaches and Supramolecular Chemistry. Acc Chem Res. 2017; 50(6):1383–1390. Copyright (2019) American Chemical Society.⁵⁰

Abbreviations: HSA, human serum albumin; IgG, immunoglobulin G.

Figure 2 Extracellular and intracellular events caused by the structural changes of corona proteins.

Notes: The interaction of NPs with proteins can induce variety of signal modulations and toxic effects in biofluids and in cells. Various physicochemical properties of NP systems basically contribute to the corona formation and structural changes of proteins. The conformational change is a dynamic process and the composition of corona proteins on NPs can be changed according to the surrounding environment. The reversible or irreversible changes of protein structures can perturb the downstream signaling, which may consequently be harmful to the host. The characteristics of corona formation may be expected to be different between extracellular and intracellular spaces. The internalized NPs by various ways such as receptor mediated internalization and endocytosis by charge can produce many toxic situations directly through their own chemical characteristics and/or indirectly through corona formation.

Abbreviations: ER, endoplasmic reticulum; ROS, reactive oxygen species.

Figure 3 The preference of secondary structural elements for MWCNT binding.

Notes: About 750 cellular proteins were monitored for binding to MWCNT by mass spectrometry-based proteomics. The secondary structures of the bound proteins were analyzed using the 3D structures deposited in the Protein Data Bank. 269 proteins out of identified 778 proteins were used for the analysis. The proportion of α-helix and β-sheet for each protein was quantified. The protein classes among the CNT-binding proteins are represented with bubbles. The most abundant protein classes contained cytoskeletal proteins, endosomal proteins, and heat shock proteins. The bubble size represents the relative binding affinity to MWCNTs. Adapted from Nanomedicine: Nanotechnology, Biology and Medicine, 9(5), Cai X, Ramalingam R, Wong HS, Cheng J, Ajuh P, Cheng SH, Lam YW. Characterization of carbon nanotube protein corona by using quantitative proteomics. Nanomedicine. 583–593, Copyright (2013), with permission from Elsevier.⁸⁷

Abbreviation: MWCNT, multi-walled carbon nanotube.

Figure 4 Secondary structures of protein-NP complexes (unpublished data).

Notes: PBS represents the reference CD curve without NPs, measured only in phosphate-buffered saline (pH 7.4). All other curves were measured with NPs in phosphate-buffered saline (pH 7.4). (A) Human serum albumin, (B) α-1-antitrypsin, (C) transferrin, (D) lysozyme, (E) fibrinogen (α, β, r).

Abbreviations: CD, circular dichroism; NP, nanoparticle.

Figure 5 The Tm curves of protein-NP complexes.

Notes: PBS represents the reference Tm curve without NPs. (A) Albumin (PDB code: 2bx8), (B) α-1-antitrypsin (PDB code: 1kct), (C) transferrin (PDB code: 2hau), (D) lysozyme (PDB code: 1lz1), (E) fibrinogen (α, β, r, PDB code: 3ghg). The structure of each protein was obtained from PDB database (http://www.rcsb.org). The Coulumb parameters were ε = 4r and thresholds ± 5.93 kcal/mol⋅e. Negative charge is indicated in red, positive in blue, and neutral in white. Surface charge was calculated using UCSF Chimera.

Abbreviations: CD, circular dichroism; NP, nanoparticle; Tm, melting temperature.

Figure 6 The advantages and disadvantages of protein corona formation.

Notes: By regulating the protein coronas through appropriate surface modification and NP selection, the biological behavior of NPs in the body could be improved. Structural studies on the corona proteins in NP systems may provide insight into how to handle the NPs.

Abbreviation: NPs, nanoparticles.

Ebrahim-Habibi MB, Ghobeh M, Aghakhani Mahyari F, Rafii-Tabar H, Sasanpour P. Protein G selects two binding sites for carbon nanotube with dissimilar behavior; a molecular dynamics study. J Mol Graph Model. 2019;87:257–267. doi:10.1016/j.jmgm.2018.12.00730594774

 PubMed Web of Science ®Google Scholar

Chen X, Wang Y, Wang P. Peptide-induced affinity binding of carbonic anhydrase to carbon nanotubes. Langmuir. 2015;31(1):397–403. doi:10.1021/la504321q25521207

 PubMed Web of Science ®Google Scholar

Vaitheeswaran S, Garcia AE. Protein stability at a carbon nanotube interface. J Chem Phys. 2011;134(12):125101. doi:10.1063/1.355877621456701

 PubMed Web of Science ®Google Scholar

Mahendra WIP, Gandhi S, Ju Nie T, et al. Protein/carbon nanotubes interaction: the effect of carboxylic groups on conformational and conductance changes. Appl Phys Lett. 2009;95(7):073704. doi:10.1063/1.3211328

 Web of Science ®Google Scholar

Liu X, Liu T, Song J, et al. Understanding the interaction of single-walled carbon nanotube (SWCNT) on estrogen receptor: a combined molecular dynamics and experimental study. Ecotoxicol Environ Saf. 2019;172:373–379. doi:10.1016/j.ecoenv.2019.01.10130731268

 PubMed Web of Science ®Google Scholar

Asuri P, Bale SS, Pangule RC, Shah DA, Kane RS, Dordick JS. Structure, function, and stability of enzymes covalently attached to single-walled carbon nanotubes. Langmuir. 2007;23(24):12318–12321. doi:10.1021/la702091c17944500

 PubMed Web of Science ®Google Scholar

Lu N, Sui Y, Tian R, Peng YY. Adsorption of plasma proteins on single-walled carbon nanotubes reduced cytotoxicity and modulated neutrophil activation. Chem Res Toxicol. 2018;31(10):1061–1068. doi:10.1021/acs.chemrestox.8b0014130207453

 PubMed Web of Science ®Google Scholar

Zeinabad HA, Zarrabian A, Saboury AA, Alizadeh AM, Falahati M. Interaction of single and multi wall carbon nanotubes with the biological systems: tau protein and PC12 cells as targets. Sci Rep. 2016;6:26508. doi:10.1038/srep2650827216374

 PubMed Web of Science ®Google Scholar

Noordadi M, Mehrnejad F, Sajedi RH, Jafari M, Ranjbar B, Lebedev N. The potential impact of carboxylic-functionalized multi-walled carbon nanotubes on trypsin: a comprehensive spectroscopic and molecular dynamics simulation study. PLoS One. 2018;13(6):e0198519. doi:10.1371/journal.pone.019851929856868

 PubMed Web of Science ®Google Scholar

Sekar G, Sivakumar A, Mukherjee A, Chandrasekaran N. Existence of hydroxylated MWCNTs demotes the catalysis effect of amylases against starch degradation. Int J Biol Macromol. 2016;86:250–261. doi:10.1016/j.ijbiomac.2016.01.07126812109

 PubMed Web of Science ®Google Scholar

Yang M, Meng J, Mao X, et al. Carbon nanotubes induce secondary structure changes of bovine albumin in aqueous phase. J Nanosci Nanotechnol. 2010;10(11):7550–7553. doi:10.1166/jnn.2010.282521137980

 PubMed Web of Science ®Google Scholar

De Paoli SH, Diduch LL, Tegegn TZ, et al. The effect of protein corona composition on the interaction of carbon nanotubes with human blood platelets. Biomaterials. 2014;35(24):6182–6194. doi:10.1016/j.biomaterials.2014.04.06724831972

 PubMed Web of Science ®Google Scholar

Zhang T, Tang M, Yao Y, Ma Y. Pu Y MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity. Int J Nanomed. 2019;14:993–1009.

 PubMed Web of Science ®Google Scholar

Worrall JW, Verma A, Yan H, Rotello VM. “Cleaning” of nanoparticle inhibitors via proteolysis of adsorbed proteins. Chem Commun (Camb). 2006;22:2338–2340. doi:10.1039/B517421J

 Google Scholar

Srivastava S, Verma A, Frankamp BL, Rotello VM. Controlled assembly of protein-nanoparticle composites through protein surface recognition. Adv Mater. 2005;17(5):617–621. doi:10.1002/adma.200400776

 Web of Science ®Google Scholar

Zarabi MF, Farhangi A, Mazdeh SK, et al. Synthesis of gold nanoparticles coated with aspartic acid and their conjugation with FVIII protein and FVIII antibody. Indian J Clin Biochem. 2014;29(2):154–160. doi:10.1007/s12291-013-0323-224757296

 PubMedGoogle Scholar

Wangoo N, Suri CR, Shekhawat G. Interaction of gold nanoparticles with protein: a spectroscopic study to monitor protein conformational changes. Appl Phys Lett. 2008;92:133104.

 Web of Science ®Google Scholar

Wangoo N, Bhasin KK, Mehta SK, Suri CR. Synthesis and capping of water-dispersed gold nanoparticles by an amino acid: bioconjugation and binding studies. J Colloid Interface Sci. 2008;323(2):247–254. doi:10.1016/j.jcis.2008.04.04318486946

 PubMed Web of Science ®Google Scholar

Tsai DH, DelRio FW, Keene AM, et al. Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods. Langmuir. 2011;27(6):2464–2477. doi:10.1021/la104124d21341776

 PubMed Web of Science ®Google Scholar

Xu C, Zhao X, Wang L, et al. Protein Conjugation with gold nanoparticles: spectroscopic and thermodynamic analysis on the conformational and activity of serum albumin. J Nanosci Nanotechnol. 2018;18(11):7818–7823. doi:10.1166/jnn.2018.15215

 Web of Science ®Google Scholar

Chaudhary A, Gupta A, Khan S, Nandi CK. Morphological effect of gold nanoparticles on the adsorption of bovine serum albumin. Phys Chem Chem Phys. 2014;16(38):20471–20482. doi:10.1039/C4CP01515K25140357

 PubMed Web of Science ®Google Scholar

Chakraborty S, Joshi P, Shanker V, Ansari ZA, Singh SP, Chakrabarti P. Contrasting effect of gold nanoparticles and nanorods with different surface modifications on the structure and activity of bovine serum albumin. Langmuir. 2011;27(12):7722–7731. doi:10.1021/la200787t21591651

 PubMed Web of Science ®Google Scholar

Gagner JE, Lopez MD, Dordick JS, Siegel RW. Effect of gold nanoparticle morphology on adsorbed protein structure and function. Biomaterials. 2011;32(29):7241–7252. doi:10.1016/j.biomaterials.2011.05.09121705074

 PubMed Web of Science ®Google Scholar

Banerjee V, Das KP. Structure and functional properties of a multimeric protein α-a-crystallin adsorbed on silver nanoparticle surface. Langmuir. 2014;30(16):4775–4783. doi:10.1021/la500700724694218

 PubMed Web of Science ®Google Scholar

Wang G, Lu Y, Hou H, Liu Y. Probing the binding behavior and kinetics of silver nanoparticles with bovine serum albumin. RSC Adv. 2017;7(15):9393–9401. doi:10.1039/C6RA26089F

 Web of Science ®Google Scholar

Kopp M, Kollenda S, Epple M. Nanoparticle-protein interactions: therapeutic approaches and supramolecular chemistry. Acc Chem Res. 2017;50(6):1383–1390. doi:10.1021/acs.accounts.7b0005128480714

 PubMed Web of Science ®Google Scholar

Cai X, Ramalingam R, Wong HS, et al. Characterization of carbon nanotube protein corona by using quantitative proteomics. Nanomedicine. 2013;9(5):583–593. doi:10.1016/j.nano.2012.09.00423117048

 PubMed Web of Science ®Google Scholar