BBA - General Subjects 1867 (2023) 130445

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BBA - General Subjects

journal homepage: www.elsevier.com/locate/bbagen

Exploring the impact of nucleic acids on protein stability in bacterial

cell lysate
Soojeong Ham , Changhan Lee *
Department of Biological Sciences, Ajou University, Suwon, South Korea

A R T I C L E I N F O A B S T R A C T

Keywords: - Addition of salt enhanced thermal stability of model substrate proteins by reducing electrostatic repulsion
Nucleic acids between protein molecules.
DNA - However, the opposite effect was observed with bacterial cell lysate, indicating that certain molecules within
RNA
the lysate could enhance protein stability via electrostatic interactions.
Chaperone
Anti-aggregation activity
- Such molecules present in cell lysate were found to be nucleic acids known to have a potent anti-aggregation
activity toward proteins involving electrostatic interactions.
- Nucleic acids showed chaperone activity in physiological salt concentration within cells and in buffer or
medium commonly used in experiments.
- The chaperone activity of nucleic acids should be taken into account when performing various in vitro assays
using cell lysate or samples containing nucleic acids.

The cell is filled with various biomolecules. Among them, proteins
play an essential role in vital phenomena such as gene expression and
enzymatic activity. Each protein has a unique structure directly related
to its specific function. Protein structure can easily undergo denatur­
ation due to external factors such as heat and acidity, which can directly
affect its function. Therefore, cells have mechanisms to protect proteins
from various stresses. The most representative system is the molecular
chaperone system [1]. Chaperones can interact with substrate proteins
to assist in protein folding, prevent aggregate formation, and dissolve
preformed aggregates [1].
In addition to chaperones, numerous biomolecules present in the cell
can also interact with proteins and moonlight as a chaperone. Nucleic
acids including DNA and RNA, sugar molecules such as trehalose, and
lipid molecules such as cardiolipin are examples of intracellular mole­
cules that possess chaperone activity [2,3]. This study investigated ef­
fects of nucleic acids present in bacterial cell lysate commonly used in
various molecular biology experiments on protein stability.
Citrate synthase (CS), malate dehydrogenase (MDH), and luciferase
are the commonly used model proteins to characterize chaperone ac­
tivity. At 43◦ C, these model protein substrates formed aggregates over
time (Fig. 1ABC, Supplement Fig. 1). However, such aggregation was
significantly reduced in buffer conditions containing 170 mM of NaCl.
Salt can stabilize proteins through a process called salting-in effect [4].
This effect is mainly due to interaction between ions of salt and polar
groups on the protein surface, leading to shielding of protein charges
and a reduction in electrostatic repulsion between protein molecules.
Therefore, 100–300 mM of salt is typically used in buffer solutions for
protein purification and various experimental uses. Presumably, this
stabilizing effect of salt could delay aggregation of model proteins.
Surprisingly, the opposite phenomenon was observed when per­
forming protein aggregation assay using cell lysate from Escherichia coli
(Fig. 1D). Protein aggregation was tightly inhibited in a buffer having no
salt. However, the addition of salt significantly induced protein aggre­
gation. This phenomenon led to the hypothesis that some molecules
present in the cell lysate could inhibit protein aggregation. In addition, it
was thought that these unknown molecules could interact with proteins
through electrostatic interaction since the addition of salt led to protein
aggregation. Among diverse molecules present in the cell, nucleic acids
such as DNA and RNA were our focus because nucleic acids could exert
potent chaperone activity [2]. In addition, electrostatic interactions are
crucial for binding of nucleic acids to proteins [5].
We tried to confirm the presence of nucleic acids in cell lysate. We
also determined their effects during a thermal aggregation assay.
Indeed, through agarose gel electrophoresis, we were able to identify the
presence of significant amounts of nucleic acids which corresponded to
260 ng/μl in the solution of cell lysate (Fig. 1E). Treatment with

* Corresponding author.
E-mail address: leec@ajou.ac.kr (C. Lee).

https://doi.org/10.1016/j.bbagen.2023.130445
Received 24 April 2023; Received in revised form 2 August 2023; Accepted 14 August 2023
Available online 15 August 2023
0304-4165/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Ham and C. Lee BBA - General Subjects 1867 (2023) 130445

benzonase that could degrade all forms of DNA and RNA confirmed the
presence of nucleic acids in crude protein lysate (Fig. 1E).
It has been suggested that nucleic acids might have chaperone ac­
tivity toward proteins. For example, domain V of 23S rRNA is capable of
refolding proteins in vitro and nucleic acids are able to alter the folding of
amyloid-forming proteins [6,7]. A systematic research study on chap­
erone activity of nucleic acids has revealed that a wide range of nucleic
acids (both DNA and RNA) with various forms and sizes exhibit
extremely efficient chaperone activity in vitro, which is 300-fold more
efficient than protein chaperone GroEL [2]. Of note, RNA can cooperate
with the DnaK chaperone system [2].
Indeed, we observed a significant increase in protein aggregation
upon degradation of nucleic acids by benzonase treatment even in a
buffer having no salt (Fig. 1F). This result confirmed that the nucleic
acids present in cell lysate contributed to thermal stability of proteins.
This phenomenon was also observed when the buffer contained salt
(Fig. 1G), suggesting that the salt concentration (170 mM of NaCl) used
in the experiment could not completely suppress the chaperone activity
of nucleic acids. Salt concentration in the cytosol of E. coli and Luria
Broth (LB) medium is approximately 150–200 mM [8]. Therefore,
nucleic acids presumably can exert chaperone activity in physiological
or in vitro experimental conditions.
In this study, the chaperone activity of nucleic acids contained in
bacterial crude extract was identified. In E. coli, total concentration of
nucleic acids is around 300 mM in base-pair equivalents, which is about
six orders of magnitude higher than the concentration of nucleic acids
exhibiting potent in vitro chaperone activity, suggesting that a small
amount of contaminated nucleic acids might affect protein stability [8].
Indeed, this phenomenon also occurs in eukaryotic cell and tissue lysate
[9]. Particularly, nucleic acids having structure with single-stranded
pyrimidine-rich bulges or loops surrounded by double-stranded re­
gions or sequences forming G-quadruplexes efficiently maintain solu­
bility of proteins [9–11]. In conclusion, since nucleic acids exhibit
potent chaperone activity both in salt-free buffer conditions and in salt-
buffered conditions that cover a range of physiological and in vitro buffer
concentrations, it is worth noting the existence of nucleic acids in
various in vitro experiments, particularly for protein folding or chap­
erone related experiments using a cell lysate.

Fig. 1. Nucleic acids present in bacterial cell lysate can stabilize proteins via electrostatic interaction. (A-D) 0.2 μM citrate synthase (CS; Sigma), 0.15 μM malate
dehydrogenase (MDH; Roche), 0.2 μM luciferase (Roche), and 400 μg of bacterial cell lysate were incubated with 40 mM HEPES pH 7.5 containing 0 or 170 mM NaCl
at 43◦ C. The amount of aggregation was monitored over time through monitoring light scattering at 360 nm. After thermal denaturation, samples were centrifuged at
16,000 x g for 20 min at 4◦ C for separation of supernatant and pellet fractions. The pellet was washed with ice-chilled 40 mM HEPES pH 7.5 and centrifuged again at
16,000 x g for 20 min at 4◦ C. The pellet was resuspended in SDS–PAGE protein sample buffer, which was 12.5-fold concentrated for CS, MDH, and luciferase and 25-
fold concentrated for cell lysate. Both supernatant and pellet fractions were analyzed by SDS–PAGE and visualized by Coomassie staining. Arrow in (C) indicates the
time point of addition of luciferase. Overnight cultured cells of E. coli K-12 MG1655 ΔhsdR strain were used to prepare cell lysate. Cells were sonicated to disrupt cells
and centrifuged at 16,000 x g for 20 min at 4◦ C. The supernatant of cell lysate was dialyzed against 40 mM HEPES pH 7.5. (E) Cell lysate was subjected to elec­
trophoresis on a 1% agarose gel containing GelRed dye to stain nucleic acids. Benzonase (Millipore) was used to treat cell lysate to cleave nucleic acids (Supplement
Materials and Methods). (F, G) The cell lysates, either treated with benzonase or not, were used in the thermal aggregation assay as described above.

2
S. Ham and C. Lee
CRediT authorship contribution statement
Soojeong Ham: Investigation, Methodology, Visualization, Writing
– review & editing. Changhan Lee: Conceptualization, Investigation,
Methodology, Writing – original draft, Writing – review & editing.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Changhan Lee reports financial support was provided by National
Research Foundation of Korea (NRF) funded by the Korea government
(MSIT). Changhan Lee reports financial support was provided by Basic
Science Research Program through the NRF funded by the Ministry of
Education. Changhan Lee reports financial support was provided by the
new faculty research fund of Ajou University.
Data availability
No data was used for the research described in the article.
Acknowledgements
Changhan Lee received funding from the National Research Foun­
dation of Korea (NRF) funded by the Korea government (MSIT) (grant
2021R1C1C1011690 and RS-2023-00217595), the Basic Science
Research Program through the NRF funded by the Ministry of Education
(grant 2021R1A6A1A10044950) and the new faculty research fund of
Ajou University.
BBA - General Subjects 1867 (2023) 130445
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbagen.2023.130445.
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