Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00206.X |
Six specific lysine
residues are crucial in maintaining the structure
and function of soluble manganese stabilizing
protein
Jin-Peng GAO1,2#, Feng
ZHANG1,3#, Li ZHANG4, Yin-Long GUO4,
Kang-Cheng RUAN5, De-An JIANG3*, and Chun-He XU1*
1 Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai 200032, China;
2 Graduate School of the
Chinese Academy of Sciences, Shanghai 200032, China;
3 College of Life Sciences,
Zhejiang University, Hangzhou 310029, China;
4 Shanghai Mass
Spectrometry Center, Shanghai Institute of Organic Chemistry, Chinese Academy
of Sciences, Shanghai 200031, China;
5 Laboratory
of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Received: May 26, 2006
Accepted: June 11, 2006
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30270347 and 30471051) and the State Key Basic Research and Development
Plan (no. G1998010100)
#
These
authors contributed equally to this work
*Corresponding
authors:
Chun-He Xu: Tel, 86-21-54924228; Fax,
86-21-54924015; E-mail, [email protected]
De-An
Jiang: Tel, 86-571-86971381;
E-mail, [email protected]
Abstract When manganese stabilizing protein (MSP)
was treated with 0.5 mM N-succinimidyl propionate (NSP), the rebinding
ability and oxygen-releasing capabilities of the modified MSP were not altered,
in spite of changes to MSP surface Lys residues. Furthermore, far-ultraviolet
circular dichroism and intrinsic fluorescence spectra analysis revealed that
0.5 mM NSP-modified MSP retained most of its native secondary and tertiary
structure. Mapping of the sites of NSP modification by Staphylococcus V8
protease digestion of the modified protein, as well as analysis by
matrix-assisted laser desorption ionization-time of flight mass spectrometry,
indicated that seven Lys residues were modified. The results suggested that
these residues are not absolutely essential to the structure and function of
MSP. However, when the NSP concentration was increased to 4 mM, the modified
MSP was unable to bind photosystem II and completely lost its reactivating
capability. Both far-ultraviolet circular dichroism and intrinsic fluorescence
spectra analysis revealed a clear conformational change in MSP after 4 mM NSP
treatment, suggesting part of the Lys residues are involved in maintaining the
structure and function of MSP. Analysis by matrix-assisted laser desorption
ionization-time of flight mass spectrometry indicated that another six Lys
residues, namely Lys20, Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys66
(or Lys76), were modified only with 4 mM NSP. Therefore, these six candidate
Lys residues are crucial in maintaining the structure and function of soluble
MSP.
Key words chemical modification; fluorescence spectrum; circular
dichroism spectrum; manganese stabilizing protein; Lys residue
As a multi-subunit protein complex embedded in the thylakoid
membrane of green plants, photosystem II (PSII) catalyzes the light-driven reduction
of plastoquinone and the oxidation of water to molecular oxygen [1]. The
detailed oxygen-evolving complex consists of a cubane-like Mn3CaO4 cluster linked to a fourth Mn using a mono-m-oxo bridge
[2,3]. The 33 kDa extrinsic protein, usually called the manganese stabilizing
protein (MSP), binds to the lumenal surface of PSII [4–6]. Removal of this protein
from PSII membranes results in the loss of two Mn2+ ions
from the oxygen-evolving complex and a sharp reduction in oxygen evolution rates
in low concentrations of Cl–.
Reconstituting MSP can cause PSII membranes to recover their oxygen-releasing
capabilities to a substantial extent [7–9].
MSP was characterized as a “natively unfolded” protein or
a “molten globule” [10,11]. Soluble MSP was characterized as having a
high content of b-sheets and turns together with a low content of a-helices [12].
The recently identified 3-D structure of oxygen-evolving PSII suggested that
MSP, located on the lumenal surface within PSII, takes on an elongated shape
with two primary domains. Domain I displays a cylindrical shape with several
hydrophobic domains that are composed of eight anti-parallel b-sheets, and
domain II is an extended head [2]. Based on a model for eukaryotic MSP, five
highly conserved regions were identified as the CC bridge, and the KL, GGER,
DPKGR and GEhhG regions. The CC bridge and GEhhG region were suggested to play
key roles in maintaining the functional structure of MSP, whereas the other
regions seem to be critical for the binding of MSP to PSII [3].
Specific amino acids have been proven to be critical for maintaining
the secondary structure of MSP in solution, and for the binding of MSP to PSII.
A single Trp in spinach MSP, buried in a strong hydrophobic b-sheet region
near the C-terminus, is suggested to be key for the structure and function of
MSP [13,14]. Two cysteines, identified as highly conserved residues in all
available amino acid sequences of MSP, are involved in a disulfide bridge that
has been shown to be important for the correct folding of MSP [15,16]. Leu245
in spinach MSP is essential for maintaining the proper conformation of MSP in
solution, allowing efficient binding to PSII [17]. A recent study using
chemically modified Tyr residues from spinach MSP showed that 1–2 superficially
buried Tyr residues are critical for maintaining the structure and function of
MSP [18].
As ionizable residues, Lys residues are importantly involved in
determining each protein’s structure, ligand binding and functional activity,
as well as the unique properties of mutants [19]. Spinach MSP contains 23 Lys
residues that are distributed throughout the entire sequence. In a study that
reported MSP with chemically-modified Lys residues could not rebind to spinach
PSII, Miura et al. [20] suggested that the modified Lys residues might
participate directly in an interaction between the protein and PSII.
In the present study, we have directly identified candidate Lys
residues involved in maintaining the structure and function of MSP.
Materials and methods
Purification of MSP
Spinach was purchased from a local market in Shanghai (China). PSII
membranes (1.0 mg chl/ml) were isolated from the spinach leaves following the
methods described by Berthold et al. [21], then treated with 1.5 M NaCl for
1 h at 4 ºC under normal room light. After centrifugation at 40,000 g
for 20 min, pellets were re-suspended in SMN medium (0.4 M sucrose, 50 mM
MES-NaOH, pH 6.2, 15 mM NaCl, 10 mM MgCl2) and
mixed with the same volume of 2 M NaCl. This suspension was immediately
centrifuged at 40,000 g for 20 min, and the pellets were further
incubated in SMN medium containing 1 M CaCl2 at 4 ºC
for 30 min in dark. The samples were subjected to further centrifugation at
40,000 g for 20 min, and the supernatant was dialyzed overnight against
5 mM MES-NaOH (pH 6.2). The resulting crude extracts were purified by column
chromatography on a diethylaminoethyl-Sepharose CL-6B column (Amersham
Pharamcia Biotech, Uppsala, Sweden), as described by Kuwabara and Murata [22],
and the purified proteins were dialyzed against 10 mM phosphate buffer (pH 6.0)
before use. The MSP concentration was calculated from ultraviolet (UV)
absorbance at 276 nm, according to the methods of Eaton-Rye and Murata [23].
Chemical modification of MSP
with N-succinimidyl propionate (NSP)
To modify the amino groups of Lys residues and the free amino
terminus of MSP, the purified protein (48 mM) was incubated in a
reaction mixture containing 20 mM phosphate buffer (pH 6.5) and 0.5 or 4 mM NSP
at 25 ºC for 90 min in dark [20,24]. The reaction was stopped by passing the
reaction mixture through a Sephadex G-25 column (Amersham Pharamcia Biotech)
equilibrated in 20 mM phosphate buffer (pH 6.5) to remove the un-reacted NSP
from the reaction mixture.
Measurement of the
fluorescence spectra
The fluorescence emission spectra of native or NSP-modified MSP were
measured with an F4010 fluorescence spectrophotometer (Hitachi, Hitachi,
Japan). The concentration of MSP was 20 mM, and the excitation
wavelength was set at 280 nm to excite both Trp and Tyr, or at 295 nm to excite
only Trp. The native or NSP-modified MSP samples were dialyzed against 10 mM
phosphate buffer (pH 6.5) for 5 h prior to measurements.
Circular dichroism (CD)
spectroscopy of MSP
For CD spectroscopy, the native or NSP-modified MSP samples were
transferred to 10 mM phosphate buffer (pH 6.5) by extensive dialysis. After the
MSP suspension had been filtered through a polyethersulfone membrane (0.2 mm), CD spectra
were measured by a Jasco J-715 spectropolarimeter (Jasco, Tokyo, Japan) at 25
ºC. The concentration of MSP was adjusted to 10 mM before each measurement.
The cell length was 1 mm. Data were collected every 0.1 nm with a 1 nm
bandwidth and a 1 s interval, at a scan speed of 10 nm/min.
Reconstitution of MSP with
PSII membranes
PSII membranes were suspended in SCNlow
solution (0.4 M sucrose, 50 mM MES-NaOH, pH 6.5, 10 mM NaCl and 10 mM CaCl2) to reach 1.0 mg chl/ml, and the suspensions were treated with 2.6 M
urea/0.2 M NaCl for 30 min at 4 ºC in dark. After centrifugation at 40,000 g
for 20 min, pellets were re-suspended in SCNhigh
solution (0.4 M sucrose, 50 mM MES-NaOH, pH 6.5, 180 mM NaCl and 10 mM CaCl2).
Prior to reconstitution, various MSPs were dialyzed against 50 mM
MES-NaOH (pH 6.5) for 3 h. Afterwards, the solution was filtered through a
polyethersulfone membrane (0.2 mm). For reconstitution, the concentrations of urea/NaCl-washed PSII
membranes were adjusted to 0.1 mg chl/ml, and MSP was added to the reaction
medium to obtain a mole ratio of protein:PSII membrane at 8:1. The mixtures
were incubated at 4 ºC for 30 min in dark, centrifuged at 40,000 g for
20 min, and the pellets were washed twice with SCNlow
solution to remove the loosely-bound MSP.
Measurement of oxygen
evolution activities
The oxygen evolution activity of PSII membranes was measured with a
Clark-type oxygen electrode in SCNlow solution at 25 ºC. The
chlorophyll concentration in the reaction medium was 10 mg/ml, and 0.8 mM
2,6-dimethyl-p-benzoquinone was used as the artificial electron
acceptor.
polyacrylamide gel
electrophoresis analysis of protein content
Protein content was analyzed with sodium dodecylsulfate-polyacrylamide
gel electrophoresis, using a Laemmli system [25] with a slab gel containing a
5% stacking gel and an 11.75% resolving gel, both containing 6 M urea. The gels
were stained with Coomassie Brilliant Blue R-250, and densitograms were
obtained with a Digital Imaging System (IS-1000; Alpha Innotech, San Leandro,
USA). The relative amounts of MSP were determined by integrating the peak
areas, with the amount of native MSP reconstituted with urea/NaCl-washed PSII
set as 100%.
Protease digestion
The MSP (3 nM) modified with 0.5 or 4 mM NSP was dried and
solubilized in 10 ml of 1 M Tris-HCl, pH 8.5, 8 M guanidine-HCl, 1 mM EDTA and 1%
dithiothreitol, and incubated at 37 ºC for 2 h to denature the MSP. Then 5 ml of 5% iodoacetamide
was added and incubated at 37 ºC for 30 min to block SH groups. After
centrifugation, cold trichloroacetic acid was added to the reaction mixture to
a final concentration of 10%, the resulting precipitates were washed twice with
acetone. The final precipitates were dried and resolubilized in 20 ml 0.1 M ammonium
bicarbonate. After 1 mg staphylococcus
V8 protease (ICN Biomedicals, Costa Mesa, USA) was added, the MSP was
digested at 37 ºC overnight then desalted by Ziptipl-C18 (Millipore, Bedford,
USA).
Mass spectroscopic analysis
The protease-digested protein was applied directly to
matrix-assisted laser desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS) (Voyager-DE STR; Applied Biosystems, Forster, USA). The matrices
for peptides were a-cyano-4-hydroxycinnamic acid, sinapic acid or 2,5-dihydroxybenzoic
acid. The calculated molecular mass of the fragments obtained by V8 protease digestion was obtained using the ExPASy PeptideMass
program (http://www.expasy.org/tools/peptide-mass.html) and the protein
digestion tool MS-Digest (http://prospector.ucsf.edu/ucsfhtml4.0/msdigest.htm).
Results
Effect of NSP modification on
the structure of MSP
Fluorescence spectroscopy was shown to be a powerful tool for studying
protein structure and function [26]. As shown in curve b of Fig. 1, the
fluorescence spectra of 0.5 mM NSP-modified MSP show a clear decrease in the
emission intensity of the fluorescence peak. However, the peak position did not
differ from that of native MSP, whether excited at 280 nm or 295 nm, suggesting
that the modification had little effect on the tertiary structure and
conformation of MSP. These observations are similar to the results obtained
using far-UV CD spectra (Fig. 2). Both native and 0.5 mM NSP-modified
MSP displayed similar CD spectral features with a positive peak at 196 nm and
flattened negative peaks at 208 nm and 222 nm, suggesting that 0.5 mM
NSP-modified MSP retains its basic secondary structure, similar to native MSP.
After the NSP concentration was increased to 4 mM, the fluorescence
spectra of 4 mM NSP-modified MSP became broader, and the peak position showed a
prominent red shift when excited at either 280 nm or 295 nm (Fig. 1,
curve c). When excited at 280 nm, the peak position showed a prominent red
shift from 310 nm to 348 nm. The 295 nm excited spectrum was significantly
red-shifted with a pronounced peak at 350 nm, compared with that of the native
protein. When selectively exciting Trp residues in a given protein by setting
the excitation wavelength at 295 nm, Trp fluorescence is extremely sensitive to
the immediate hydrophilic/hydrophobic microenvironment. As the sole Trp
residue in MSP, Trp241 is buried in a hydrophobic region, thus the red shift of
the 295 nm spectrum of 4 mM NSP-modified MSP revealed that the hydrophobic
micro-region of Trp241 had become strongly hydrophilic. The changes in the
fluorescence spectra indicated that 4 mM NSP-modified MSP lost most of its
native tertiary structure. Similarly, a clear conformational change was
observed after MSP had been modified by 4 mM NSP as indicated in far-UV CD
spectrum. The elliptical curve of 4 mM NSP-modified MSP was more negative than
that of native MSP, and the CD spectrum contained a negative peak at 202 nm, suggesting
that the a-helices and b-sheets of MSP were dramatically altered into random coils.
Effects of NSP modification on
the binding and function of MSP
It was shown that a modification of amino groups on the MSP with 0.5
mM NSP affected neither its binding ability (Fig. 3, lane 4) nor its
influence on oxygen production (Table 1). MSP modified with 4 mM NSP can
not rebind PSII (Fig. 3, lane 5) and completely loses its reactivating
capability (Table 1). These data
are consistent with the previous work by Miura and colleagues who asserted that
MSP lost its ability to bind PSII following NSP modification primarily because
the modified Lys residues directly participate in the interaction between MSP
and PSII [20]. However, in the present study, a clear conformational change in
4 mM NSP-modified MSP was identified, suggesting that the modified Lys residues
not only directly participate in the interaction between MSP and PSII, but also
participate in maintaining the structure of MSP.
Identification of NSP-modified
Lys residues in MSP
MALDI-TOF MS analysis coupled with enzyme digestion was considered
to provide the necessary information to identify the extent and the precise
sites of NSP modification in MSP. In ammonium bicarbonate buffer, V8 protease selectively cleaves peptide bonds at the C-terminal side
of Asp and Glu and yields simpler mass spectra than other proteases such as
trypsin and chymotrypsin [27]. From the increase in the molecular weight of
peptides derived from the N-propionyl group (molecular
weight=56), the sites of modifications can be deduced.
In the present study, 0.5 mM NSP-modified MSP was completely
digested with V8 protease in the presence of iodoacetamide, and
the digested segments were analyzed with MALDI-TOF MS. To detect more complete
peptide fragments obtained from V8 protease digestion of the
modified MSP, three different matrices, including a-cyano-4-hydroxycinnamic
acid, sinapic acid and 2,5-dihydroxybenzoic acid, were used. Calculated and
observed masses of various peptide fragments (within a 0.1% mass error) of 0.5
and 4 mM NSP-modified MSP obtained after V8
digestion are summarized in Tables 2 and 3, respectively. As
shown in the tables, the same peptide fragment could be modified with a
different number of N-propionyl groups, implying that MSP, after NSP
modification, is composed of various protein products with various Lys
modifications. This situation is similar to the results obtained by modification
of the MSP with N-hydrosuccinimidobiotin (NHS-biotin) [28], NSP and
2,4,6-trinitrobenzen sulfonic acid [20].
In the MSP modified with 0.5 mM NSP, 26 different peptides were
identified, ranging in mass from 573.3 to 4578.1 Da (Table 2). Among
these peptides, seven Lys residues were found to be modified by NSP. Three of
these residues are Lys105, Lys159 and Lys186; one is Lys66 or Lys76; one is
Lys130 or Lys137; and two are Lys230, Lys233, or Lys236. Additionally, as the
N-terminus of Glu1–Asp9 was susceptible to modification with NSP, as was Lys4, either
Glu1 or Lys4 could be modified by NSP. Modifying the above-listed Lys residues
in MSP did not alter its structure or function, suggesting that at least seven
Lys residues on the surface of MSP are not necessary to maintain the structure
and function of MSP in solution.
When MSP was modified in 4 mM NSP, 35 different peptides were
identified with masses ranging from 512.8 Da to 4581.5 Da. As shown in Table
3, among all known MSP Lys residues, the potential sites modified by NSP
were the amino group at the amino terminus (Glu1) and the e-amino groups of
Lys20, Lys66, Lys76, Lys101, Lys130, Lys137, Lys159, Lys186, Lys196, Lys207,
Lys230 and Lys233. The Lys sites of MSP identified after 4 mM NSP modification
contain most of the Lys sites modified by 0.5 mM NSP except for Lys105. This
can be explained by assuming that the peptide fragment where Lys105 is located
is too small to be detected by MALDI-TOF MS. In addition, NSP is a hydrophilic
reagent and might not be efficient in modifying residues located in the
hydrophobic part of the protein at the same time, taking into consideration
some peptide fragments were not identified. Therefore, spinach MSP contains 23
Lys residues that are distributed throughout the entire sequence. However, our
mass spectrometric analysis indicated that there were only 12 Lys residues that
were modified.
Discussion
In comparing the Lys sites of 4 mM NSP-modified MSP with those of
0.5 mM NSP-modified MSP, six additional Lys sites were identified. CD and
fluorescence spectra showed the structure of MSP modified by 4 mM NSP in
solution changed significantly. Furthermore, the rebinding and oxygen evolution
activities of MSP were lost. These results indicated that these Lys residues
might be critically required for maintaining the structure and function of MSP
of PSII. Among the six Lys sites, four, including Lys20, Lys101, Lys196 and
Lys207, could be directly determined, but the other two Lys sites remain
unknown, although one is located at 66 or 76 and the other is located at 130 or
137.
It seems that these additional six Lys residues were less reactive
with NSP than the free Lys residues modified by 0.5 mM NSP. There are two
possible mechanisms that might account for the differential sensitivity to NSP
modification of these additional six Lys residues: these Lys residues form
hydrogen bonds with neighboring residues [29]; or these Lys residues are
superficially buried on the interior aspect of the MSP, which might shield them
from NSP modification through steric effects.
Among the six candidate Lys residues involved in maintaining the
structure and function of MSP identified in this study, Lys101, Lys196, Lys207,
Lys76 (or Lys66) and Lys130 (or Lys137) are located in the regions predicted to
contain b-strands in full-length MSP. The observation that the hydrophobic
region changes to hydrophilic in 4 mM NSP-modified MSP suggested that several
Lys residues participate in forming the hydrophobic compact region of MSP in solution.
Furthermore, current evidence suggests that charges on the surface might do
very little to stabilize the protein, but superficially buried charges are
likely to be extremely destabilizing [19]. In line with this view, the seven
Lys residues in MSP modified with 0.5 mM NSP are predicted to be located on the
surface of MSP in solution. Based on our findings that further modification of
additional six Lys residues destabilizes the native MSP, it is therefore
reasonable to assume that these Lys residues, as charged amino acids, are
superficially buried in hydrophobic regions. These buried Lys residues would be
of importance to the structure of MSP because of their hydrogen bonds with
nearby polar or charged groups. At the same time, NSP modifications have
blocked these buried Lys residues that are essential for formation of
charge-pair interactions to give high affinity binding of the protein to PSII.
Based on a model for eukaryotic MSP, five highly-conserved regions
were identified as the CC bridge, and the KL, GGER, DPKGR and GEhhG regions.
The CC bridge and GEhhG region were suggested to play key roles in maintaining
the functional structure of MSP, whereas the other regions seem to be critical
for the binding of MSP to PSII [3]. Our results indicated that six Lys
residues, namely Lys20, Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys76
(or Lys66) residues, are crucial in maintaining the structure and function of
solution MSP. Among these Lys residues, Lys76 is located in the KL region,
whereas Lys101 and Lys130 are located in the GGER region (Fig. 4). These
results indicated that Lys76, Lys101 and Lys130 residues are critical for the
binding of MSP to PSII. however,
Lys20 and Lys207 are close to the CC bridge or GEhhG region (Fig. 4),
which are considered important in determining the structure of MSP [3]. Lys66,
Lys196, and Lys137 residues are not involved to the five highly conserved
regions (Fig. 4), and we can not presume their function in MSP.
Because the Lys residues of MSP were heterogeneously modified, it is
impossible to determine the NSP concentration at which MSP begins to lose its
structure and function. Thus, the exact Lys sites involved in maintaining the
structure and function of MSP could not be conclusively identified. However,
the present study clearly showed the structure of MSP modified by 4 mM NSP in
solution changed significantly. At the same time, the rebinding and oxygen
evolution activities of MSP were lost. Several Lys residues, including Lys20,
Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys66 (or Lys76), might play a
crucial role in maintaining the function and structure of MSP in solution.
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