Editorial

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Nanoparticle-induced conformational
changes in protein corona revealed by
circular dichroism spectroscopy
Juliana Sakamoto Yoneda*,1 & Mateus Borba Cardoso1
1
Brazilian Synchrotron Light Laboratory (LNLS), Brazilian Center for Research in Energy & Materials (CNPEM), Campinas, Sao Paulo,
13083-970, Brazil
*Author for correspondence: juliana.yoneda@lnls.br

“CD spectroscopy is a suitable technique for understanding protein–NP interaction, providing

crucial nanobiointerface information which might likely be used in the rational development of
nanomedicines”

First draft submitted: 20 April 2023; Accepted for publication: 15 May 2023; Published online:
1 June 2023

Keywords: circular dichroism • nanomedicine • nanoparticle • nanoparticle surface • nanoparticle–protein

interaction • protein corona • protein secondary structure • synchrotron radiation circular dichroism

Clinical translation of target-specific nanoparticles (NPs) has been mainly hampered due to their lack of colloidal
stability as well as mistargeting-related issues. Consequently, these nanocarriers’ reliability depends on how the NPs
interact with the biological environment, and the disturbances associated with these interactions may cause severe
consequences in vivo. Among the biological entities, proteins stand out as one of the critical body fluid components
because their nonspecific adsorption on the NPs’ surface – the so-called protein corona (PC) – will confer a new
NP biological identity which may impact colloidal stability [1]. The maintenance of colloidal stability is a critical
aspect to be considered because the formation of irreversible NP aggregates compromises biodistribution, drug
delivery and targetability [2]. Furthermore, protein–NP interaction may induce protein conformational changes and
unfolding that might produce undesirable biological responses. A recent review highlights important progress in PC
research and its impact on advancing nanobiotechnology [3]. To evaluate protein conformation in the biomolecular
corona layer effectively, the nanomedicine community needs new methodologies and improvements to current
approaches [3].
A pool of different and complementary techniques has been used to elucidate distinct aspects of the PC.
Dynamic light scattering, fluorescence microscopies and spectroscopies, electron microscopy-based strategies and
electrophoretic techniques have successfully shown that binding parameters, structural features and composition
might be reliably obtained. However, studies that elucidate whether the conformation of proteins can be affected
by NP–protein interaction and whether this is related to nanomaterial-induced responses are still sparse. As an
alternative approach, electronic circular dichroism (CD) spectroscopy stands out as a valuable tool that can probe
PC formation while examining the conformational changes of proteins upon interaction with NPs. Although not
yet widespread in the nanomedicine community, this powerful technique can unveil structural protein aspects of
NP adsorption and impact the rational design of nanosized structures for medical applications.
CD spectroscopy is a well-established method based on the differential absorption of left and right circularly
polarized light emerging from an optically active material, providing information about the absolute configuration
of chiral molecules. In the case of proteins, the amide group of peptide bonds is the chromophore, located in a chiral
environment dictated by different peptide backbone torsion angles. Different protein secondary structures give rise
to characteristic CD spectra in the far-UV region of the electromagnetic spectrum (260–170 nm). Consequently,
quantitative analyses are able to determine the secondary structure content in terms of α-helical, β-strand and
unordered structures. In addition, information about the tertiary structure can be obtained due to the signals in the
near-UV range arising from the aromatic residues tryptophan, tyrosine and phenylalanine or disulfide chains [4–6].

10.2217/nnm-2023-0115 
C 2023 Future Medicine Ltd Nanomedicine (Lond.) (2023) 18(9), 709–711 ISSN 1743-5889 709
Editorial Yoneda & Cardoso

It is an appropriate technique to detect and quantify conformational changes of proteins due to interactions with
other components or disturbances in the surrounding environment, such as pH, temperature and ionic strength.
However, one disadvantage is that CD measurements of protein mixtures give rise to spectra which are a convolution
of all protein contributions. When NPs are in contact with complex biofluids, miscellaneous proteins are involved
and may be adsorbed on the NPs surface. CD spectroscopy would give an average result related to the protein
composition mixture, because this technique cannot reveal the identity of proteins. On the other hand, CD is the
ideal tool to unveil structural changes of a single protein component in solution. Therefore, the best strategy is
to start with low-complexity systems (e.g., purified proteins encountered in biological fluids) to understand their
behavior from the point of view of an individual protein, and then gradually increase the complexity.
It is essential to point out that CD spectroscopy results can be significantly enhanced when synchrotron radiation
(SR) is used as the light source. Recent developments in bioinformatics tools to promote advances in data analysis,
validation, deposition and sharing have boosted the use of this technique in the last few years [6]. Concerning NPs,
we can take advantage of this progress to study NP–PC complexes.
Although the CD technique can be used to probe the PC formation, a previous separation step of adsorbed
and free proteins in solution is required, usually achieved by centrifugation and subsequent washes. Otherwise, a
inconclusive CD result would be caused by the convolution of possible bound and unbound proteins. However, it
is challenging to detect weakly adsorbed proteins (soft corona) because vigorous separation procedures can induce
their detachment, and only information from a tightly bound fraction (hard corona) is usually obtained. In specific
cases, it is possible to investigate both soft and hard corona, as in one study where the authors found that the
strength of the protein–NP interaction was dependent on the pH of the surrounding medium. In that work, SRCD
was applied to detect changes in protein secondary structure in situ after forming either soft or hard PC [7].
The quantitative analysis of conformational changes in secondary structure content can be successfully completed
from either CD or SRCD results, albeit a significant enhancement of CD spectroscopy is achieved using SRCD.
The extended energy range obtained through SRCD allows the assessment of high-energy transitions, increasing
the amount of information derived from the CD spectrum and consequently allowing for better discrimination of
more types of secondary structures [8].
At a quantitative level, the analysis relies on the deconvolution of the CD spectra to determine the contributions
of each type of secondary structure in a protein molecule by combining reference datasets composed of spectra from
proteins with known structures and empirical algorithms. Freely online programs are available for the community,
such as the web server Dichroweb [9,10], which includes different reference databases and algorithms for the analysis,
allowing the choice of a representative dataset for the examined protein and enabling comparison among different
analysis methods.
The advantage of extending the vacuum UV spectrum with SR is clearly exemplified for proteins with mixed α-
helix and β-strand contents. More accurate results are obtained for α-helical-rich proteins because of their higher
regularity compared with β-sheets. The latter present greater structural diversity, including orientations and twisting,
which implies variable CD spectra. The peaks of α-helices and β-strands appear at similar wavelengths in the far
UV, and the magnitude of β-strand peaks is smaller than that of α-helices. Consequently, the peaks from β-sheets
will be hidden if a protein mixture contains two types of structures with a higher proportion of α-helical content.
Thus the determination of β-strands is more challenging and less accurate. The more comprehensive wavelength
range, including the vacuum UV region, can alleviate this issue because, at low wavelengths, helices and sheets
can have bands with opposite signs, and the discrimination of these structures can be facilitated [8].
The SR light source can also be advantageous if the conformational change is not very pronounced and detection
by observing and analyzing the spectrum is not easily achievable. Then, high-quality spectra can be obtained in a
reduced time because SR can be orders of magnitude more intense than conventional lamps. Therefore, the less noisy
spectrum enables the discrimination of subtle differences, increasing the technique’s sensitivity. Another strategy
to overcome this challenge is measuring the thermal stability of a protein in the presence and absence of NPs. If
the protein’s thermostability changes in the presence of NPs, it provides a shred of experimental evidence that the
protein interacts with NPs. For instance, lower resistance to conformational modifications induced by temperature
increments suggests that a protein is more prone to undergo secondary structural changes after interaction with
NPs, which can restrict their safe use in vivo [7,11].
In summary, CD spectroscopy is a nondestructive method that can effectively analyze structural changes in
proteins. Additionally, the preparation of the sample for this technique is a simple and cost-effective procedure.
However, to obtain reliable data with either conventional CD or SRCD, attention must be paid to the sample

710 Nanomedicine (Lond.) (2023) 18(9) future science group

 Nanoparticle-induced conformational changes in protein corona revealed by circular dichroism spectroscopy Editorial

preparation [6], choice of sample holders, running parameters [12], data analysis and results interpretation. Moreover,
caution must be given to scattering artifacts, as these can provoke distortion on CD spectra, leading to data
misinterpretation in the context of NP suspensions [13]. In conclusion, CD spectroscopy is a suitable technique for
understanding protein–NP interaction, providing crucial nanobiointerface information which might likely be used
in the rational development of nanomedicines.

Author contributions
J Sakamoto Yoneda and M Borba Cardoso contributed both literature review and manuscript writing.

Financial & competing interests disclosure

Authors are acknowledged to Fapesp (grant no. 2021/12071-6). The authors have no other relevant affiliations or financial in-
volvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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