The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone and ATPase Activities at Elevated Temperatures
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Article
The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone
and ATPase Activities at Elevated Temperatures
Jose A. Mendoza * , Julian L. Ignacio and Christopher M. Buckley
Department of Chemistry and Biochemistry, California State University, San Marcos, CA 92096, USA;
ignac010@cougars.csusm.edu (J.L.I.); buckl015@cougars.csusm.edu (C.M.B.)
* Correspondence: jmendoza@csusm.edu
Abstract: The heat-shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori.
Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would
be expected to function as a molecular chaperone in this organism. H. pylori is a type of bacteria
that grows on the gastric epithelium, where the pH can fluctuate between neutral and 4.5, and the
intracellular pH can be as low as 5.0. We previously showed that Hsp60 functions as a chaperone
under acidic conditions. However, no reports have been made on the ability of Hsp60 to function as
a molecular chaperone under other stressful conditions, such as heat stress or elevated temperatures.
We report here that Hsp60 could suppress the heat-induced aggregation of the enzymes rhodanese,
malate dehydrogenase, citrate synthase, and lactate dehydrogenase. Moreover, Hsp60 was found
to have a potassium and magnesium-dependent ATPase activity that was stimulated at elevated
temperatures. Although, Hsp60 was found to bind GTP, the hydrolysis of this nucleotide could not
be observed. Our results show that Hsp60 from H. pylori can function as a molecular chaperone
under conditions of heat stress.
Citation: Mendoza, J.A.; Ignacio, J.L.; Keywords: Hsp60; molecular chaperone; protein aggregation; heat stress
Buckley, C.M. The Hsp60 Protein of
Helicobacter Pylori Exhibits
Chaperone and ATPase Activities at
Elevated Temperatures. BioChem 2021, 1. Introduction
1, 19–25. https://doi.org/
Helicobacter pylori is a Gram-negative, microaerophilic bacterium present in the stom-
10.3390/biochem1010002
ach of approximately half of the human population [1]. Chronic infection by this mi-
croorganism can, in certain individuals, give rise to gastric and duodenal ulcers, gastric
Academic Editor: Yehia Mechref
adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma [2,3]. H. pylori sur-
vives transient exposure to extreme acid prior to adherence and growth on the gastric
Received: 2 March 2021
Accepted: 30 March 2021
epithelium, where the pH can fluctuate between neutral and 4.5, and the intracellular pH
Published: 3 April 2021
can be as low as 5.0 [4,5]. Under neutral and moderately acidic conditions, the heat-shock
protein Hsp60 is one of the most abundant proteins in H. pylori. [4,6]. Given its sequence
Publisher’s Note: MDPI stays neutral
homology to the Escherichia coli Hsp60 or GroEL [7], Hsp60 from H. pylori would be ex-
with regard to jurisdictional claims in
pected to function as a molecular chaperone in this organism. The molecular chaperones
published maps and institutional affil- are a class of proteins that have been shown to facilitate the folding of nascent polypeptides,
iations. activation of other proteins, and protection of native proteins against the effects of heat,
and oxidative and acid stress [8–13]. The co-expression of H. pylori urease, Hsp60, and
Hsp10 in E. coli was shown to substantially increase the activity of urease [14], which
suggested that urease activity was protected by the heat-shock proteins. Furthermore,
Copyright: © 2021 by the authors.
we previously demonstrated that Hsp60 from H. pylori had a chaperone activity under
Licensee MDPI, Basel, Switzerland.
acidic conditions [15]. No reports, however, have been made on the potential chaperone
This article is an open access article
activity of Hsp60 from H. pylori under heat-shock conditions. It is well known that protein
distributed under the terms and denaturation can be induced at elevated temperatures and molecular chaperones, such
conditions of the Creative Commons as Hsp60 may be needed to stabilize them against heat-induced aggregation. This study
Attribution (CC BY) license (https:// was performed to determine if Hsp60 could function as a molecular chaperone at elevated
creativecommons.org/licenses/by/ temperatures. The enzymes rhodanese, malate dehydrogenase (MDH), citrate synthase
4.0/). (CS), and lactate dehydrogenase (LDH) were used as substrate proteins for Hsp60 given
BioChem 2021, 1, 19–25. https://doi.org/10.3390/biochem1010002 https://www.mdpi.com/journal/biochemBioChem 2021, 1 20
their propensity to aggregate at elevated temperatures and because they were previously
used as protein models to investigate the function of other heat-shock proteins [16–19].
Here, we report that Hsp60 prevented the heat-induced aggregation of these enzymes at
elevated temperatures. The aggregation of Hsp60 alone was not observed under these
conditions. It is also reported here that Hsp60 was able to hydrolyze ATP in a potassium
and magnesium-dependent manner. Furthermore, Hsp60’s ATPase activity was found to
be highly stimulated at elevated temperatures. Thus, our results show that Hsp60 from H.
pylori can function as a molecular chaperone with an increased ability to hydrolyze ATP
under conditions of heat stress.
2. Materials and Methods
All the reagents used here were of analytical grade. IPTG was purchased from Gold
Biotechnology and benzonase nuclease from Novagen. The protease inhibitor cocktail,
rhodanese, MDH, CS, and LDH were purchased from Sigma Co. The H. pylori Hsp60
gene was synthesized by GenScript (Piscataway, NJ, USA) and inserted into the expression
vector pET-22b (+). The resulting plasmid pET-Hsp60 was transformed into competent E.
coli BL21 (DE3) cells using ampicillin resistance for selection. The expressed protein was
purified using His GraviTrap chromatography (GE Healthcare, Chicago, IL, USA) followed
by dialysis. Purification of Hsp60 was confirmed by SDS-PAGE [20] and its concentration
determined by using a Bradford assay (BioRad, California, CA, USA).
Protein aggregation assay: the aggregation of rhodanese, MDH, CS, and LDH was
monitored by using light scattering. Protein (1 µM) samples were incubated in 50 mM
Tris-HCl (pH 7.5). Measurement of the light scattering of each enzyme, alone or in the
presence of Hsp60 (0.5–2 µM), was made with a Fluoromax-3 spectrophotometer using an
excitation wavelength of 350 nm. The light scattered intensity at 90◦ was recorded at the
same wavelength. The appropriate blanks were subtracted.
ATPase activity assay: Hsp60 (1 µM) was incubated in a reaction mixture containing
50 mM Tris-HCl (pH 7.5), 1 mM KCl, 1 mM MgCl2 , and 250 µM ATP at 25 ◦ C. Every 5 min,
aliquots were removed and the released phosphate was detected using a Malachite Green
Assay Kit (Cayman, UK) by measuring the absorbance at 620 nm.
All the experiments were independently performed twice (n = 2), and the results are
the average of both. Error bars were used to show the range or variability of the data.
3. Results and Discussion
3.1. Hsp60 Reduces Protein Thermal Aggregation
Given the sequence homology of H. pylori Hsp60 to the E. coli Hsp60 protein or
GroEL [7,21], Hsp60 from H. pylori would be expected to function as a molecular chap-
erone. Here, we tested the ability of Hsp60 from H. pylori to prevent the heat-induced
aggregation of several heat-sensitive enzymes. Thus, to test the ability of Hsp60 to prevent
the aggregation of these enzymes, each enzyme was incubated at 48 ◦ C in the absence
or presence of Hsp60, and the samples light scattering was measured after 60 min. As
shown in Figure 1, Hsp60 was able to reduce aggregation of the tested enzyme, resulting
in decreased scattered light intensities in the case of rhodanese (96.2%), MDH (91.9%), CS
(94.2%), and LDH (99.9%). The potential aggregation of Hsp60 alone was monitored by
incubating the protein at the same temperature and measuring the sample light scattering
with time. No significant changes in light scattering were detected (not shown) that would
indicate either Hsp60 aggregation or disassembly by an increase or decrease in the light
scattering, respectively.decreased scattered light intensities in the case of rhodanese (96.2%), MDH (91.9%), CS
(94.2%), and LDH (99.9%). The potential aggregation of Hsp60 alone was monitored by
incubating the protein at the same temperature and measuring the sample light scattering
BioChem 2021, 1, FOR PEER REVIEW
with time. No significant changes in light scattering were detected (not shown) that would 3 of 7
BioChem 2021, 1 indicate either Hsp60 aggregation or disassembly by an increase or decrease in the light 21
scattering, respectively.
% Suppression by Hsp60 of Light Scattered
100
% Suppression by Hsp60 of Light Scattered
100
80
80
60
60
40
40
20
20
0
0 Rhod
1 MDH
2 CS
3 LDH
4
Rhod
1 MDH
2 CS
3 LDH
4
Figure 1. Hsp60 reduced the aggregation of proteins as measured by the reduction in light scattered
Figure 1.
intensity. Hsp60
Hsp60 (2reduced
µM) wasthe aggregation
incubated in 50of
mM proteins as measured
Tris-HCl by◦the
pH 7.5 at 48 reduction
C for in light
10 min and thenscat-
1 µM
oftered intensity.
rhodanese Hsp60 dehydrogenase
or malate (2 μM) was incubated
(MDH)inor50citrate
mM Tris-HCl
synthasepH 7.5or
(CS) at lactate
48 °C for 10 min and
dehydrogenase
then 1 μM of rhodanese or malate dehydrogenase (MDH) or citrate synthase (CS) or lactate dehy-
(LDH) was added and the light scattering was recorded after 60 min. n = 2 biologically independent
drogenase (LDH) was added and the light scattering was recorded after 60 min. n = 2 biologically
experiments, and the result is the average of both.
independent experiments, and the result is the average of both.
Figure 2 shows the time course of the light scattering of MDH (panel A) and CS (panel
Figure 2 shows the time course of the light scattering of MDH (panel A) and CS (panel
B) when these enzymes were incubated at 48 ◦ C in the absence or presence of Hsp60. Light
B) when thesedetected
scattering was enzymestowere incubated
increase withinata 48
few°Cminutes
in the absence oraddition
after the presenceofofMDH
Hsp60.
orLight
CS
toscattering
the bufferwas detected
without to increase within a few minutes after the addition of MDH or CS
Hsp60.
to the buffer without Hsp60.
5
3 x 10
Light Scattering (A.U.)
Light Scattering (A.U.)
5
2.5 x 10 1 x 10
7
2 x 10
5 MDH alone
CS alone
5
1.5 x 10 5 x 10
6
5
1 x 10
MDH + Hsp60 CS + Hsp60
4
5 x 10
0
0
0 2 4 6 8 10 12 0 5 10 15 20 25 30 35 40
Time (min) Time (min)
(A) (B)
Figure
Figure 2. 2. Time
Time course
course of of
thethe light
light scattering
scattering ofof MDH
MDH (A)
(A) and
and CSCS(B)(B) without
without (closed
(closed circles)
circles) oror with
with Hsp60
Hsp60 (open
(open circles).
circles).
For samples containing Hsp60, this was incubated at 2 μM in 50 mM Tris-HCl pH 7.5 at ◦
48 °C
For samples containing Hsp60, this was incubated at 2 µM in 50 mM Tris-HCl pH 7.5 at 48 C for 10 min and then MDH for 10 min and then MDH
(1 μM) or CS (1 μM) was added at t = 0. n = 2 biologically independent experiments, and the result is the
(1 µM) or CS (1 µM) was added at t = 0. n = 2 biologically independent experiments, and the result is the average of the two. average of the
two.
As shown in Figure 2 no increase in the intensity of scattered light was observed when
MDH or AsCSshown in Figure 2with
were incubated no Hsp60.
increaseThus,
in the intensity
these resultsofdemonstrate
scattered light
that was
Hsp60 observed
from
when MDH or CS were incubated with Hsp60. Thus, these results
H. pylori can function as a molecular chaperone at elevated temperature by preventing demonstrate that Hsp60
from
the H. pylori can
heat-induced function asofa these
aggregation molecular
model chaperone
proteins.atOurelevated temperature
findings by prevent-
are consistent with
aning the heat-induced
expected chaperone role aggregation
for Hsp60ofgiven
theseits model proteins.
high degree Our findings
of homology are consistent
to GroEL [7,21].
with that
Given an expected
Hsp60 from chaperone
H. pylori rolehas
forbeen
Hsp60 given its
reported to high
exist degree of homology
as a mixture of dimersto GroEL
and
[7,21]. Given
tetramers that the
[22], and Hsp60 from
tested H. pylori
enzymes hasmonomeric
were been reported to exist asdimeric
(rhodanese), a mixture
(MDH of dimers
and
CS), and tetrameric (LDH), in these aggregation assays, Hsp60
and tetramers [22], and the tested enzymes were monomeric (rhodanese), dimeric (MDHand enzymes were mixed in
a 1:1
andtetrameric
CS), and to monomeric
tetrameric molar
(LDH), inratio.
these aggregation assays, Hsp60 and enzymes were
Figure
mixed in a3 1:1
shows that sub-stoichiometric
tetrameric to monomeric molar amounts of Hsp60 did not suppress completely
ratio.
the observed
Figure 3light
shows scattered by rhodanese. The
that sub-stoichiometric suppression
amounts of Hsp60of 42%didlight scattered com-
not suppress by
rhodanese
pletely thewas observed
observed with
light a 1:0.5 by
scattered tetrameric
rhodanese. Hsp60
Thetosuppression
rhodanese stoichiometric ratio.
of 42% light scattered
Asbyshown in the
rhodanese was figure,
observedcomplete
with asuppression
1:0.5 tetrameric of light
Hsp60 scattered by rhodanese
to rhodanese in thera-
stoichiometric
presence of Hsp60 occurred only with a 1:1 tetrameric Hsp60 to
tio. As shown in the figure, complete suppression of light scattered by rhodanese in the monomeric rhodanese
stoichiometric ratio. Our
presence of Hsp60 light only
occurred scattering
with ameasurements
1:1 tetramericfor Hsp60
Hsp60 to could not detect
monomeric any
rhodanese
structural changes
stoichiometric at the
ratio. Ourtested
lighttemperatures suggesting thatfor
scattering measurements Hsp60
Hsp60 retained
couldits
notquaternary
detect any
structure during
structural changes these at experiments. Functional suggesting
the tested temperatures lower oligomers have been
that Hsp60 reported
retained for
its quater-
the Hsp60 protein from M. tuberculosis [23]. The unusual quaternary
nary structure during these experiments. Functional lower oligomers have been reported structure of H. pylori
Hsp60 raises the question if, under in vivo or certain in vitro conditions, Hsp60 couldBioChem 2021, 1 22
undergo oligomerization like the Hsp60 from M. tuberculosis into a double ring structure
BioChem 2021, 1, FOR PEER REVIEW
like that of GroEL [24]. Whereas, in the experiments reported here, Hsp60 by itself
5 of 8
was
an effective chaperone, it has been shown that the folding process of newly synthesized
proteins in E. coli involves several molecular chaperones [8]. Thus, it remains to be seen
BioChem 2021, 1, FOR PEER REVIEW whether Hsp60 from H. pylori can support the folding of other proteins and interact5 of with
8
other chaperones. Moreover, the nature of the Hsp60-protein complexes detected here and
Scattering (A.U.)
Rho alone
the 100
mechanism of release of the bound proteins remains to be elucidated.
80
Light(A.U.)
60
100 Rho alone
+ 0.5 Hsp60
Scattering
40
80
Relative
20
60
+ 0.5 Hsp60+ 1.0 Hsp60
Relative Light
0
40
1 2 3 Hsp604 alone
20
+ 1.0 Hsp60
0
1 2 3 Hsp604 alone
Figure 3. Effect of Hsp60 on the light scattered by rhodanese. Rhodanese (1 μM) was incubated
without Hsp60
Figure or with
3. Effect of Hsp60
Hsp60 (0.5 μM)light
on the or with Hsp60by
scattered (1 rhodanese.
μM) in Tris-HCl, pH 7.5(1atµM)
Rhodanese 48 °Cwas
andincubated
then
the light
withoutscattering
Hsp60was recorded
or with Hsp60after
(0.5 60
µM)min.
or n = 2 Hsp60
with biologically independent
(1 µM) in Tris-HCl,experiments, ◦ C and
pH 7.5 at 48 and the then
Figureis3.the
result Effect of Hsp60
average of theon the light scattered by rhodanese. Rhodanese (1 μM) was incubated
two.
the light scattering was recorded after 60 min. n = 2 biologically independent experiments, and the
without Hsp60 or with Hsp60 (0.5 μM) or with Hsp60 (1 μM) in Tris-HCl, pH 7.5 at 48 °C and then
result is the average of the two.
the light scattering was recorded after 60 min. n = 2 biologically independent experiments, and the
3.2. ATPase Activity of Hsp60
result is the
3.2. average
ATPase of theoftwo.
Activity Hsp60
Given the ability of H. pylori Hsp60 to function as a molecular chaperone, it would
be expectedGiven the ability
to have an of H. pylori Hsp60 to function as a molecular chaperone, it would
3.2. ATPase Activity of ATPase
Hsp60 activity like its homologue the E. coli Hsp60 protein or
GroELbe expected
[25]. It hastobeenhavereported
an ATPasethat activity like its homologue
both potassium and magnesium the E.are
coli Hsp60 for
required protein
the or
Given[25].
GroEL the ability
It has of H.reported
been pylori Hsp60
that to function
both potassium as aand
molecular chaperone,
magnesium are it would
required for the
ATPase activity of the E. coli Hsp60 or GroEL protein [26]. Potassium appeared to increase
be expected
ATPase to haveofan
activity theATPase
E. coli activity
Hsp60 orlike
GroEL its homologue
protein [26]. the E. coli Hsp60
Potassium appeared protein
to or
increase
the affinity of GroEL for ATP, while magnesium is required since the magnesium-bound
GroEL [25]. It has been reported that both potassium and magnesium are required for the
formthe ofaffinity of GroELbinds
the nucleotide for ATP,
GroEL.while magnesium
Thus, the ability is required
of Hsp60since the magnesium-bound
to hydrolyze ATP was
ATPase
form activity
of the of the E. colibinds
nucleotide Hsp60 or GroEL
GroEL. Thus,protein
the [26]. Potassium
ability of Hsp60 appeared
to to increase
hydrolyze ATP4was
tested in the absence or presence of potassium and/or magnesium. The results in Figure
the tested
affinityinoftheGroEL for or
ATP, while of magnesium isand/or
required since the magnesium-bound
show that potassium and magnesium are required by Hsp60 to hydrolyze ATP. We Figure
absence presence potassium magnesium. The results in also 4
form of the
show that nucleotide binds GroEL. Thus, the ability of Hsp60 to hydrolyze ATP wasalso
examined the potassium and magnesium
potential GTPase activity ofare required
Hsp60. by Hsp60
However, to hydrolyze
as shown ATP.4,We
in Figure the
tested in
examined the absence
the by or presence
potential GTPase of potassium
activity and/or
of not
Hsp60. magnesium.
However, The results in
Figure 4,4 the
Figure
hydrolysis of GTP Hsp60 from H. pylori was observed in the as shownofinpotassium
presence
show that potassium
hydrolysis of GTP and magnesium
by Hsp60 from H. are required
pylori was not by observed
Hsp60 to in hydrolyze ATP.ofWe
the presence also
potassium
and/or magnesium.
examined the potential
and/or magnesium.B GTPase activity of Hsp60. However, as shown in Figure 4, the
hydrolysis of GTP by Hsp60 from H. pylori was not observed in the presence of potassium
and/or magnesium.
(%)
100 B
Hydrolysis
80
(%)
100
60
Hydrolysis
80
40
Relative Relative
60
20
40
0
+ 2+ + 2+ + 2+ + 2+
K /ATP Mg /ATP K /Mg ATP K /GTP Mg /GTP K /Mg /GTP
20
Figure 04. ATP or GTP hydrolysis by Hsp60. Hsp60 (1 µM) was incubated with 50 mM Tris-HCL,
pH 7.5 at 25 ◦Mg
K /ATP C in
/ATPthe presence
K /Mg ATP K /GTP of
Mg1 /GTP
mMK KCl or 1 mM MgCl2 or both and 250 µM ATP, and in the
+
/Mg /GTP
2+ + 2+ + 2+ + 2+
Figure 4. ATP or GTP hydrolysis by Hsp60.
presence of 1 mM potassium or 1 mM magnesium Hsp60 (1 μM) wasand
or both incubated
250 µM with
GTP. 50 mMhydrolysis
Then, Tris-HCL,of ATP
pH 7.5 at 25 °C in the presence of 1 mM KCl or 1 mM MgCl2 or both and 250 μM ATP, and in the
or GTP was determined, as described in Materials and Methods. n = 2 biologically independent
presence of 1 mM potassium or 1 mM magnesium or both and 250 μM GTP. Then, hydrolysis of
experiments,
Figure and the result is by
theHsp60.
average of the(1two.
ATP or 4.
GTPATPwas or determined,
GTP hydrolysisas described inHsp60
Materials μM)
andwas incubated
Methods. n = 2with 50 mM Tris-HCL,
biologically
pH 7.5 at
independent 25 °C in the
experiments,presence
and of
the 1 mM
result KCl
is theor 1 mM MgCl
average of 2 or both and 250 μM ATP, and in the
the two.
3.3. Binding of GTP to Hsp60
presence of 1 mM potassium or 1 mM magnesium or both and 250 μM GTP. Then, hydrolysis of
ATPBinding
or GTPTo determine
was if GTPaswas
determined, not hydrolyzed
described in Materialsbyand Hsp60 because
Methods. n = 2ofbiologically
the protein’s inability to
3.3. of GTP to Hsp60
bind the experiments,
independent nucleotide, we andused the fluorescent
the result is the average nucleotide
of the two.analog TNP-GTP [27]. An increase
Tothe
in determine if GTP
fluorescence of was not hydrolyzed
TNP-GTP, by Hsp60
in the presence of a because
protein,of
is the protein’sa inability
considered reliable test
to bind
3.3. for the nucleotide,
the assessment
Binding of GTP to ofwe used the fluorescent nucleotide analog TNP-GTP [27].5 An
the nucleotide-binding capacity of the protein [28]. Figure
Hsp60 shows
increase in the fluorescence of TNP-GTP, in the presence of a protein, is considered a
To determine if GTP was not hydrolyzed by Hsp60 because of the protein’s inability
reliable test for the assessment of the nucleotide-binding capacity of the protein [28].
to bind the nucleotide, we used the fluorescent nucleotide analog TNP-GTP [27]. An
Figure 5 shows that Hsp60 had a significant effect on the fluorescence spectrum of TNP-
increase in the fluorescence of TNP-GTP, in the presence of a protein, is considered a
GTP, suggesting that Hsp60 has a GTP-binding site. The significance of the binding of
reliable test for the assessment of the nucleotide-binding capacity of the protein [28].ATP or GTP was determined, as described in Materials and Methods. n = 2 biologically independ-
ent3.3. Binding ofand
experiments, GTPthe
toresult
Hsp60is the average of the two.
To determine if GTP was not hydrolyzed by Hsp60 because of the protein’s inability
3.3. Binding
to bind the of nucleotide,
GTP to Hsp60 we used the fluorescent nucleotide analog TNP-GTP [27]. An in-
To determine
crease if GTP was
in the fluorescence not hydrolyzed
of TNP-GTP, in theby Hsp60 of
presence because of the
a protein, protein’s inability
is considered a reliable
BioChem 2021, 1 23
totest
bindforthethe
nucleotide,
assessment weofused
the the fluorescent nucleotide
nucleotide-binding capacityanalog
of the TNP-GTP [27]. Figure
protein [28]. An in- 5
crease
shows in that
the fluorescence
Hsp60 had aofsignificant
TNP-GTP,effect
in theonpresence of a protein,
the fluorescence is considered
spectrum a reliable
of TNP-GTP, sug-
test for thethat
gesting assessment
Hsp60 has of athe nucleotide-binding
GTP-binding site. Thecapacity of the
significance of protein [28]. Figure
the binding of GTP5to
shows
Hsp60 that
that in Hsp60 hada asignificant
the function
Hsp60 had significant
and effect
structure ofon
effect onthe
the the fluorescence
chaperone remains
fluorescence spectrum ofofTNP-GTP,
TNP-GTP,suggesting
to be determined.
spectrum sug-
gesting
thatthat Hsp60
Hsp60 has has a GTP-binding
a GTP-binding site.significance
site. The The significance
of the of the binding
binding of GTP of GTP toin the
to Hsp60
Hsp60 in the function
function and structure
and structure of the chaperone
of the chaperone remains remains to be determined.
to be determined.
Fluorescence Intensity (A.U.)
20 TNP-GTP + Hsp60
Fluorescence Intensity (A.U.)
20 15 TNP-GTP + Hsp60
TNP-GTP
15 10
TNP-GTP
10 5
5 0
500 520 540 560 580 600
Wavelength (nm)
0
500 520 540 560 580 600
Wavelength (nm)
Figure 5. Binding of GTP to Hsp60 analyzed by fluorescence spectroscopy. The fluorescence of
Figure at
TNP-GTP Binding
5. 50 of GTP
μM in 600 μL ofto50Hsp60
mM trisanalyzed
buffer, by pHfluorescence
7.8, with andspectroscopy.
without Hsp60The wasfluorescence
measured of
TNP-GTP
using5.an
Figure at 50
excitation
Binding µM in 600
wavelength
of GTP µL of 50
to Hsp60ofanalyzedmM
409 nm and tris buffer, pH 7.8, with
its emission spectroscopy.
by fluorescence and without
recorded fromThe Hsp60
500fluorescencewas= measured
to 600 nm. n of 2 bio-
logically
usingatindependent
TNP-GTP an
50 excitation
μM in 600experiments,
wavelength
μL of 50 mM and the
oftris
409 result
nm
buffer, and
pHis its
the average
with andof
7.8,emission the two.Hsp60
recorded
without from 500wastomeasured
600 nm. n = 2
using biologically
an excitationindependent
wavelengthexperiments,
of 409 nm and its the
and emission
result recorded fromof
is the average 500
thetotwo.
600 nm. n = 2 bio-
3.4. Temperature
logically independent Dependence of the
experiments, andATPase
the resultActivity of Hsp60
is the average of the two.
3.4. Temperature Dependence of the ATPase Activity of Hsp60
Given that the ATPase of GroEL was previously shown to be stimulated at elevated
3.4. Given[29,30],
Temperature
temperatures that thewe
DependenceATPase
of the of GroEL
ATPase
investigated was
Activity
the previously
ability Hsp60shown
ofofHsp60 from H.topylori
be stimulated
to hydrolyze at elevated
ATP
temperatures
Given that
at elevated [29,30],
the ATPase of
temperatures. we investigated
GroEL
Figure the ability
was previously
6 shows the ATPase of Hsp60
shown from
to beof
activities H. pylori
stimulated
Hsp60 in at to hydrolyze
theelevated
22–67 °C ATP
at elevated temperatures. Figure 6 shows the ATPase activities of Hsp60 in the ◦
temperatures
range in the[29,30], we of
presence investigated
potassiumthe andability of Hsp60As
magnesium. from
shownH. pylori
in thetofigure,
hydrolyze ATP C
22–67
maximum
range in
at ATPase
elevated the presenceFigure
temperatures. of potassium and magnesium. As shown in the
in figure, maximum
activity was observed 6atshows 62 °C.the ATPase
◦Thus, it wouldactivities of Hsp60
be expected that the 22–67
ATPase °Cof
range ATPase
in the activity
presence was
of observed
potassium at
and62 C. Thus,
magnesium. it would
As shown be expected
in the that
figure, the ATPase of
maximum
Hsp60 from H. pylori observed here in that temperature range, would be needed to trigger
ATPase Hsp60 fromwas
activity H. pylori observed
observed 62here in that it
temperature range, would be
theneeded to trigger
the release of bound proteinsatfrom °C. Thus,
Hsp60, likewould be expected
in GroEL-mediated that
protein ATPase
foldingof [8].
the release of bound proteins from Hsp60, like in GroEL-mediated protein folding [8].
Hsp60 from H.
However, thispylori observed
question awaitshere in that
further temperature range, would be needed to trigger
experimentation.
However, this question awaits further experimentation.
the release of bound proteins from Hsp60, like in GroEL-mediated protein folding [8].
However, this question awaits further experimentation.
12
(micromol/min/micromol Hsp60)
Released
10
12
8
PhosphateHsp60)
Phosphate Released
10
6
(micromol/min/micromol
8
4
6
2
4
0
2 20 30 40 50 60 70
o
Temperature ( C)
0
20 30 40 50 60 70
Figure 6. Temperature dependence(of o the ATPase activity of Hsp60 in the presence of potassium and
Temperature C)
magnesium in the 22–67 ◦ C range. Hsp60 (1 µM) was incubated at the indicated temperature with
Figure 6. Temperature dependence of the ATPase activity of Hsp60 in the presence of potassium
and50magnesium
mM Tris-HCl,in the 22–67
pH 7.5, 1°CmM
range.
KCl,Hsp60
1 mM(1MgCl
μM) 2was
, andincubated at theand
250 µM ATP, indicated temperature
the ATPase activity was
Figuredetermined
6. Temperature dependence
as described of the ATPase
in Materials activityn of
and Methods. = 2Hsp60 in the presence
biologically of potassium
independent experiments, and
and magnesium
the result isinthe
theaverage
22–67 °C
of range. Hsp60 (1 μM) was incubated at the indicated temperature
the two.
4. Conclusions
Our results demonstrate that Hsp60 from H. pylori can function as a molecular chaper-
one at heat-shock temperatures. Given that that Hsp60 from H. pylori contains a significant
exposure of hydrophobic surfaces [15] and that these are also present in proteins subjected
to elevated temperatures, our results suggest that the interaction between Hsp60 and
other proteins undergoing heat-induced denaturation may be mediated by hydrophobic
interactions. The ability of Hsp60 to protect a protein exposed to elevated temperaturesBioChem 2021, 1 24
is significant in light of the fact that under in vivo conditions of heat shock, like those in
which the synthesis of the Hsp60 proteins is stimulated, partially thermally-denatured
proteins may actually exist.
Author Contributions: J.A.M. designed the study, directed the project, and drafted the manuscript.
J.L.I. and C.M.B. carried out the experiments and made the graphs and figures. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was supported by a development grant to J.A.M from the California State
University Program for Education and Research in Biotechnology (CSUPERB).
Institutional Review Board Statement: The study did not involve humans or animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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