Probing the Lysine Proximal Microenvironments within Membrane

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Probing the lysine proximal microenvironments within membrane protein complexes by active dimethyl labeling and mass spectrometry Ye Zhou, Yue Wu, Mingdong Yao, Zheyi Liu, Jin Chen, Jun Chen, Lirong Tian, Guangye Han, Jian-Ren Shen, and Fangjun Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02502 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Analytical Chemistry

Probing the lysine proximal microenvironments within membrane protein complexes by

1

active dimethyl labeling and mass spectrometry

2 #,†,‡

#,$,†

, Yue Wu

, Mingdong Yao§, Zheyi Liu†,‡, Jin Chen†,‡, Jun Chen§, Lirong Tianǁ, ‡,

3

Ye Zhou

4

Guangye Han||, Jian-Ren Shen||, , Fangjun Wang*,†

5 6 7 8 9 10

⊥

†

Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian

Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China §

State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian

National Laboratory for Clean Energy, Dalian, 116023, China ||

Key Laboratory of Photobiology, Institute of Botany, the Chinese Academy of Sciences, Beijing, 100093, China.

‡

University of Chinese Academy of Sciences, Beijing, 100049, China

11

⊥

12 13 14 15 16

Naka 3-chome, Tsushima, Okayama, 700-8530, Japan # $

Photosynthesis Research Center, Graduate School of Natural Science and Technology, Okayama University, 1-1,

Authors contributed equally Current address: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA,

30332-0400, USA *

Correspondence and requests for materials should be addressed to F.W. (email: [email protected])

17

ACS Paragon Plus Environment


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ABSTRACT：：Positively-charged lysines are crucial to maintaining the native structures of pro-

19

teins and protein complexes by forming hydrogen bonds and electrostatic interactions with their

20

proximal amino acid residues. However, it is still a challenge to develop an efficient method for

21

probing the active proximal microenvironments of lysines without changing their biochemi-

22

cal/physical properties. Herein, we developed an active covalent labeling strategy combined with

23

mass spectrometry to systematically probe the lysine proximal microenvironments within mem-

24

brane protein complexes (~700 kDa) with high throughput. Our labeling strategy has the ad-

25

vantages of high labeling efficiency and stability, preservation of the active charge states as well as

26

biological activity of the labeled proteins. In total, 121 lysines with different labeling levels were

27

obtained for the photosystem II complexes from cyanobacteria, red algae and spinach, and pro-

28

vided important insights for understanding the conserved and non-conserved local structures of

29

PSII complexes among evolutionarily divergent species that perform photosynthesis.

30 31

Lysines are usually positively charged in the native environments of living organisms and play

32

important roles in regulating the protein’s structure, interaction, function and stability1. Because of

33

the large energy needed to bury charged residues, most of the lysines are located on the protein

34

surfaces in water environment. Some of the lysines appear at the interfaces of protein-protein in-

35

teraction to form hydrogen bonds or electrostatic interactions with their proximal units. For exam-

36

ple, the positively charged lysines are important for the electrostatic interaction between spinach

37

PsbQ and photosystem II (PSII)2, whereas several specific lysines are crucial in maintaining the

38

native structure and function of PsbO3,4. Moreover, lysines could interact with the specific oxygen

39

atoms of the phospholipid headgroups, reflecting the preferred localization of these residues at the

40

lipid/water interface5. Lysine methylation in the nucleosome core histones plays important role in

41

regulating gene transcription, and mono-, di-, and tri-methylated lysines are differentially distrib-

42

uted across chromatin fibers6 and show cell-specific regulation during cell development7. There-

43

fore, probing the native status of lysine microenvironments is important for understanding the na-

44

tive structures of proteins and protein complexes, especially for the membrane protein complexes

45

that are difficult to crystalize.

46

Mass spectrometry (MS) is a powerful tool in providing protein structure information with

47

much less restrictions in protein size, solubility and purity8-11, compared with other methods such

48

as X-ray crystallography and nuclear magnetic resonance (NMR). Usually chemical labeling is

49

needed before the protein structure investigation by MS, such as hydrogen-deuterium exchange

50

(HDX) and chemical covalent labeling strategies12. The HDX is an active labeling strategy without

51

changing the biophysical properties of proteins, yet the fast back-exchange of deuterium to hy-

52

drogen and gas-phase “hydrogen-deuterium scrambling” within MS compromise its accuracy13. In

53

contrast, covalent labeling strategies usually modify the side chains of amino acid residues with

54

high stability, among which the primary amine on the lysine side chain is the most commonly

55

modified one due to its high reactivity. However, most of the covalent labeling strategies for ly-


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56

sines

neutralize

their

positive

charges

by

utilizing

highly

reactive

reagents

like

57

N-acetyl-succinimide, sulfosuccinimidyl acetate and S-methylthioacetimidate, which might per-

58

turb the native structures and induce biological activity loss for the labeled proteins14. It is still a

59

challenge to develop an active covalent labeling strategy without changing the biophysical proper-

60

ties and biological activity of the labeled proteins.

61

Reductive dimethyl labeling of the primary amine on lysine side chain by reacting with formal-

62

dehyde and sodium cyanoborohydride is a highly efficient covalent labeling method15 and unlikely

63

to induce protein’s structure changes and artificial protein-protein interactions, since the charge

64

states of lysines are not changed by the dimethyl labeling16-20. In this study, we developed an al-

65

ternative strategy by coupling active dimethyl labeling with MS to globally probe the lysine

66

proximal microenvironments within active macromolecular membrane protein complexes, based

67

on the fact that free lysines on protein exterior surfaces are much easier and faster to be accessed

68

and labeled than those that are either buried or engaged in intra/intermolecular interactions (Figure

69

1). The reactivity or labeling levels of lysines determined by MS reveals the interaction strength

70

between the lysines with their proximal microenvironments.

71 72

EXPERIMENTAL SECTION

73

Materials and Reagents. Horse heart myoglobin, chymotrypsin and trypsin were purchased

74

from Sigma (St. Louis, MO, USA). Acetonitrile (ACN, HPLC grade) was provided from

75

Merck (Darmstadt, Germany). 2-butanone was analytical grade from Bonuo (Dalian, China).

76

Formaldehyde (HCHO), formaldehyde-D2 (DCDO), sodium cyanoborohydride (NaBH3CN),

77

formic acid (FA), dithiothreitol (DTT), iodoacetamide (IAA) and other chemical reagents

78

were purchased from Sigma (St. Louis, MO, USA). All of the water utilized in the experi-

79

ments was purified by a Milli-Q system from Millipore Company (Bedford, MA, USA).

80

Fused silica capillaries with 75 µm i.d. were provided from Polymicro Technologies (Phoenix,

81

USA), and with 200 µm i.d. were purchased from Yongnian Optical Fiber Factory (Hebei,

82

China). Daisogel ODS-AQ (3 µm, 120 Å) HPLC packing materials were purchased from

83

DAISO Chemical CO., Ltd. (Osaka, Japan).

84

Dimethyl labeling of holo- and apo-myoglobin. Apo-myoglobin was obtained from ho-

85

lo-myoglobin by removing the heme group using the 2-butanone extraction method21. The

86

apo-myoglobin solution was dialyzed with HEPES buffer (20 mM, pH 7.0), and the ho-

87

lo-myoglobin was dissolved into the same buffer. An aliquot of 0.1 µg/µl holo- or apo-myoglobin

88

was dimethylated by adding 2 mM HCHO and NaBH3CN at 25 °C for 30 min, followed by

89

quenching with addition of 20 mM NH4HCO3 and incubation for 20 min. Then, the labeled pro-

90

teins were boiled for 3 min and digested overnight with chymotrypsin at 30 °C at an enzyme to

91

substrate ratio 1/25 (w/w).

92

Preparation of PSII complex samples. The spinach PSII core complex was prepared by using

93

0.4% (w/v) n-octyl-β-D-thioglucoside (OTG) from fresh spinach leaves as described in the previ-



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ous work22. The PSII complex was finally suspended into a SMN buffer containing 50 mM

95

MES-NaOH (pH 6.0), 400 mM sucrose and 15 mM NaCl to obtain 0.24 mg chlorophyll/mL. The

96

total chlorophyll concentration of PSII complex was calculated using the optical absorption coef-

97

ficients of chlorophyll a and b in 80% acetone aqueous solution23 (Chl (mg/mL) = 7.15 × A663.2 +

98

18.71 × A646.8, and A is the measured absorbance at the indicated wavelength). Based on the num-

99

bers of chlorophyll molecules per PSII reaction center in the complex, we estimated that the con-

100

centration of spinach PSII complex was about 2.7 mg/mL. The sample was stored at -80 °C in the

101

dark until use.

102

Steady-state O2 evolution activity of purified PSII complex was monitored with a Clark-type

103

oxygen electrode at 25 °C under saturated red light illumination24. The PSII complex correspond-

104

ing to 5 mg chlorophyll were dispersed in a buffer containing 0.60 mM recrystallized 2,

105

6-dichlorobenzoquinone and 10 mM CaCl2 . The O2 evolution rate was estimated from the O2

106

concentration change.

107

Cyanobacterial and red algal PSII core complexes were isolated from a thermophilic cyanobac-

108

terium Thermosynechococcus vulcanus25 and a red alga Cyanidioschyzon merolae26, respectively.

109

The final PSII samples were suspended in a buffer containing 30 mM Mes (pH 6.0), 20 mM NaCl

110

and 3 mM CaCl2.

111

Dimethyl labeling of PSII complex. An aliquot of 100 µg PSII complex was suspended into a

112

20 mM HEPES (pH=7.0) buffer and diluted to a concentration of 0.1 µg/µl. Reductive dimethyl

113

labeling was performed by adding 5 or 10 mM DCDO and NaBH3CN, followed by incubation at

114

37 °C in darkness for the required time (30 min or 2h). The reaction was stopped by addition of 50

115

mM NH4HCO3 and a subsequent incubation for 20 min. We also performed control experiments of

116

BSA dimethyl labeling with and without 400 mM sucrose, which demonstrated that the high con-

117

centration of sucrose has little influence on dimethyl labeling because it does not contain an amino

118

group and will not alter the pH of reaction solution (Table S7).

119

Protein digestion using trypsin or chymotrypsin. The labeled PSII complex was digested by

120

trypsin or chymotrypsin. Briefly, the proteins were denatured by 6 M guanidine, and then reduced

121

by 10 mM dithiothreitol (DTT) at 60 °C for 1 h, followed by alkylation with 20 mM iodoacetam-

122

ide (IAA) in darkness at 25 °C for 40 min. The denatured proteins were digested by trypsin (at

123

37 °C) or chymotrypsin (at 30 °C) overnight with an enzyme to substrate ratio of 1/25 (w/w), and

124

the resulted peptides mixtures were purified by a C18 solid-phase extraction column before

125

LC-MS/MS analysis.

126

LC-MS/MS analysis. All of the protein samples were separated using in-house packed capil-

127

lary column (75 µm, i.d., 15 cm length) packed with C18 particles (3 µm, 120 Å). The flow rate

128

was 300 nL/min. The solution of 0.1% (v/v) formic acid (FA)/H2O was used as mobile phase A,

129

and 0.1% (v/v) FA/acetonitrile (ACN) was used as mobile phase B. The reversed phase (RP) bi-

130

nary separation gradient was set as follows: from 1-10% solvent B in 2 min, 10-35% in 220 min,

131

35-90% in 2 min, after washing with 90% solvent B for 3 min, the system was equilibrated with


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100% solvent A for 13 min.

133

An LTQ-Orbitrap Velos (Thermo, San Jose, CA) or Q-Exactive mass spectrometer was utilized

134

for MS analyses. The MS parameters were set as follows: ion transfer capillary 250 °C, spray

135

voltage 1.8 kV, full MS scan from m/z 400 to 2000 with a resolution of 60000 in centroid mode.

136

Data-dependent MS/MS scans were performed by selecting the 20 most intense ions in the full MS

137

scan for collision induced dissociation (CID) with 35.0% normalized collision energy. The dy-

138

namic exclusion was set as follows: repeat count 1, repeat duration 30 s, and exclusion duration

139

120 s.

140

Data analysis. The sequences of cyanobacterial, red algal and spinach PSII complex were ob-

141

tained from UniProt (http://www.uniprot.org/). All of the *.raw files were converted to *.mgf files

142

by Protein Discovery (version 1.4). The generated *.mgf files were searched against the PSII da-

143

tabase via Mascot (version 2.3.0). Cysteine (+57.021464 Da) was set as fixed modification. Di-

144

methylation of lysine and protein N-terminus (+32.056407 Da), oxidation of methionine

145

(+15.994915 Da), deamidation of asparagine and glutamine (+0.984016 Da), acetylation of

146

N-terminus (+42.010565 Da), phosphorylation of threonine, threonine and tyrosine (+79.96633 Da)

147

and N-formylation (+27.994915 Da) were set as variable modifications. Peptides were processed

148

using fully trypsin or chymotrypsin cleavage and up to 3 (trypsin) or 5 (chymotrypsin) missed

149

cleavage sites were allowed. Peptide mass tolerance was 20 ppm, and fragment mass tolerance

150

was 0.8 Da (if LTQ used) or 0.05 Da (if Orbitrap used). Peptides having Mascot score ≥ 20 (rank 1,

151

P ≤ 0.05) were retained for further analyses.

152

The amino acid sequences of PSII complex of cyanobacteria, red algae and spinach were com-

153

pared by using Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) to identify conserved ly-

154

sines among these three species. The dimethylated lysines within conserved sequences among cy-

155

anobacteria, red algae and spinach were mapped onto the existing cyanobacterial PSII crystal

156

structure27 by using PYMOL (The PyMOL Molecular Graphics System, v1.3).

157 158

RESULTS

159

Examining the proximal microenvironments of lysines during subtle protein conformation

160

change. To assess the efficiency of our MS-based active labeling strategy in probing the proximal

161

microenvironments of lysines, we firstly utilized myoglobin as a model protein for examining the

162

subtle conformational changes. Holo-myoglobin is a globular protein with an α-helical fold28, and

163

apo-myoglobin is generated upon the dissociation of heme prosthetic group. According to the

164

NMR data, the majority of apo-myoglobin adopts a well deﬁned structure similar to ho-

165

lo-myoglobin, except that loop EF, helix F and loop FG become disordered29. We prepared apo-

166

and holo-myoglobin as described by Teale et al.21, and performed the dimethyl labeling with 2

167

mM labeling reagents for 30 min at 25 °C. The concentration of the labeling reagents, reaction

168

time and reaction temperature were optimized for slowing down the reaction speed, as all of the

169

lysines are relatively easy to be accessed and fully labeled for myoglobin due to relatively simple



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170

structure (Figure S1). As a result, the covalent labeling levels of most lysines within holo- and

171

apo-myoglobin were highly consistent (Table S1), which is in agreement with the fact that the

172

majority of protein structure remains unchanged and the distribution of all lysines identified was

173

shown in Figure 2a. A subset of the lysines also exhibited different labeling levels between these

174

two conformations. For example, the labeling level of Lys-46 was 49% in holo-myoglobin due to

175

the formation of a hydrogen bond with the carbonyl group of heme (2.5 Å, Figure 2b). In contrast,

176

this interaction does not exist after the removal of heme in apo-myoglobin, thus Lys-46 was nearly

177

fully labeled with a labeling level of 99% at the identical labeling conditions. Similarly, the dime-

178

thyl labeling level of Lys-78 was 69% in holo-myoglobin but 96% in apo-myoglobin as this resi-

179

due forms a hydrogen bond with the side chain carbonyl group of Glu-19 (2.9 Å, Figure 2c) within

180

holo-myoglobin, whereas this interaction is destroyed in apo-myoglobin as Lys-78 is located ex-

181

tremely close to loop EF30. In general, our MS-based dimethyl labeling strategy can feasibly probe

182

the lysine proximal microenvironments even in proteins only with subtle conformational changes.

183 184

Dimethyl labeling and MS analysis of spinach PSII complex. Photosystem II (PSII) is the

185

first membrane-pigment protein complex in the light-dependent reactions of photosynthesis, and

186

catalyzes the light-driven oxidation of water to O2 and reduces bound plastoquinones. The PSII

187

core complex is embedded in the thylakoid membranes mainly as a dimer from cyanobacteria to

188

higher plants, and each monomer consists of over 20 different subunits with the total molecular

189

weight about 350 kDa. We purified spinach PSII core complex with high oxygen-evolving activity

190

(about 1300 µmol O2 (mg of chlorophyll)-1h-1) as described before22. Then, the active covalent

191

labeling reaction was carried out at the optimized reaction condition of 5 mM labeling reagents for

192

30 min at 37 °C (pH 7.0), which was harder than the myoglobin labeling as the dimeric PSII com-

193

plex is much larger (~700 kDa) and more complicated. As a result, 40 lysines were highly labeled

194

with labeling levels over 85%, and 24 residues were partially labeled with relative lower labeling

195

levels (Table S2). Most of the partially labeled lysines were located in PsbB and some parts of

196

PsbO, and almost all lysines located in PsbP and PsbQ were nearly fully labeled, which might im-

197

ply that the PsbP and PsbQ are located at the outside of PSII and much easier to be accessed and

198

labeled than PsbO and PsbB. Further increasing the labeling reagents to 10 mM and labeling time

199

to 2 h led to elevated labeling (labeling level > 85%) of 100% lysines (Table S3), yet high biolog-

200

ical activity (about 1000 µmol O2 (mg of chlorophyll)-1h-1) was still remained for this highly di-

201

methylated PSII complex.

202 203

Comparison of dimethyl labeling of lysines in cyanobacterial, red algal and spinach PSII

204

complexes. We further performed identical MS-based labeling analyses for the purified cyanobac-

205

terial and red algal PSII complexes at the optimized labeling conditions, and obtained 54 (19 par-

206

tially and 35 highly) and 62 (28 partially and 34 highly) covalently labeled lysines for the cyano-

207

bacterial and red algal PSII, respectively (Tables S4 and S5). The amino acid sequences of PSII


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208

complexes from cyanobacteria, red algae and spinach were compared by using Clustal W2. A total

209

of 121 lysines (the conserved lysines among different species were considered as the same one)

210

were detected from the three organisms with different labeling levels and 49 lysines within con-

211

served sequences in at least two species were obtained in our results, among which 18 lysines ex-

212

hibited significant difference in labeling levels (P < 0.05 in student ttest, Table S6). We further

213

obtained 72 PSII lysines within non-conserved sequences of the three species, including 21 par-

214

tially and 51 highly labeled ones (Table S6). For lysines existing only in cyanobacteria and red

215

algae, half of them were located in PsbU, PsbV and PsbQ’ subunits. For spinach, most of the

216

unique lysines were from PsbP and PsbQ, which only exist in spinach PSII complex.

217 218

Evaluating the proximal microenvironments of lysines based on the crystal structure. We

219

examined the proximal microenvironments of the 54 (19 partially and 35 highly dimethyl labeled)

220

cyanobacterial PSII lysines in the crystal structure. The labeling levels of most lysines (96%)

221

could be explained by their proximal microenvironments in crystal structure, where residues ex-

222

posed or closed to the exterior surfaces were highly labeled whereas those either buried or en-

223

gaged in intra/intermolecular interactions were partially labeled (Figure S2a and Table S6). Then,

224

the proximal microenvironments of the 5 lysines within highly conserved sequences among all of

225

the three species were examined in more detail. Briefly, the 4 partially labeled lysines were all

226

involved in intra/intermolecular interactions, forming either hydrogen bonds or electrostatic inter-

227

actions with their proximal units. The cyanobacterial PsbB-Lys-320 (red algal PsbB-Lys-321 and

228

spinach PsbB-Lys-321) was relatively difficult to be covalently labeled, with a labeling level of

229

only 13% in cyanobacteria, 19% in red algae and 32% in spinach (Table S6). This is consistent

230

with the location of this residue near the interaction interface of PsbB and PsbD (Figure 3a). The

231

side chain amino group of cyanobacterial PsbB-Lys-320 forms a hydrogen bond with

232

PsbB-Phe-362 (distance between N and O is 2.8 Å) and may also interact with PsbB-Met-358

233

(distance between N and S is 3.4 Å, Figure 3b). With these strong interactions, PsbB-Lys-320 is

234

not solvent exposed, thus preventing its reaction with labeling reagents, even at the elevated la-

235

beling condition (Table S3, the spinach PsbB-Lys-321 labeling level reached to 88% after 2 h la-

236

beling time with 10 mM labeling reagents). Similarly, cyanobacterial PsbB-Lys-307 (red algal

237

PsbB-Lys-308 and spinach PsbB-Lys-308) is embedded inside PsbB with its side chain amino

238

group forming hydrogen bonds with PsbB-Leu-173 and PsbB-Glu-265 (Figure 3c,d). Thus, the

239

labeling level of PsbB-Lys-307 was 47% in cyanobacteria, 13% in red algae and 0% in spinach

240

PSII complex, and the labeling level of cyanobacteria was significantly different from the other

241

two species (Table S6), which might imply structural difference in cyanobacteria. Cyanobacterial

242

PsbC-Lys-435 (red algal PsbC-Lys-442 and spinach PsbC-Lys-457) is located at the interaction

243

interface of PsbC and PsbD, forming a hydrogen bond with PsbD-Glu-217 (Figure 3e,f), and the

244

labeling level was 74% in cyanobacteria, 72% in red algae and 44% in spinach (Table S6). Fur-

245

thermore, the cyanobacterial PsbD-Lys-307 (red algal PsbD-Lys-316 and spinach PsbD-Lys-318)



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246

is located at the interaction interface of PsbA and PsbD. Due to the electrostatic interaction with

247

PsbA-Asp-61 (Figure 3g, h), PsbD-Lys-307 was partially labeled with labeling levels of 53% in

248

cyanobacteria, 55% in red algae and 61% in spinach, which exhibited consistent labeling level

249

among the three species (Table S6). We further examined the sequences around the proximal in-

250

teracting sites of these 4 lysines in cyanobacteria, red algae and spinach, and these sequences were

251

all highly conserved, except that the interaction between PsbC-Lys-435 and PsbD-Glu-217 in cy-

252

anobacteria was replaced by PsbC-Lys-457 and PsbC-Glu-456 in spinach (Table S6). Finally, for

253

cyanobacterial PsbO-Lys-158 (red algal PsbO-Lys-236 and spinach PsbO-Lys-243), although the

254

hydrogen bond was observed between the lysine with its proximal units, it was located near the

255

protein complex outer surface and easily to be accessed and labeled (Figure 3i, j). Thus it was ful-

256

ly labeled in both cyanobacteria and spinach (Table S6). Our results suggested that the local struc-

257

tures of most of the sequence conserved lysines and their proximal microenvironments are highly

258

conserved between different PSII complexes, while some structural differences were also probed

259

(supporting information text).

260 261

DISSCUSION

262

MS is an excellent tool for investigating the protein structures and protein-protein

263

interactions31-33. Recently, the crosslinking mass spectrometry analyses (XL-MS) revealed the lo-

264

cation of the PsbQ subunit in cyanobacterial PSII complex34, and generated evidence for multiple

265

interactions between PsbP and PsbQ subunits in spinach PSII complex35,36. Although XL-MS is

266

extremely useful in providing information of protein structure and protein-protein interaction, the

267

MS datasets of crosslinked peptides are relatively difficult to interpret37, and the XL strategy pre-

268

fers to generate distance restraints of amino acid pairs locating onto the external surfaces. Probing

269

the lysine proximal microenvironments in their active states is important for understanding the

270

local structures of macromolecular proteins and protein complexes. Covalent labeling of lysines

271

by using N-acetyl-succinimide, sulfosuccinimidyl acetate and S-methylthioacetimidate were al-

272

ready reported for protein structure analyses, but the proteins charge states and biological activity

273

were usually altered by these reactions14. We combined the active dimethyl labeling with MS to

274

systematically probe whether the lysines within the macromolecular membrane protein complexes

275

are free or in interactions with other proximal units. Compared with the other MS-based protein

276

structure investigating methods, the dimethyl labeling analysis strategy exhibits advantages of

277

high labeling efficiency and stability (compared with HDX-MS) and reservation of the active

278

charge states as well as biological activity of the labeled proteins (compared with other covalent

279

labeling strategies like acetylation). We have checked the biological activity of spinach PSII com-

280

plex after elevated dimethylation of all lysines (100% lysines with labeling level > 85%), and

281

found that high activity (about 1000 µmol O2 (mg of chlorophyll)-1h-1, 77% of the original activity)

282

was still remained.

283

Our MS-based labeling analysis of PSII complex can systematically probe the proximal micro-


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284

environments of positively charged lysines under nearly native condition. The lysines with con-

285

sistent covalent labeling levels among cyanobacteria, red algae and spinach were mainly involved

286

in the region of PSII membrane lumen-side surface and located onto the interaction interfaces of

287

core subunits (PsbB, PsbC, PsbD and PsbO, Figure 4 and Table S6), which is also the core region

288

for oxygen evolution. Our results therefore suggested that this region may have been conserved

289

during evolution due to its important role in maintaining the PSII function. In addition, PsbB,

290

PsbC and PsbD are intrinsic subunits in PSII complex, and PsbO acts as an organizational tem-

291

plate for the PSII reaction center38. Taking PsbB as an example, most of the lysines were validated

292

in crystal microenvironments are highly conserved from prokaryotic to eukaryotic organisms

293

(Figure S3). However, the local structure within transmembrane helices could not be investigated

294

by dimethyl labeling in the structures investigated here due to the absence of lysines in these re-

295

gions of PSII complexes.

296

Although we have achieved active covalent labeling of detergent solubilized PSII complexes

297

through dimethyl labeling, performing the reaction on the membrane protein complex embedded

298

in the biological membrane prior to isolation is a future direction. Further, besides the pro-

299

tein-protein interactions, the essential interactions among other types of biomolecules, such as the

300

interactions of lysines with lipids5, should be also investigated in the future by MS-based

301

high-throughput strategies.

302 303

CONCLUSION

304

In this study, dimethyl labeling was performed in nearly native condition with high efficiency to

305

keep the active structure and biological activity of labeled proteins and protein complexes. Our

306

results provided important insights in the active statuses of lysine proximal microenvironments as

307

well as the conserved and non-conserved local structures among cyanobacterial, red algal and

308

higher plants PSII complexes. To the best of our knowledge, this is the first time that active lysine

309

proximal microenvironments within macromolecular membrane protein complexes could be sys-

310

tematically probed in high-throughput via the MS-based labeling analyses.

311 312

ASSOCIATED CONTENT

313

Supporting Information

314

Additional information as noted in text. This material is available free of charge via the Internet at

315

http://pubs.acs.org.

316 317

AUTHOR INFORMATION

318

Corresponding Authors

319

*Tel: +86-411-84379576. Fax: +86-411-84379620. E-mail: [email protected].

320

Notes

321

The authors declare no competing financial interest



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322 323

ACKNOWLEDGEMENTS

324

This work was supported by the China State Key Basic Research Program Grant

325

(2013CB911203), the China State Key Research Grant (2016YFA0501402), the National Natural

326

Science Foundation of China (21675152, 21535008, 21305139 and 21573223), the Youth

327

Innovation Promotion Association CAS (2014164), and the Key Research Program of the Chinese

328

Academy of Sciences (Grant No. KGZD-EW-T05).

329 330

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383

Figure legends

384 385 386

Figure 1. The schematic diagram of the active dimethyl labeling of intact protein or protein com-

387

plex. The red and blue arrows indicate labeled and unlabeled lysines, respectively.

388 389 390

Figure 2. (a) All lysines identified in holo-myoglobin were colored by labeling levels accordingly

391

(Green 30% - 40%, Chartreuse 40% - 50%, Lemon 50% - 60%, Yellow 60% - 70%, Orange 70% -

392

80%, Warm red 80% - 90%, Red 90% - 100%). The proximal microenvironments of Lys-46 (b)

393

and Lys-78 (c) in holo-myoglobin, which are consistent with the active labeling levels. The image

394

was made using PDB file 1YMB.

395 396 397

Figure 3. The proximal microenvironments of cyanobacterial PsbB-Lys-320 (a and b),

398

PsbB-Lys-307 (c and d), PsbC-Lys-435 (e and f), PsbD-Lys-307 (g and h) and PsbO-Lys-158 (i

399

and j) in crystal structure. The image was prepared from the cyanobacterial PSII structure PDB

400

file 3WU2. PsbA, PsbB, PsbC, PsbD and PsbO are in orange, firebrick, marine, deep teal and ma-

401

gentas, respectively.

402 403 404

Figure 4. The lysines with consistent covalent labeling levels among cyanobacteria, red algae and

405

spinach were mainly involved in the region of PSII membrane lumen-side surface and located on-

406

to the interaction interfaces of core subunits (PsbB, PsbC, PsbD and PsbO). The image was pre-

407

pared from the cyanobacterial PSII structure PDB file 3WU2. PsbB, PsbC, PsbD and PsbO are in

408

firebrick, marine, deep teal and magentas, respectively. The lysines were colored by labeling lev-

409

els accordingly (Blue 0 - 10%, Slate 10% - 20%, Cyan 20% - 30%, Green 30% - 40%, Chartreuse

410

40% - 50%, Lemon 50% - 60%, Yellow 60% - 70%, Orange 70% - 80%, Warm red 80% - 90%,

411

Red 90% - 100%)..


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Figure 1

413 414

415



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Figure 2

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Figure 3

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Figure 4

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Table of Contents graphic (TOC)

429 430

431



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Figure 1. The schematic diagram of the active dimethyl labeling of intact protein or protein complex. The red and blue arrows indicate labeled and unlabeled lysines, respectively. 80x48mm (300 x 300 DPI)


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Figure 2. (a) All lysines identified in holo-myoglobin were colored by labeling levels accordingly (Green 30% - 40%, Chartreuse 40% - 50%, Lemon 50% - 60%, Yellow 60% - 70%, Orange 70% - 80%, Warm red 80% - 90%, Red 90% - 100%). The proximal microenvironments of Lys-46 (b) and Lys-78 (c) in holo-myoglobin, which are consistent with the active labeling levels. The image was made using PDB file 1YMB. 104x56mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. The proximal microenvironments of cyanobacterial PsbB-Lys-320 (a and b), PsbB-Lys-307 (c and d), PsbC-Lys-435 (e and f), PsbD-Lys-307 (g and h) and PsbO-Lys-158 (i and j) in crystal structure. The image was prepared from the cyanobacterial PSII structure PDB file 3WU2. PsbA, PsbB, PsbC, PsbD and PsbO are in orange, firebrick, marine, deep teal and magentas, respectively. 105x157mm (300 x 300 DPI)


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Figure 4. The lysines with consistent covalent labeling levels among cyanobacteria, red algae and spinach were mainly involved in the region of PSII membrane lumen-side surface and located onto the interaction interfaces of core subunits (PsbB, PsbC, PsbD and PsbO). The image was prepared from the cyanobacterial PSII structure PDB file 3WU2. PsbB, PsbC, PsbD and PsbO are in fire-brick, marine, deep teal and magentas, respectively. The lysines were colored by labeling levels accordingly (Blue 0 - 10%, Slate 10% - 20%, Cyan 20% - 30%, Green 30% - 40%, Chartreuse 40% - 50%, Lemon 50% - 60%, Yellow 60% - 70%, Orange 70% - 80%, Warm red 80% - 90%, Red 90% - 100%). 82x40mm (300 x 300 DPI)



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Table of Contents graphic 77x46mm (300 x 300 DPI)


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Probing the Lysine Proximal Microenvironments within Membrane

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