Modulation and Salt-Induced Reverse Modulation ... - ACS Publications

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Modulation and Salt-induced Reverse-Modulation of the Excited State Proton Transfer Process of Lysozymized Pyranine : The Contrasting Scenario of the Ground State Acid-Base Equilibrium of the Photoacid Ishita Das, Sudipta Panja, and Mintu Halder J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 29 Jun 2016 Downloaded from http://pubs.acs.org on June 29, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

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

The Journal of Physical Chemistry

1

Modulation and Salt-induced Reverse-Modulation of the Excited State Proton Transfer

2

Process of Lysozymized Pyranine : The Contrasting Scenario of the Ground State Acid-

3

Base Equilibrium of the Photoacid

4

Ishita Das, Sudipta Panja, and Mintu Halder*

5

Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

6

*Corresponding Author: E-mail: [email protected]

7

Phone: +91-3222-283314, FAX: +91-3222-282252

8 9 10

Abstract

11

Here we report on the excited state behavior in terms of Excited State Proton Transfer

12

(ESPT) reaction as well as ground state acid-base property of Pyranine [8-hydroxypyranine-

13

1,3,6-trisulfonate (HPTS)] in presence of an enzymatic protein, human lysozyme (LYZ).

14

HPTS forms a 1:1 ground state complex with LYZ having the binding constant, KBH =

15

(1.4±0.05)×104 M-1 , and its acid-base equilibrium gets shifted toward the deprotonated

16

conjugate base (RO-), resulting in a downward shift in pKa. This suggests that the conjugate

17

base (RO-) is thermodynamically more favored over the protonated (ROH) species inside

18

lysozyme matrix, resulting in an increased population of the deprotonated form. But on the

19

other hand, for the release of the proton from the excited photoacid, interestingly the rate of

20

proton transfer gets slowed down due to ‘slow’ acceptor biological water molecules present

21

in the immediate vicinity of the fluorophore binding-region inside the protein. The observed

22

ESPT time constants, ~140 ps and ~750 ps, of protein-bound pyranine are slower than in bulk

23

aqueous media (~100 ps, single exponential). Molecular docking study predicts that the most

24

probable binding location of the fluorophore is in a region near to the active site of the

25

protein. Here we also report on the effect of external electrolyte (NaCl) on the reverse

26

modulation of ground state prototropy as well as ESPT process of the protein-bound

27

pyranine. It is found that there is a dominant role of electrostatic forces in the HPTS-LYZ

28

interaction process, since increase in ionic strength by the addition of NaCl dislodges the

29

fluorophore from the protein pocket to the bulk again. The study shows a considerable

30

different perspective of the perturbation offered by the model macromolecular host used

31

unlike the available literature reports on the concerned photoacid.

ACS Paragon Plus Environment


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

32

1. Introduction

33

Proton transfer reactions are fundamental and play a key role in various chemical and

34

biological processes 1-9. Intermolecular proton transfer in the excited state (ESPT) has drawn

35

great attention and has been extensively studied to extract information about the mechanism

36

and several parameters that control such reactions. Biological processes like DNA damage

37

due to disordering of base pair in presence of UV radiation, some enzymatic reactions,

38

reactions at photosynthetic centres etc. are induced by ESPT10,11. Molecules, having enhanced

39

acidity in the excited electronic state consequently undergo proton transfer upon

40

photoexcitation, are known as photoacids12. There are many photoacids including fluorescent

41

dyes and some drug molecules which are known to exhibit intermolecular or intramolecular

42

proton transfer. Such systems have been explored to gain information about their acid-base

43

reaction, ultrafast photodynamics in excited state, and effect of their confinement in different

44

chemical or biological environments12-14. Pyranine (8-hydroxypyranine-1,3,6-trisulfonate,

45

HPTS, Figure 1a) is also a photoacid and upon photoexcitation, its pKa downshifts from ~7.7

46

to ~0.6. ESPT reaction of HPTS has already been studied in several confined assemblies like

47

micelles, reverse micelles, liposomes, proteins, cyclodextrins, ionic liquids, and more9, 15-23.

48

Those studies reveal that proton-transfer rate of HPTS gets affected because of different

49

environmental effects offered by various host systems, and the excited state behavior of the

50

fluorophore has been exploited to probe the environment and local territory of host systems.

51

Hence considering the important role of proton-transfer process in several biological

52

processes and to probe the local environment that modulates proton-transfer processes, the

53

study of ESPT reactions in protein environments appears to be quite implicative. Besides

54

being a model photoacid, HPTS is also used as an intracellular pH indicator. Intracellular pH

55

plays many critical roles in cell, enzyme, and tissue activities, including proliferation and

56

apoptosis24,

57

contraction30, 31. Monitoring of pH alterations inside living cells is also important for studying

58

cellular internalization pathways, such as phagocytosis32, endocytosis33, and receptor ligand

59

internalization34.

25

, multidrug resistance26, ion transport27-29, endocytosis25, and muscle

60

HPTS has two distinct absorption maxima corresponding to the two different

61

protomers (Scheme 1), one centered at 404 nm for the protonated form and the other at 454

62

nm due to the deprotonated conjugate base. With increase of pH, absorbances at those two

63

wavelengths, decreases and increases, respectively, and this alteration is utilized (absorbance

64

ratiometric approach) to measure intracellular pH35. In this case the Henderson–Hasselbalch


Page 2 of 33

Page 3 of 33

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


36

65

equation

can be employed, which utilizes the ground-state pKa of the probe. If interaction

66

of the probe with any biomolecule affects the absorbance ratio of the two protomers, an

67

apparent pKa is obtained which will be different from the actual value. Hence, the knowledge

68

of the apparent pKa in presence of any biomolecule will be essential for the estimation of the

69

actual pH of the environment. In most of the available studies on ESPT process of HPTS in

70

several supramolecular assemblies, the fate of the ground state acid-base equilibrium of the

71

fluorophore was either not accounted or very little change was noticed.

72

In a recent study on ESPT reaction of HPTS with human serum albumin (HSA), it

73

was found that the ground state acid-base equilibrium of the photoacid shifts toward the

74

protonated form, and in the excited state the proton transfer occurs to biological water

75

molecules, gets slowed down inside the protein pocket23. It was suggested that, inside the

76

HSA pocket, HPTS binds in the subdomain IIIA which is known to bind ligands

77

predominantly by dipole-dipole, van der Waals, and hydrogen-bonding interactions. The

78

older literature reports suggest that the lower the pKa* (as well as the pKa), the faster the

79

ESPT rate would be37. In this work, we have chosen a model antibacterial enzymatic protein

80

human lysozyme (LYZ, Figure 1b), which is smaller than the serum protein and has only one

81

water exposed ligand binding catalytic active site. We intend to study how this protein affects

82

the ground state acid-base equilibrium as well as excited state proton transfer process of the

83

model photoacid pyranine.

84

Lysozyme is an enzyme found in various tissues and in some protecting secretions

85

like tears, saliva, mucus, milk etc. It can damage bacterial cell wall by cleaving the β-

86

glycosidic

linkage

between

N-acetylmuramic

acid

and

N-acetylglucosamine

in

38-40

87

peptidoglycan, and thereby protecting against bacterial infections

. It is a single chain

88

globular protein consisting of 130 amino acid residues. It has isoelectric point at pH ~11.35.

89

The active site of the protein has a crevice that separates the protein into two domains

90

connected by an α-helix. One domain consists mostly of β-sheet conformations (40-80 amino

91

acids), whereas the other domain (89-99 amino acids) is more α-helical in nature41. Lysozyme

92

has also been chosen as a model protein to study and understand folding and dynamics,

93

structure-function relationships, and ligand-protein interactions due to its small size, high

94

stability, natural abundance42,

95

endogenous and exogenous compounds including some important drug molecules44.

43

. Lysozyme is capable of binding reversibly a number of

96

Besides the studies involving confinement of the photoacids in different host

97

macromolecular systems, there are reports on ESPT process of HPTS in the presence of



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

Page 4 of 33

98

electrolytes only or in presence of acetate base17, 45. Leiderman et al.45 showed that at a high

99

concentration of salt MgCl2 or NaCl (~5 M), proton-transfer rate gets slowed down, whereas

100

at concentration below 0.5 M of MgCl2 and 0.8 M of NaCl, there was no alteration in proton-

101

transfer rate. A recent report shows that ESPT rate of pyranine slows down in niosome

102

compared to bulk water, and in presence of NaCl (in concentration range 1 M to 4 M) a

103

further decrease in proton-transfer rate was observed22. However, there are a very limited

104

number of reports on the role of any external additive that could further affect ESPT process

105

of the confined photoacid. It is well known that ionic strength of medium is a parameter that

106

sometime can act as a modulator of protein-ligand complexation process affecting the binding

107

interaction46,

108

electrolyte, like NaCl. So, presence of NaCl may affect the HPTS-LYZ binding, which in

109

turn may further modify ESPT process of the photoacid in the complex.

47

. Ionic strength of meduim could be varied by adition of an external

110 (a)

(b)

111 112 113 114 115 116 117

Figure 1 (a) Molecular structure of pyranine, and (b) 3D representation of lysozyme

118 119 120 121

pKa ~ 7.7, pKa* ~ 0.6 +

H2O

+

H3O+

122 123 124

Scheme 1. Acid-base equilibrium of pyranine in water

125 126

Therefore, in the present study, we investigated the ground state prototropy and the

127

excited state proton-transfer behavior of HPTS in the presence of LYZ. We have also

128

examined the role of NaCl as an external addititive on the further modification of ground

129

state and excited state behavior of lysozymized HPTS. In order to find the most probable

130

binding location of HPTS in the protein, molecular docking study was performed. Circular


Page 5 of 33

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


131

dichroism spectral study was monitored to investigate if there is any conformational

132

alteration of LYZ upon binding the fluorophore.

133 134

2. Experimental section

135

2.1. Materials

136

Human lysozyme (>90%, recombinant, expressed in rice), HPTS were purchased

137

from Sigma-Aldrich. Other chemicals are of analytical grade (AR). Buffer solutions of 0.01

138

M phosphate buffer in the pH range 5.2 to 8.7 were used for pKa estimation. All other

139

experiments were carried out in 0.01 M phosphate buffer solution of pH 7.0. Ultrapure water

140

was used for all solution preparation. The pH of buffer solutions were measured with

141

EUTECH pH 510 ion pH-meter.

142 143

2.2. Instrumentation and methods

144

UV−Vis absorption spectra were recorded on a Shimadzu UV-2450 absorption

145

spectrophotometer against a solvent blank reference in the wavelength range of 350−550 nm.

146

The concentration of HPTS used was 1×10-5 M.

147

All steady-state fluorescence emission and steady-state emission anisotropy

148

measurements were taken on a Jobin Yvon-Spex Fluorolog-3 spectrofluorometer, using 1 cm

149

path length quartz cuvette. HPTS concentration kept at 2.5×10-6 M for all the fluorescence

150

measurements. Excitation of HPTS was done at wavelength of 380 nm. Emission spectra

151

were collected from 395 to 650 nm, keeping excitation slit at 1.5 nm and emission slit at 1

152

nm.

153

Time resolved fluorescence intensity decays were collected by using a time-correlated

154

single-photon counting picosecond spectrophotometer (LifeSpec II, Edinburgh Instruments,

155

U.K.). Samples were excited by a pulsed laser (EPL-375) centered at 375 nm, and emission

156

signals were collected at magic angle (54.7˚), using a photomultiplier tube (H10720-01

157

Photosensor module from Hammamatsu). The laser repetition rate was kept at 5 MHz, and

158

excitation peak power was ~70 mW48. The instrument response function (IRF) was ~220 ps.

159

The decay analyses were performed by using F 900 software from Edinburgh Instruments49.

160

The

161

r ( t ) = ( I ( t ) − G I ⊥ ( t )) ( I + 2 G I ⊥ ( t ))

162

at parallel ( I (t ) ) and perpendicular ( I ⊥ (t ) ) polarizations of the emission with respect to the

time-resolved

anisotropy

was

constructed

using

the

expression,

where G is the ratio between the fluorescence intensity



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

Page 6 of 33

163

excitation beam. The value of G was measured at a gating window, in which the fluorescence

164

is almost completely depolarized (tail-matching technique). The quality of the fits was judged

165

in terms of the residual distribution and reduced χ2 values. All experiments were performed at

166

298K.

167

Time resolved area normalized emission spectra (TRANES) were constructed through

168

the following steps50. (a) Fluorescence intensity decays [(I (λ,t)] were collected at emission

169

wavelengths in the range of 420 nm to 580 nm, in 5 nm interval. Then, each emission

170

intensity decay was fitted to a multi-exponential function as: I (λ , t ) = ∑α i (λ ) exp(−t / τ i (λ )) .

n

i =1

171

(b) Time resolved emission spectra (TRES) were constructed at different times (tk) using the

172

equation: I (λ , tk ) = I ss (λ ). ∑ α i (λ ) exp(−tk / τ i (λ )) ∑ α i (λ )τ i (λ ) . Here, Iss(λ) is the steady-

n i =1

n i =1

173

state emission intensity at wavelength λ. (c) TRANES is then generated by area

174

normalization of each TRES.

175 176

2.3 Molecular docking

177

Crystal structure of Human lysozyme (PDB ID: 1IX0)51 was taken from protein data

178

bank for docking study. To get the software specific files, polar hydrogens were added and

179

water molecules were removed from the PDB file. The tri-negative protonated form of HPTS

180

was optimized by PM3 prescription using MOPAC 200252. That optimized structure of HPTS

181

was docked onto 3D crystal structure of LYZ using AutoDock 4.253, which uses Lamarckian

182

genetic algorithm to search optimum binding site of small molecules in protein. AutoDock

183

reports a docked energy that includes a solvation-free energy term and intermolecular

184

interaction energy term of the ligand. Blind docking of HPTS was run in LYZ crystal. For

185

performing docking analysis the grid centre was set at 60, 60, and 60 along X-, Y- and Z-

186

axes, respectively, with a grid spacing of 0.375 Å. The lowest energy conformation, out of 50

187

conformations (from 40 runs) was taken as the most preferred binding position of HPTS in

188

LYZ. Uncomplexed protein and HPTS docked conformation of lowest energy were used for

189

calculation of solvent accessible surface area (SASA) using the Discovery Studio Visualizer

190

4 from Accelrys Software Inc.54. The change in SASA for residue is estimated as ∆SASA =

191

SASALYZ - SASALYZ–HPTS.

192 193


Page 7 of 33

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

194


2.4. Circular Dichroism spectra

195

Circular dichroism (CD) spectra were recorded on a Jasco-810 automatic recording

196

spectropolarimeter at 298 K over a wavelength range of 200–250 nm with a scan speed 50

197

nm/min under constant nitrogen flushing. A quartz cell having path length 0.1 cm was used

198

and two successive scans were accumulated for each spectrum. Baseline was corrected with

199

buffer solution running under the same condition as blank and subtracted from the

200

experimental spectra. The concentration of LYZ was taken as 20 µM.

201 202

3. Results

203 204

3.1. Steady-state spectra in lysozyme, and in lysozyme with added NaCl

205

UV-Vis absorption study:

206

In aqueous buffer of pH 7, the UV-Vis spectrum of HPTS shows an intense absorbance

207

maximum at 403 nm due to the protonated form (ROH) and another weaker band at 454 nm

208

for its deprotonated form (RO- ). Addition of LYZ shifts the ground state prototropic

209

equilibrium of HPTS toward the deprotonated form, since the absorbance at 403 nm

210

decreases and that at 454 nm increases (Figure 2a). The increased population of RO- in

211

presence of LYZ suggests a preference for the deprotonated form in the ground state which

212

results in an altered pKa of HPTS. In order to estimate ground state pKa value of HPTS in the

213

absence and in presence of LYZ, absorbance monotored at 454 nm in different buffered

214

solutions were plotted as a function of pH of the medium (Figure 2b). The ground state pKa of

215

HPTS was estimated to be (7.30±0.04) in only aqueous media, that matches quite well with

216

literature reported value55 and a pKa value (6.90±0.06) was estimated in the presence of LYZ,

217

i.e., pKa of HPTS has been shifted by ~0.4 unit in the presence of the protein. Approximate

218

pKa* values of HPTS in the absence and in presence of LYZ were also estimated from the

219

Förster cycle56 and are tabulated in Table T1, Supporting information. The shift in pKa* of

220

HPTS in the presence of LYZ was found to be same as the shift in pKa (∆pKa*~0.4).

221

Absorbance spectra in different pH in the absence and presence of lysozyme are also

222

provided in Supporting information, Figure S1.

223

Ground state acid-base behavior of HPTS in the presence of lysozyme was studied

224

upon addition of salt (NaCl). It was found that with the maximum NaCl concentration used

225

here (0.2 M), the acid-base equilibrium of HPTS shifts slightly toward the deprotonated form



and a very small downward shift in pKa (by ~0.15 unit) was obtained (the measured pKa was

227

(7.15±0.07)). The addition of 0.2 M NaCl with the HPTS in the presence of LYZ changes the

228

pKa from (6.90±0.06) to (7.15±0.08), which matches the value obtained for HPTS-only in the

229

presence of the same concentration of NaCl. The pKa values and relative shift in pKa in

230

different systems along with pKa* (with respect to aqueous medium) are listed in Table T1,

231

Supporting information. The corresponding graphical plots for the determination of pKa are

232

shown in the Supporting information, Figure S2. The observed reversed behavior of ground

233

state acid-base equilibrium of HPTS upon salt addition to HPTS-LYZ system indicates that

234

the presence of salt most likely dislodges HPTS from lysozyme-pocket.

236

238 239

0Μ

0.01

[LYZ]

(A-A0)

237

(a)

0.00

−4

1.4 ×10 Μ

-0.01

-5

0.0

5.0x10

-4

1.0x10

[LYZ] (M)

0.06

−4

1.4 ×10 Μ

[LYZ] 0Μ

0.00 400

242

At 40 3n m

0.12

240 241

0.08

450

Absorbance at λ = 454 nm

0.18

At 45 4

235

nm

226

Absorbance

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

Page 8 of 33

(b)

In absence of LYZ In presence of LYZ

0.06

0.04

0.02

∆pKa~0.4

0.00 500

5.5

6.0

Wavelength (nm)

6.5

7.0

7.5

8.0

8.5

9.0

pH

243

Figure 2. (a) Absorbance spectra of HPTS in absence and in presence of increasing lysozyme

244

concentration in aqueous phosphate buffer of pH 7. Inset shows change in absorbance (A-A0)

245

upon addition of lysozyme monitored at 403 nm and 454 nm. (b) Determination of pKa of

246

HPTS: Absorbance at 454 nm is plotted as a function of pH of the medium, in the absence

247

and in presence of lysozyme, [LYZ] =1.0 ×10-4 M.

248

Emission spectral study:

249

For a photoacid, the steady state emission spectra can provide a good qualitative

250

account of the ESPT process. In aqueous buffer of pH 7, fluorescence emission spectrum of

251

HPTS shows an intense band at 510 nm due to the deprotonated species (RO-*) in the excited

252

state and a very weak band at 437 nm from the photo-excited protonated species (ROH*)

253

(Figure 3). In aqueous phosphate buffer, the emission intensity of ROH* is very weak

254

compared to RO-* (IROH* /IRO-* is ~0.03, displayed in Figure 4b, Table 1) because of ESPT

255

reaction. Successive addition of LYZ to the aqueous buffered solution of HPTS increases the

256

emission intensity at ROH* with a concomitant decrease in intensity at the RO-*


Page 9 of 33

257

accompanying an iso-emissive point at 470 nm (Figure 3). In 2.4×10-4 M LYZ, when almost

258

saturation of such inter-conversion is observed, a ~3.5 fold increase in IROH* /IRO-* (~0.10,

259

Figure 4b) was obtained (the magnitude of IROH* /IRO-* as a function of lysozyme

260

concentration is provided in the Supporting information, Figure S3). The emission spectral

261

profile in the presence of LYZ indicates shifting of the equilibrium between the two

262

concerned protomeric species toward the protonated form. This could have resulted due to

263

increase in population of the ROH exhibiting an upshift of both pKa and pKa*. But, contrarily,

264

it was observed in the UV-Vis spectral study that the pKa as well as pKa* are downshifted in

265

presence of LYZ resulting in some increased population of the RO form. Hence, the most

266

probable cause for this observed feature of emission spectra could be due to a progressive

267

slowing down of ESPT reaction rate with increasing lysozyme concentration as compared to

268

that in the neat aqueous buffer9, 22. A clear and quantitative picture could be obtained only

269

from the time-resolved studies, discussed later.

-

270 6

1.0x10

0M

271 272 273 274

Fluorescence Intensity (a.u.)

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


[LYZ] −4

2.4×10 M 5

5.0x10

275 276 277 278

−4

2.4×10 M [LYZ] 0M 0.0 400

470 nm 450

500

550

600

Wavelength (nm)

279

Figure 3. Emission spectra of HPTS in the absence and presence of increasing lysozyme

280

concentration, in aqueous phosphate buffer of pH 7. λexc = 380 nm.

281

Then, to examine whether salt has any effect on further modulation of the excited

282

state proton transfer process of HPTS in the presence of lysozyme, NaCl was varied in the

283

concentration range of 0.02 M to 0.2 M. No change was noticed in ESPT behavior of the

284

fluorophore in the presence of salt only, as evident from steady state emission spectral feature

285

and in ESPT time constant (Supporting information, Figure S4, S5, Table T2). Interestingly,

286

upon addition of salt in the HPTS-LYZ complex the ratio (IROH* /IRO−*) is found to decrease

287

from 0.10 (without NaCl) to ~0.035 (with 0.2 M NaCl), which is similar to that observed in

288

neat aqueous buffer only (IROH* /IRO−*is ~0.03) (Figure 4a, 4b; Table 1). The reversal of



289

emission spectral profile (Figure 4a) in increasing salt concentration also passes through an

290

iso-emissive point at 470 nm. Such reversal in steady state emission profile of LYZ-bound

291

HPTS with increase of ionic strength is also an indication of removal of the fluorophore from

292

the protein environment to the bulk water, and this naturally shows a bulk-like faster proton-

293

transfer rate again compared to the retarded proton-switching process in its protein-bound

294

state.

296 297 298 299 300

6

1.05x10

0.12

(a)

5

7.00x10

5

3.50x10

0.00 400

(b) (ii)

In aqueous buffer only With LYZ With LYZ + 0.02 N NaCl With LYZ + 0.05 N NaCl With LYZ + 0.1 N NaCl With LYZ + 0.2 N NaCl

IROH* / IRO-*

295

Fluorescence Intensity (a.u.)

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

Page 10 of 33

0.10

0.08

(iii)

0.06

(vii) 0.04

(i) (viii)

0.02 450

500

550

600

0.0

Wavelength (nm)

0.1

0.2

[NaCl] (M)

301 302

Figure 4. (a) Emission spectra of HPTS with 2.4×10-4 M lysozyme in increasing NaCl

303

concentration, in aqueous phosphate buffer of pH 7. (b) IROH* /IRO-* plotted against different

304

NaCl concentration: (i) in aqueous buffer only (0 M NaCl), (ii) in presence of 2.4×10-4 M

305

lysozyme (0 M NaCl), (iii)→(vii) in presence of 2.4×10-4 M lysozyme with increasing NaCl

306

(0.02 M to 0.2 M), and (viii) in 0.2 M NaCl only. λexc = 380 nm.

307 308

Estimation of binding constants:

309

For the estimation of the binding constant of HPTS-LYZ complex, the emission

310

intensity enhancement of ROH in presence of LYZ has been utilized. The binding constant

311

(KBH) of the complex has been estimated using the Benesi-Hildebrand equation (equation 1)57

312

(I∞ − I0 ) 1 = 1+ ........... (1) ( It − I0 ) K BH [ protein]n

313

Here, I0, It and I∞ are emission intensities of ROH (monitored at λem= 437 nm) in the absence

314

of LYZ, at any intermediate concentration of LYZ and at maximum LYZ concentration that

315

has been used here, respectively; and n is the number of binding sites. The plot of


(I∞ − I0 ) ( It − I0 )

Page 11 of 33

1 , using the Benesi-Hildebrand equation has been shown in Figure 5. The linear [ protein]n

316

vs.

317

nature of the plot (Figure 5) suggests a 1:1 stoichiometry (n = 1) of the complex. Binding

318

constant (KBH) estimated from the linear plot is found to be (1.4±0.05) ×104 M-1.

319

As observed from the fluorescence data that the presence of salt affects pyranine

320

binding to LYZ, the binding constants of HPTS-LYZ complex were determined in the

321

presence of different NaCl concentration also, using the above mentioned equation 1. The

322

estimated values of binding constants are listed in Table 2, which shows that the binding

323

constant decreases as salt concentration is increased. At 0.2 M NaCl, binding constant

324

reduces to ~2.5 fold from the initial value. This decrease in binding constant also suggests

325

that HPTS is getting dissociated from the complex and returns to the bulk water.

326

. 30

327 25

328

(I∞-I0) / (It-I0)

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


20

329

HPTS-LYZ + 0 M NaCl HPTS-LYZ + 0.02 M NaCl HPTS-LYZ + 0.1 M NaCl HPTS-LYZ + 0.2 M NaCl

15

330

10 5

331

0

332

0.0

4

4.0x10

4

8.0x10

5

1.2x10

5

1.6x10

-1

333

1/[LYZ] (M )

334

Figure 5. Benesi-Hildebrand plot for HPTS-LYZ complexation in the absence and in

335

presence of NaCl.

336

Fluorescence Excitation spectra:

337

The fluorescence excitation spectrum collected at 510 nm in aqueous buffer

338

(Supporting information, Figure S6) is found to be similar in shape to that of the absorption

339

spectrum, having maximum intensity at 403 nm (for the protonated form) along with a

340

weaker band at 454 nm (for the deprotonated form). Addition of LYZ increases the band

341

intensity at 454 nm with a concomitant decrease in intensity at 403 nm. This also

342

substantiates the aforementioned ground state equilibrium shifting toward the deprotonated

343

conjugate base, as was revealed from absorption spectral study in the presence of LYZ.

344

Steady-state anisotropy:



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

345

Steady-state anisotropy gives a quantitative measure of local fluidity in the immediate

346

vicinity of a fluorophore. Steady-state anisotropy of HPTS was monitored upon addition of

347

lysozyme in the absence and in presence of salt and they are highlighted in Figure 6.

348

Anisotropy (r) of the probe was found to increase gradually with lysozyme concentration

349

followed by saturation. The protein scaffold renders a motional restriction to the bound

350

fluorophore due to local environmental rigidity compared to bulk aqueous buffer medium.

351

Steady-state anisotropy of the fluorophore was measured upon the addition of lysozyme in

352

the presence of added 0.2 M NaCl also. It was found that in the presence of 0.2 M NaCl, the

353

increase in anisotropy (r) of the probe as a function of lysozyme concentration was almost

354

negligible compared to that observed in the absence of added salt. Hence, steady-state

355

anisotropy data also supports that the presence of salt impairs the aforementioned HPTS-LYZ

356

binding (in the presence of 0.2 M NaCl in aqueous buffer, anisotropy (r) of HPTS was almost

357

same to that found in aqueous buffer only). Steady-state anisotropy measurements can also

358

provide the binding constant of protein-ligand complexation process. The detailed method

359

and the representative plot (Figure S7) for binding constant estimation has been discussed in

360

the Supporting information under the section titled “Binding constant estimation from steady-

361

state anisotropy”. The binding constant (Kan) evaluated from anisotropy data for HPTS-LYZ

362

complex, was found to be (1.5±0.07)×104 M-1, and (7.6±0.05)×103 M-1; in absence and in

363

presence of salt, respectively. These binding constant values are also in good agreement with

364

those obtained from the Benesi-Hildebrand method (Table 2).

365 366 367 368 369 370 371 372 373 374


Page 12 of 33

Page 13 of 33

0.04

With LYZ only With LYZ + 0.2 M NaCl

375 376 377 378

Anisotropy (r)

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


0.03

0.02

379 0.01

380

0.0

5.0x10

-5

-4

1.0x10

-4

1.5x10

[LYZ] (M) 381

Figure 6. Variation of steady-state fluorescence anisotropy of HPTS with increasing

382

lysozyme concentration (in absence and in presence of NaCl) in aqueous buffer of pH 7;

383

emission collected at λem = 510 nm. λexc = 380 nm.

384 385

3.2 Time-resolved fluorescence emission studies

386

Time resolved emission studies provide a quantitative picture of proton-transfer

387

process in the excited state. Fluorescence decays of ROH* were recorded at 435 nm and those

388

of the RO-* were recorded at 520 nm in a picoseconds TCSPC setup. Figures 7a, 7b show

389

emission decays of HPTS at 435 nm (for ROH* emission) and at 520 nm (for RO-* emission),

390

respectively, in different systems. The ESPT rate of HPTS could be tracked either by

391

monitoring the decay time of the excited protonated form (ROH*) at its emission maximum

392

~435 nm, or the rise-time of the conjugate base (RO-*) emission at ~520 nm. The time

393

constants of multi-exponential fits of emission decays at both the observation wavelengths

394

(435 nm and 520 nm) are presented in Table 3. In aqueous buffer, the decay fits with a tri-

395

exponential function with time constants ~100 ps, ~550 ps and ~3.25 ns at 435 nm. The

396

shortest time constant (100 ps) has a contribution ~92%, the other picosecond component has

397

~7%, and the nanosecond component contributes to ~1%. At 520 nm, emission decay was

398

fitted by a bi-exponential function with two time constants, ~100 ps corresponding to a rising

399

component and ~5.5 ns, a decay component. This rise-time (~100 ps) at 520 nm correlates

400

with the decay component of 100 ps observed at 435 nm. Several earlier reports indicate

401

nearly same value (~90 ps) of proton-transfer time constant of HPTS in water9. Hence, the

402

100 ps rise-time could be attributed to the ESPT time constant of HPTS in aqueous buffer. As

403

reported by Cohen et al.23, a ~260 ps component has been assigned to the lifetime of ROH* in

404

0.1 M aqueous phosphate buffer of pH 7.0. Here the ~550 ps component at the ROH* form



405

could be assigned to the lifetime of ROH*, and ~5.5 ns component to the lifetime of RO-*, in

406

0.01 M aqueous phosphate buffer. The difference is presumably due to different buffer

407

strengths, as the presence of phosphate ions are known to affect the lifetimes of the species

408

under observation23. In aqueous phosphate buffer, excited state equilibrium (due to geminate

409

recombination) between the protonated and deprotonated forms is not observed because of

410

the phosphate anions available in low concentration in the system, can act as scavengers of

411

protons, thus affecting the recombination process without any effect on the proton-transfer

412

rate2, 58. Here also geminate recombination was not observed as evident from Time Resolved

413

Area Normalized Emission Spectra (TRANES) (Figure 8) which shows that the protonated

414

form is practically non-existent after ~500 ps (lifetime value assigned for ROH).

415 10.0k

417

8.0k

418

(a)

IRF In aqueous buffer only With LYZ With LYZ + 0.02 M NaCl With LYZ + 0.05 M NaCl With LYZ + 0.1 M NaCl With LYZ + 0.2 M NaCl

6.0k

10.00k

(b)

IRF In aqueous buffer only With LYZ With LYZ + 0.2 M NaCl

7.50k

Counts

416

Counts

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

Page 14 of 33

5.00k

419

4.0k

420

2.0k

421

0.0

2.50k

5

6

7

Time (ns)

8

9

5

6

7

8

9

10

Time (ns)

422 423

Figure 7. Fluorescence emission decay of HPTS in different conditions, in aqueous

424

phosphate buffer of pH 7. Decays collected at (a) 435 nm (for ROH*), and (b) at 520 nm (for

425

RO-*). λexc = 375 nm, [LYZ] =1.8×10-4 M.

426

In 1.8×10-4 M lysozyme, fluorescence decay at 435 nm and the rise obtained at 520

427

nm are found to be markedly slower compared to aqueous buffer. Emission decays of HPTS

428

are found to fit by three exponentials at both the observation wavelengths (435 nm and 520

429

nm). At 520 nm (due to RO-*), the transient is fitted by three exponential components

430

showing two different rise-times, and one decay time. Between the two time constants, the

431

shorter one comes at ~140 ps, and the other comes at ~780 ps. In pure aqueous buffer,

432

relative amplitude of the 100 ps rising component (emission monitored at 520 nm) was found

433

to be ~52%, with ~48% contribution from the 5.5 ns which is the lifetime of excited anionic

434

species. In presence of lysozyme, the tri-exponential fit of the decay collected at 520 nm,


Page 15 of 33

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


435

gives relative amplitudes of ~13% corresponding to the 140 ps component and ~9% for 780

436

ps component, and 78% of the RO-*. Both of the rise-times, in the presence of LYZ, are

437

slower compared to that in aqueous media suggesting a slower ESPT process of lysozymized

438

HPTS. In order to substantiate the findings of salt addition study from steady state emission

439

measurements and to get a quantitative picture in terms of proton-transfer time constants

440

upon salt addition to HPTS-LYZ system, further time resolved emission measurements were

441

attempted. The retarded decay (at 435 nm) and rise (at 520 nm) of HPTS, due to binding with

442

lysozyme, both turned faster with increasing salt concentration (Figure 7a, 7b). Here also the

443

emission transient at 520 nm is found to fit by tri-exponential decay function with two rise

444

components and one decay component. Now, the shorter 140 ps rise component, found in the

445

presence of LYZ, goes progressively faster with increasing NaCl concentration, and with 0.2

446

M NaCl it becomes ~99 ps which is close to that found in aqueous buffer media only, while

447

the other slower ~780 ps rise component survives with nearly same magnitude. In 0.2 M

448

NaCl, among the three components, the 99 ps component contributes to ~52%, the other

449

longer rising component contributes to ~2% and the decay component contributes to ~46%. If

450

we consider the relative weightage between the two growing components, it is found that in

451

the presence of lysozyme only, the 140 ps component has a contribution of ~58%, and the

452

other has ~42% weightage (Table 4). The addition of salt (increase of ionic strength) makes

453

the 140 ps time constant faster and its relative contribution also increases, while the relative

454

weightage of the longer component is found to reduce largely. In 0.2 M NaCl, relative

455

weightage of the shorter rising component is found to be ~95%, while the other one has a

456

very little contribution of only ~5%. TRANE spectra (Figure 8) also complements the

457

observed proton-transfer rates (or, formation rate of RO-*) in different systems as the intensity

458

ratio of ROH* and RO-* at any instant in different systems matches well the observed proton-

459

transfer time constant (Supporting information, T3). Hence, the results from time resolved

460

decays with increasing ionic strength also suggest that the probe comes out from the protein

461

pocket to bulk aqueous medium.

462 463 464 465 466 467 468



5

469 470 471 472 473 474

3x10

Fluorescence intensity (a.u.)

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

-*

(a)

0 ps 10 ps 20 ps 50 ps 100 ps 200 ps 500 ps 1 ns 5 ns 10 ns 15 ns

[RO ] 0 ps *

[ROH] 5

2x10

15 ns

5

1x10

0 420

(b)

-*

[RO ]

5

2x10

0 ps *

[ROH] 15 ns 5

1x10

15 ns

15 ns

480

510

Wavelength (nm)

-*

(c)

[RO ]

0 ps

5

3x10

540

570

0 420

450

480

510


*

[ROH] 15 ns 5

2x10

15 ns

5

1x10

0 ps

0 ps 450


Page 16 of 33

0 ps

540

570

Wavelength (nm)

0 420

450

480

510

540

Wavelength (nm)

475

Figure 8. Time resolved area normalized emission spectra of HPTS in (a) aqueous buffer

476

only (b) with 1.8×10-4 M LYZ (c) with 1.8×10-4 M LYZ + 0.2 M NaCl. λexc = 375 nm.

477

In an ESPT reaction, where proton is transferred to water molecules, the rate will

478

strongly depend on dynamics of the acceptor water molecules. The water molecules confined

479

in a supramolecular assembly like protein are a bit immobilized in nature and, moreover, the

480

probe attached to protein also experiences some specific or non-specific interactions with the

481

nearby charge-containing amino acid side-chains23. An earlier work on ESPT of confined

482

HPTS in γ-cyclodextrin reports about two proton-transfer time constants ~140 ps and ~1.4 ns

483

which are due to the change in properties of water in the vicinity of cyclodextrin cavity21.

484

Two different proton-transfer time constants of HPTS (250 ps and 2.4 ns) were reported in

485

P123-CTAC aggregate due to two different locations of dye, one that is a buried location

486

inside the hydrophobic core (2.4 ns component assigned) and other is a water exposed

487

location (250 ps component)59. Cohen et al. have reported proton-transfer time constant of

488

HPTS spanning from 150 fs to 1.22 ns while bound to human serum albumin23, where the

489

slower 1.22 ns decay was assigned to proton transfer to biological water molecules in the

490

vicinity of the protein. Considering the molecular structure and electrical charge properties of

491

the probe, they suggested that it binds primarily in the subdomain IIIA cavity of site II of

492

HSA. Here the small protein lysozyme has only one, a bit water exposed, binding cavity

493

which is its catalytic site and binds small molecules60. We investigated a most probable

494

binding location of the probe in LYZ by molecular docking study. The factors those are most

495

likely to be responsible in modulating the proton-transfer process in LYZ only and also upon

496

salt addition to lysozymized pyranine, have been addressed in discussion part.

497 498


570

Page 17 of 33

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

499


3.3. Time resolved anisotropy decay

500

Time resolved anisotropy measurement reports about the rigidity of the environment

501

around a probe. Rotational relaxation time of free probe molecule and probe molecule

502

confined to any macromolecular rigid environment differs considerably and it gives some

503

idea about rotational restriction imposed on the probe after its interaction with the host

504

macromolecule. In the aqueous buffer only, fluorescence anisotropy decay of HPTS (Figure

505

9) fits with a single exponential function with a rotational relaxation time constant of ~154

506

ps, similar values in aqueous medium has already been reported in the literature9. In presence

507

of 1.8×10-4 M LYZ, the emission anisotropy of HPTS becomes bi-exponential, with

508

rotational time constants ~199 ps (α1rot ~13%) and ~2.17 ns (α2rot ~87%). The longer

509

nanosecond component could be assigned to be due to highly restricted rotational motion of

510

HPTS inside the protein matrix, and the other picosecond component is probably due to some

511

contribution coming from loosely protein-bound HPTS.

512

In order to find how salt affects rotational relaxation time of the probe in the complex,

513

time resolved anisotropy measurements were monitored upon salt addition. Anisotropy decay

514

shows bi-exponential nature upto 0.05 M NaCl (Table 5). The contribution of the shorter

515

component increases with a concomitant decrease in the longer component indicating the

516

contribution of larger population of free HPTS in aqueous phase. The average rotational time

517

constant decreases with increasing salt concentration and at some higher NaCl concentration

518

anisotropy decay shows single-exponential behavior. A rotational time constant ~190 ps is

519

found in the presence of 0.2 M NaCl. This decrease in rotational time constant in the presence

520

of salt suggests that HPTS gets untied from the environmental rigidity of the protein. Hence,

521

transient anisotropy measurement in the presence of salt also suggests that HPTS gets

522

dislodged from the protein and returns to the bulk water, and this has already been

523

corroborated by steady state data.

524 525 526 527 528 529



530

0.25

531

0.20

532 533 534

Anisotropy, r (t)

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

In aqueous buffer only With LYZ With LYZ + 0.02 M NaCl With LYZ + 0.05 M NaCl With LYZ + 0.1 M NaCl With LYZ + 0.2 M NaCl

0.15

0.10

0.05

535 0.00 5

6

7

8

9

536

Time (ns)

537

Figure 9. Emission anisotropy decay of HPTS collected at 520 nm in different systems in

538

aqueous buffer of pH 7. λexc = 375 nm. [LYZ] =1.8×10-4 M.

539

3.4. Molecular docking study

540

In order to find a preferred location of HPTS (ligand) in the HPTS-LYZ complex, docking of

541

the fluorophore into 3D crystal structure of LYZ was modelled by molecular docking

542

simulation using Auto Dock 4.2. Several literatures are available regarding binding of

543

different ligands and antigens to the catalytic active site of LYZ61-63. The energetically

544

favorable docked pose of HPTS to LYZ is depicted in Figure 10. The ligand was found to be

545

docked near the substrate binding region of lysozyme and the amino acid residues in close

546

vicinity of docked HPTS (within 4 Å) are: Trp 34, Glu 35, Asn 44, Asp 53, Gln 58, Trp 109,

547

Val 110, Ala 111, Asn 114, and Arg 115. Considering the chemical nature of the amino acid

548

residues in close proximity of the ligand, these could interact with the ligand via different

549

modes as follows. The amino acid residues Asp 53, Asn 114, Arg 115, and Val 110 make

550

hydrogen-bond with the sulfonate groups of HPTS. Presence of some polar amino acid

551

residues like Gln 35, Asn 114,44 and Trp 34,109 can provide van der Waals interaction. The

552

Arg 115, having a positively charged side chain is expected to interact via electrostatic mode

553

with the negatively charged sulfonate groups of HPTS. The π-π interaction may also take

554

place between the aromatic ring of Trp 35, 109 and the pyrene moiety of HPTS. The Val 110

555

and Ala 111 can provide some hydrophobic interaction also. So overall, a combination of H-

556

bonding, van der Waals forces, π-π and electrostatic interactions are playing role to host the

557

ligand. Upon NaCl addition, mainly electrostatic interaction becomes feeble with some

558

possible weakening of van der Waals forces, π-π interaction, and hydrogen-bonding as well.

559

While hosting a ligand, some amino acid residues of the protein get involved in binding the

560

ligand through different kind of interaction modes as discussed and this results in a decrease


Page 18 of 33

Page 19 of 33

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


561

in solvent accessible surface area (SASA) of the interacting amino acid residues54. Here we

562

calculated the change in SASA values of the interacting nearby amino acid residues and the

563

data are tabulated in Table 6. There are noticeable changes in SASA values of participating

564

amino acid residues, suggesting substantial binding of the probe with LYZ.

565 566 567 568 569 570

Pyranine

571 572

Figure 10. Interacting amino acid residues around HPTS (within 4 Å) in binding region of

573

lysozyme.

574

3.5. Circular dichroism spectral study

575

Circular dichroism spectral study of a protein provides information about its

576

secondary structure. Any structural changes in the protein due to ligand binding could be

577

monitored from CD spectral behavior. CD spectra of LYZ shows two negative peaks at 208

578

nm and 222 nm characteristics of n→π* transition in amide bond of α-helix64.

579

Estimated α-helix content of LYZ was found to be 21.7% (The details regarding

580

corresponding equations for α-helix content calculation and the CD spectra (Figure S8) are

581

provided in Supporting information under the section titled “Circular dichroism spectra”).

582

Upon binding of HPTS, α-helix content slightly decreases to 19.97% at 1:1 protein-ligand

583

ratio. This decrease in α-helix content suggests an alteration of lysozyme secondary structure

584

upon HPTS binding. On adding NaCl (1.6 M) in the HPTS-LYZ complex, α-helix content

585

was found to be increased again to 20.9%, which indicates that the protein again recovers its

586

secondary structure to some extent.

587 588



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

589 590 591

4. Discussion The experimental results show that the ground state as well as the excited state behavior of HPTS is markedly modified upon binding with lysozyme.

592

As observed from UV-Vis absorption study, the deprotonated tetra-negative anion has

593

more preference over the protonated tri-negative anion for lysozyme causing a downshifted

594

ground state pKa of the protein-bound HPTS. If we consider the overall charge of the protein

595

(LYZ) at the experimental pH condition (pH 7) it has net positive charge (as isoelectric point

596

of LYZ is ~11.35) and that is why most likely it prefers the deprotonated anionic protomer

597

presumably on the electrostatic ground. The effect of salt on HPTS-LYZ complex in the

598

ground state as well as in excited state also suggest about a major role of electrostatic

599

interaction in the complexation process. While attempting for a local pH estimation using

600

HPTS (the intracellular pH indicator) utilizing the Henderson–Hasselbalch equation, the pKa

601

value corresponding to aqueous solution is utilized. But, interestingly it was found that the

602

presence of lysozyme alters the absorbance ratio of those two protomers of HPTS and

603

consequently gives a shifted pKa. In that case, using the same pKa value obtained for aqueous

604

medium will end up in erroneous pH estimation. Hence, to probe the actual pH of the

605

environment where the probe interacts with some other biomolecule (here LYZ), the shifted

606

pKa (as stated earlier) of the probe should be plugged in.

607

Strikingly, although there is a preference of the deprotonated form of HPTS over its

608

protonated one in lysozyme, resulting a downshifted pKa as well as pKa*, the excited state

609

proton-transfer becomes slower than in bulk water (only 100 ps). To have a scenario about

610

the slowing down of proton-transfer of the probe in LYZ matrix, we have focused on the

611

factors that control the kinetics of proton-transfer in the excited state. An ESPT process in

612

which a proton is transferred to a suitable acceptor molecule (in aqueous solution, a proton is

613

donated to water molecule) depends on dielectric constant, solvation dynamics, and

614

availability of sufficient water molecules to solvate the ejected proton and the conjugate base.

615

The dielectric constant determination for a macromolecule like protein is a complicated task

616

due to the presence of many charged side groups and simulation studies have shown that it

617

depends also on the model used 65

65

. For hen egg white lysozyme in water, static dielectric

618

constant was reported

to be ~30. Hen egg white lysozyme, consisting of 129 amino acid

619

residues, has ~59% sequence homology with human lysozyme66. So, a comparable value of

620

the static dielectric constant for human lysozyme is also expected. Considering a nearly

621

similar value it is evident that there is a reduction in dielectric constant in lysozyme pocket


Page 20 of 33

Page 21 of 33

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


622

and hence the solvation energy of the ejected proton as well as the population of the

623

conjugate base will be less as compared to that in bulk water. The other very important factor

624

is solvation dynamics. For a polar reaction, the activation barrier of its polar transition state

625

depends on solvation and any slow component of solvation will retard the reaction due to

626

insufficient or incomplete solvation67. In many natural macromolecular systems, water is

627

confined in such environment where its free movement is restricted and its three-dimensional

628

H-bonded network gets disrupted due to the interaction with the macromolecule. Such

629

confined water molecules are different in their dynamical nature and show some ultraslow

630

component of solvation compared to bulk. For water in bulk, solvation occurs in ~1 ps67. A

631

slow component like 530 ps was detected in eosin covalently attached to lysozyme68, whereas

632

a 38 ps component was reported for tryptophan located near the surface of a protein subtilisin

633

Carlsberg69. These suggest that water molecules available in the protein cavity or on the

634

surface are more constrained in nature and their dynamical property also differs depending on

635

the location in the protein. Here in our system, between the two ESPT time constants

636

observed in presence of LYZ, the slower component of 780 ps could be attributed to proton-

637

transfer occurring to water molecules that are deep-seated (tightly-bound) inside the protein

638

scaffold and showing relatively slower dynamics; while the other 140 ps component could be

639

ascribed to water molecules that are loosely associated to the protein and shows nearly bulk-

640

like behavior. This has also been suggested by time resolved anisotropy measurements that

641

some of the population of HPTS remains there as loosely attached to the protein (having a

642

rotational time constant ~199 ps), and the rest of the population is tightly bound to the protein

643

(rotational time constant ~2.17 ns). The other governing factors of ESPT process is the

644

requirement of sufficient number of water molecules to solvate the ejected proton and the

645

conjugate base. In the vicinity of the active site of protein, the water molecules present are

646

smaller in number, and those can also contribute to slowing down the ESPT process in

647

lysozyme.

648

Next we discuss the most likely causes for further modification in ESPT process of

649

LYZ-bound pyranine upon NaCl addition. First we consider how the factors, which proton-

650

transfer depends on, are expected to get affected and influence the proton-transfer rate in the

651

presence of NaCl only. Presence of small ions in water decreases the dielectric constant of the

652

medium, and solvation energy of proton and the conjugate base will be reduced to some

653

extent, making proton-transfer process slower. In high ionic strength, solvation becomes slow

654

and the number of free water molecules also gets reduced. These three factors should retard



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

655

the ESPT process in presence of NaCl. But, as mentioned previously, the steady state

656

emission and time resolved data show that the presence of NaCl in our studied concentration

657

range (upto 0.2 M) does not affect the proton-transfer rate directly by slowing it down.

658

Instead, the presence of salt makes the retarded ESPT process faster again in the complex.

659

Importantly, the concentration range of NaCl that we used here does not salt out LYZ 70 and

660

hence protein salting-out from the medium should not be the cause for the observed proton

661

transfer rate alterations. It is well known that electrostatic interaction between two interacting

662

partners (like protein and ligand) can be screened with increased ionic strength46, 47. Hence, it

663

is most probable that the increased ionic strength of medium results in weakening of the

664

binding interaction between lysozyme and pyranine, i.e. in other words, electrostatic

665

interaction has a dominant role in the complexation process. As previously mentioned, by

666

considering charges on the two interacting partners it is quite likely that the electrostatic

667

interaction is the major mode of binding. Therefore, as observed from the steady-state and

668

excited-state spectroscopic data, the salt addition throws HPTS from interaction zone of LYZ

669

to bulk water. As a consequence, the 140 ps time constant, assigned to be due to the loosely

670

associated water molecules in the protein, disappears and consequently the 99 ps time

671

constant reappears and this is very similar to the behavior of the probe in bulk water.

672

Whereas, the other longer component (780 ps), assigned to be due to the deep-seated water

673

molecules inside the protein matrix, survives with very negligible contribution.

674

5. Conclusion

675

The present work shows that the ground state and the excited state behavior of the photoacid

676

pyraine gets markedly modified while bound in lysozyme cavity compared to bulk aqueous

677

media. In spite of the preference for the conjugate base of HPTS inside the protein, the ‘slow’

678

biological water associated with the protein matrix retards the proton-transfer process in the

679

excited state causing a slowing down of the proton transfer rate. HPTS forms a 1:1 complex

680

with the protein in the ground state with a binding constant (KBH) of (1.4±0.5)×104 M-1.

681

Transient emission anisotropy measurements confirm the said complexation as rotational

682

time constant of HPTS increases in the presence of the protein. The docking simulation study

683

shows a possible binding region near the active site of the protein, and there is a little

684

conformational change in the protein upon ligand binding as observed in the circular

685

dichroism spectra. Since there is a major dominance of electrostatic forces of interaction

686

between HPTS and lysozyme, salt addition interrupts the fluorophore-protein complex,

687

ultimately throwing the fluorophore away from lysozyme cavity to the bulk. As a


Page 22 of 33

Page 23 of 33

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


688

consequence, binding constant of the complex gets reduced and rotational time constant

689

decreases again. Importantly, the shifted prototropic equilibrium of HPTS toward the

690

conjugate base due to interaction with lysozyme has been found to be back-shifted toward the

691

protonated species in the presence of salt and consequently the excited state proton transfer

692

rate has been observed to follow bulk-like feature ultimately. The study shows a considerable

693

different perspective of the perturbation offered by the model bio-macromolecular host with

694

substantial water exposure at the interaction site unlike the available literature reports

695

involving bio-macromolecular pockets and some other macromolecular cavity associated

696

with much less water exposure and different electrical nature on the concerned photoacid. It

697

is indeed worth noting that modulation of the prototropy in the presence of different

698

biomacromolecular host can lead to distinctive behavioral pattern that need to be explored in

699

detail to understand the intricacies of physico-chemical processes.

700 701

Supporting information

702

Absorption spectra with varying pH, in absence and in presence of LYZ; pKa estimation in

703

presence of 0.2 M NaCl and in presence of LYZ + 0.2 M NaCl, IROH* /IRO−* with increasing

704

LYZ concentration, excitation spectra of HPTS in absence and in presence of LYZ, intensity

705

ratio of ROH and RO- obtained from time resolved area normalized emission spectra at

706

different times, steady state emission spectra, time resolved emission decay with decay

707

parameters at maximum NaCl concentration used (0.2 M), binding constant evaluation from

708

steady-state anisotropy measurements, and Circular dichroism spectral study in detail are

709

presented in supporting information.

710 711

Acknowledgement

712

MH thanks DST SERB-India (Fund no. SB/S1/PC-041/2013) and IIT Kharagpur for financial

713

supports. ID thanks IIT Kharagpur and SP thanks UGC-India for their fellowship. We would

714

like to thank the anonymous reviewers for their critical comments and suggestions.

715 716 717



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

718

References

719 720

1.

Agmon, N. Elementary steps in excited-state proton transfer. J. Phys. Chem. A 2005, 109, 13-

721

35.

722

2.

723

A 2000, 104, 6689-6698.

724

3.

Kuhlbrandt, W. Bacteriorhodopsin - the movie. Nature 2000, 406, 569-570.

725

4.

Rini, M.; Magnes, B. Z.; Pines, E.; Nibbering, E. T. J. Real-time observation of bimodal

726

proton transfer in acid-base pairs in water. Science 2003, 301, 349-352.

727

5.

728

transfer rates in diffusion-controlled reactions. Chem. Phys. Lett. 1997, 281, 413-420.

729

6.

730

(HPTS). J. Phys. Chem. A 2007, 111, 230-237.

731

7.

732

1955-1976.

733

8.

734

surfaces. J. Am. Chem. Soc. 1984, 106, 265-273.

735

9.

736

transfer of pyranine in a gamma-cyclodextrin cavity. Chem. Phys. Lett. 2005, 412, 228-234.

737

10.

738

absence of the DNA backbone. Nature 2000, 408, 949-951.

739

11.

740

bacterial reaction centers. FEBS Lett. 2003, 555, 45-50.

741

12.

742

intermolecular reactions. J. Photochem. Photobiol., A 1993, 75, 1-20.

743

13.

744

reactions. J. Photochem. Photobiol., A 1993, 75, 21-48.

745

14.

746

cyclodextrins and human serum albumin protein. Langmuir 2012, 28, 6746-6759.

747

15.

748

cell membranes. J. Phys. Chem. B 2009, 113, 10210-10221.

749

16.

750

cell membranes. Langmuir 2008, 24, 3690-3698.

751

17.

752

from pyranine to acetate in gamma-cyclodextrin and hydroxypropyl gamma-cyclodextrin. J. Phys.

753

Chem. A 2006, 110, 13646-13652.

Genosar, L.; Cohen, B.; Huppert, D. Ultrafast direct photoacid-base reaction. J. Phys. Chem.

Pines, E.; Manes, B. Z.; Lang, M. J.; Fleming, G. R. Direct measurement of intrinsic proton

Spry, D. B.; Goun, A.; Fayer, M. D. Deprotonation dynamics and stokes shift of pyranine

Douhal, A. Ultrafast guest dynamics in cyclodextrin nanocavities. Chem. Rev. 2004, 104,

Politi, M. J.; Fendler, J. H. Laser pH-jump initiated proton transfer on charged micellar

Mondal, S. K.; Sahu, K.; Sen, P.; Roy, D.; Ghosh, S.; Bhattacharyya, K. Excited state proton

Nir, E.; Kleinermanns, K.; de Vries, M. S. Pairing of isolated nucleic-acid bases in the

Paddock, M. L.; Feher, G.; Okamura, M. Y. Proton transfer pathways and mechanism in

Arnaut, L. G.; Formosinho, S. J. Excited-state proton-transfer reactions I. Fundamentals and

Formosinho, S. J.; Arnaut, L. G. Excited-state proton-transfer reactions II. Intramolecular

Martin, C.; Gil, M.; Cohen, B.; Douhal, A. Ultrafast photodynamics of drugs in nanocavities:

Spry, D. B.; Fayer, M. D. Proton transfer and proton concentrations in protonated nafion fuel

Moilanen, D. E.; Spry, D. B.; Fayer, M. D. Water dynamics and proton transfer in nafion fuel

Mondal, S. K.; Sahu, K.; Ghosh, S.; Sen, P.; Bhattacharyya, K. Excited-state proton transfer


Page 24 of 33

Page 25 of 33

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


754

18.

Mondal, T.; Das, A. K.; Sasmal, D. K.; Bhattacharyya, K. Excited state proton transfer in

755

ionic liquid mixed micelles. J. Phys. Chem. B 2010, 114, 13136-13142.

756

19.

757

proton transfer of pyranine in an ionic liquid microemulsion. J. Chem. Phys. 2010, 132, No. 194505,

758

DOI : 10.1063/1.3428669.

759

20.

760

microenvironment of a binding site. Eur. J. Biochem. 1982, 121, 637-642.

761

21.

762

antenna effect of the gamma-cyclodextrin outer surface, measured by excited state proton transfer. J.

763

Phys. Chem. B 2006, 110, 26354-26364.

764

22.

765

ultrafast proton transfer in niosome. J. Phys. Chem. B 2012, 116, 8105-8112.

766

23.

767

reaction dynamics within the human serum albumin protein. J. Phys. Chem. B 2011, 115, 7637-7647.

768

24.

769

monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J. Immunol.

770

Methods 1998, 221, 43-57.

771

25.

772

by several agents is preceded by intracellular acidification. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,

773

654-658.

774

26.

775

Proc. Natl. Acad. Sci. USA 1994, 91, 4101-4101.

776

27.

777

evaluation of the use of endoplasmic reticulum-targeted "cameleons". Endocrinology 2004, 145,

778

4540-4549.

779

28.

780

intracellular pH of Caco-2 cells. Int. J. Pharm. 2007, 338, 104-109.

781

29.

782

PK1 cells - recovery from an acid load via basolateral Na+/H+ exchange. J. Membr. Biol. 1987, 97, 63-

783

78.

784

30.

785

pH buffering power in smooth muscle. Pflugers Arch. 1998, 435, 575-577.

786

31.

787

different intensifies in mammalian single muscle fibres. J. Physiol. 1998, 512, 831-840.

788

32.

789

engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester. J. Immunol. Methods

790

2009, 342, 71-77.

Sen Mojumdar, S.; Mondal, T.; Das, A. K.; Dey, S.; Bhattacharyya, K. Ultrafast and ultraslow

Gutman, M.; Huppert, D.; Nachliel, E. Kinetic studies of proton transfer in the

Gepshtein, R.; Leiderman, P.; Huppert, D.; Project, E.; Nachliel, E.; Gutman, M. Proton

Mondal, T.; Ghosh, S.; Das, A. K.; Mandal, A. K.; Bhattacharyya, K. Salt effect on the

Cohen, B.; Alvarez, C. M.; Carmona, N. A.; Organero, J. A.; Douhal, A. Proton-transfer

Ishaque, A.; Al-Rubeai, M. Use of intracellular pH and annexin-V flow cytometric assays to

Gottlieb, R. A.; Nordberg, J.; Skowronski, E.; Babior, B. M. Apoptosis induced in Jurkat cells

Simon, S.; Roy, D.; Schindler, M. Intracellular pH and the control of multidrug-resistance. Varadi, A.; Rutter, G. A. Ca2+-induced Ca2+ release in pancreatic islet beta-cells: critical

Liang, E.; Liu, P. C.; Dinh, S. Use of a pH-sensitive fluorescent probe for measuring

Montrose, M. H.; Friedrich, T.; Murer, H. Measurements of intracellular pH in single LLC-

Bullock, A. J.; Duquette, R. A.; Buttell, N.; Wray, S. Developmental changes in intracellular

Chin, E. R.; Allen, D. G. The contribution of pH-dependent mechanisms to fatigue at

Miksa, M.; Kornura, H.; Wu, R. Q.; Shah, K. G.; Wang, P. A novel method to determine the



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

791

33.

Lakadamyali, M.; Rust, M. J.; Babcock, H. P.; Zhuang, X. W. Visualizing infection of

792

individual influenza viruses. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9280-9285.

793

34.

794

Michael, N. P.; Milligan, G.; Game, S. A pH-sensitive fluor, CypHer 5, used to monitor agonist-

795

induced G protein-coupled receptor internalization in live cells. Biotechniques 2002, 33, 1152-1157.

796

35.

797

2709-2728.

798

36.

799

Chem. Educ. 2001, 78, 1499-1503.

800

37.

801

ultrafast pH jump. Chem. Phys. Lett. 1979, 64, 522-527.

802

38.

803

yesterday. Mol. Cell. Biochem. 1984, 63, 165-189.

804

39.

805

Structure of hen egg-white lysozyme - a 3-dimensional Fourier synthesis at 2Å resolution. Nature

806

1965, 206, 757-761.

807

40.

808

J. M.; Zhang, L.; et al. Characterization of bioactive recombinant human lysozyme expressed in milk

809

of cloned transgenic cattle. PLoS ONE 2011, 6, e17593.

810

41.

811

distortion of an N-acetylmuramic acid residue bound in site D. J. Mol. Biol. 1991, 220, 401-424.

812

42.

813

partly folded protein by heteronuclear NMR-spectroscopy - assignment and secondary structure-

814

analysis of hen egg-white lysozyme in trifluoroethanol. Biochemistry 1995, 34, 13219-13232.

815

43.

816

probing the process of unfolding. Biophys. J . 2007, 92, 2523-2535.

817

44.

818

X.; He, Q. The targeting of 14-succinate triptolide-lysozyme conjugate to proximal renal tubular

819

epithelial cells. Biomaterials 2009, 30, 1372-1381.

820

45.

821

the excited-state proton transfer and geminate recombination. J. Phys. Chem. A 2006, 110, 5573-5584.

822

46.

823

in the pH-dependent interaction of bovine serum albumin with cochineal red A: a combined view

824

from spectroscopy and docking simulations. J. Agric. Food. Chem. 2013, 61, 4606-4613.

825

47.

826

on binding of tartrazine with two homologous serum albumins: quantification by use of the Debye-

Adie, E. J.; Kalinka, S.; Smith, L.; Francis, M. J.; Marenghi, A.; Cooper, M. E.; Briggs, M.;

Han, J. Y.; Burgess, K. Fluorescent indicators for intracellular pH. Chem. Rev. 2010, 110,

Po, H. N.; Senozan, N. M. The Henderson-Hasselbalch equation: its history and limitations. J.

Smith, K. K.; Kaufmann, K. J.; Huppert, D.; Gutman, M. Picosecond proton ejection-

Jolles, P.; Jolles, J. What’s new in lysozyme research - always a model system, today as

Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R.

Yang, B.; Wang, J. W.; Tang, B.; Liu, Y. F.; Guo, C. D.; Yang, P. H.; Yu, T. A.; Li, R.; Zhao,

Strynadka, N. C. J.; James, M. N. G. Lysozyme revisited - crystallographic evidence for

Buck, M.; Schwalbe, H.; Dobson, C. M. Characterization of conformational preferences in a

Ghosh, A.; Brinda, K. V.; Vishveshwara, S. Dynamics of lysozyme structure network:

Zhang, Z. R.; Zheng, Q.; Han, J.; Gao, G. P.; Liu, J.; Gong, T.; Gu, Z. W.; Huang, Y.; Sun,

Leiderman, P.; Gepshtein, R.; Uritski, A.; Genosar, L.; Huppert, D. Effect of electrolytes on

Bolel, P.; Mahapatra, N.; Datta, S.; Halder, M. Modulation of accessibility of subdomain IB

Bolel, P.; Datta, S.; Mahapatra, N.; Halder, M. Spectroscopic investigation of the effect of salt


Page 26 of 33

Page 27 of 33

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


827

Hückel limiting law and observation of enthalpy-entropy compensation. J. Phys. Chem. B 2012, 116,

828

10195-10204.

829

48.

LifeSpec-II User guide, rev. 2, copyright © 2012 Edinburgh Instruments Ltd. UK

830

49.

Maity, B.; Chatterjee, A.; Ahmed, S. A.; Seth, D. Interaction of the nonsteroidal anti-

831

inflammatory drug indomethacin with micelles and its release. J. Phys. Chem. B 2015, 119, 3776-

832

3785.

833

50.

834

spectroscopy (TRANES): a novel method for confirming emission from two excited states. J. Phys.

835

Chem. A 2001, 105, 1767-1771.

836

51.

837

conformational stability of a protein. Protein Eng. 2003, 16, 5-9.

838

52.

839

Comput. Chem. 2011, 32, 174-182.

840

53.

841

A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy

842

function. J. Comput. Chem. 1998, 19, 1639-1662.

843

54.

844

serum proteins toward individual response to specific ligands: HSA-pocket resembles flexible

845

biological slide-wrench unlike BSA. J. Phys. Chem. B 2014, 118, 6071-6085.

846

55.

847

internal aqueous hydrogen-ion concentration in phospholipid-vesicles. Biochemistry 1981, 20, 1534-

848

1538.

849

56.

850

physical-chemistry experiment that explores acid-base properties in the excited-state. J. Chem. Educ.

851

1992, 69, 247-249.

852

57.

853

of human serum albumin on the modulation of pKa of warfarin and structurally similar acidic guests: a

854

possible implication on biological activity. J. Photochem. Photobiol., B 2014, 130, 76-85.

855

58.

856

recombination processes. Chem. Phys. Lett. 1992, 192, 77-81.

857

59.

858

proton transfer of pyranine in a supramolecular assembly: PEO-PPO-PEO triblock copolymer and

859

CTAC. J. Phys. Chem. B 2007, 111, 13504-13510.

860

60.

861

alkanolamine forms of sanguinarine to lysozyme: spectroscopic analysis, thermodynamics, and

862

molecular modelling Studies. J. Phys. Chem. B 2014, 118, 13077-13091.

Koti, A. S. R.; Krishna, M. M. G.; Periasamy, N. Time-resolved area-normalized emission

Takano, K.; Yamagata, Y.; Yutani, K. Buried water molecules contribute to the

Allouche, A. R. Gabedit- A graphical user interface for computational chemistry softwares. J.

Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson,

Datta, S.; Halder, M. Detailed scrutiny of the anion receptor pocket in subdomain IIA of

Clement, N. R.; Gould, J. M. Pyranine (8-Hydroxy-1,3,6-Pyrenetrisulfonate) as a probe of

Marciniak, B.; Kozubek, H.; Paszyc, S. Estimation of pKa* in the first excited singlet-state - a

Datta, S.; Halder, M. Effect of encapsulation in the anion receptor pocket of sub-domain IIA

Goldberg, S. Y.; Pines, E.; Huppert, D. Proton scavenging in photoacid geminate

Ghosh, S.; Dey, S.; Mandal, U.; Adhikari, A.; Mondal, S. K.; Bhattacharyya, K. Ultrafast

Jash, C.; Payghan, P. V.; Ghoshal, N.; Kumar, G. S. Binding of the iminium and



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

863

61.

Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Catalysis by hen egg-white lysozyme

864

proceeds via a covalent intermediate. Nature 2001, 412, 835-838.

865

62.

866

human lysozyme revealed by affinity labeling. Biochemistry 1996, 35, 13562-13567.

867

63.

868

directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg-white lysozyme.

869

Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 133-137.

870

64.

871

Proc. Natl. Acad. Sci. USA 1971, 68, 517-522.

872

65.

873

inhibitor and lysozyme calculated from molecular dynamics simulations. J. Phys. Chem. 1993, 97,

874

2009-2014.

875

66.

876

lysozyme: importance of ligand-induced perturbation of the secondary structure on the mode of

877

binding of exogenous ligand and possible consequences. J. Photochem. Photobiol., B 2016, 161, 253-

878

265.

879

67.

880

Chem. Res. 2003, 36, 95-101.

881

68.

882

environments studied by photon echo spectroscopy. J. Phys. Chem. B 1999, 103, 7995-8005.

883

69.

884

macromolecular hydration. J. Phys. Chem. B 2002, 106, 12376-12395.

885

70.

886

Methods Enzymol. 2014, 541, 85-94.

Muraki, M.; Harata, K.; Sugita, N.; Sato, K. Origin of carbohydrate recognition specificity of

Malcolm, B. A.; Rosenberg, S.; Corey, M. J.; Allen, J. S.; Debaetselier, A.; Kirsch, J. F. Site-

Halper, J. P.; Latovitzki, N.; Bernstein, H.; Beychok, S. Optical activity of human lysozyme.

Smith, P. E.; Brunne, R. M.; Mark, A. E.; Vangunsteren, W. F. Dielectric properties of trypsin

Panja, S.; Halder, M. Exploration of electrostatic interaction in the hydrophobic pocket of

Bhattacharyya, K. Solvation dynamics and proton transfer in supramolecular assemblies. Acc.

Jordanides, X. J.; Lang, M. J.; Song, X. Y.; Fleming, G. R. Solvation dynamics in protein

Pal, S. K.; Peon, J.; Bagchi, B.; Zewail, A. H. Biological water: femtosecond dynamics of

Duong-Ly, K. C.; Gabelli, S. B. Salting out of proteins using ammonium sulfate precipitation.

887 888 889 890 891 892 893 894 895 896 897 898


Page 28 of 33

Page 29 of 33

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

899


Table 1. IROH* /IRO-* of HPTS at different conditions in aqueous phosphate buffer of pH 7

900 System In aqueous buffer only With 2.4×10-4 M LYZ only With 2.4×10-4 M LYZ + 0.02 M NaCl With 2.4×10-4 M LYZ + 0.05 M NaCl With 2.4×10-4 M LYZ + 0.08 M NaCl With 2.4×10-4 M LYZ + 0.10 M NaCl With 2.4×10-4 M LYZ + 0.15 M NaCl With 0.02 M NaCl only

IROH* /IRO−* 0.033 0.102 0.064 0.052 0.044 0.041 0.038 0.031

901 902 903 904 905 906 907

Table 2. Binding constant of HPTS-LYZ complex in aqueous buffer of pH 7 and at different

908

NaCl concentration

[NaCl] (M) 0

Binding constant, KBH (M-1) (From Benesi-Hildebrand plot) (1.4±0.4)×104

Binding constant, Kan (M-1) (From Steady-state anisotropy data) (1.5±0.07)×104

0.05

(7.6±0.6)×103

--

0.1

(6.7±0.5)×103

--

0.2

3

(6.2±0.6)×10

(7.6±0.05)×103

909 910 911 912 913 914



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

Page 30 of 33

915

Table 3. Picosecond decay parameters# of ROH* emission (at 435 nm) and RO-* emission (at

916

520 nm) in different systems, at pH 7. λexc=375 nm

917

Observation System

τ1 (ns)

τ2 (ns)

τ2 (ns)

α1

α2

α3

437

0.100

0.550

3.25

0.92

0.073

0.007

520

0.100

5.46

-

(-)0.52

0.48

-

437

0.139

0.736

3.64

0.82

0.12

0.06

520

0.139

0.780

5.77

(-)0.13

(-)0.09

0.78

437

0.134

0.840

3.63

0.86

0.093

0.047

520

0.134

0.737

5.66

(-)0.13

(-)0.10

0.77

437

0.110

0.820

3.56

0.89

0.078

0.032

520

0.110

0.770

5.57

(-)0.20

(-)0.06

0.74

437

0.103

0.785

3.54

0.91

0.064

0.026

520

0.103

0.744

5.51

(-)0.44

(-)0.02

0.54

437

0.099

0.763

3.51

0.90

0.063

0.017

520

0.099

0.760

5.48

(-)0.52

(-)0.02

0.46

wavelength (nm)

In aqueous buffer only With 1.8×10-4 M LYZ only With 1.8×10-4 M LYZ + 0.02 M NaCl

With 1.8×10-4 M LYZ + 0.05 M NaCl With 1.8×10-4 M LYZ + 0.1 M NaCl With 1.8×10-4 M LYZ + 0.2 M NaCl

918

#

th

Error in measuring/fitting lifetime parameters is ~4%. αi is the normalized weightage of i component.

919 920 921 922 923 924 925 926 927 928


Page 31 of 33

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


929

Table 4. Relative amplitudes of the two rise-time components# at RO-* emission (at 520 nm)

930

in the presence of LYZ at different NaCl concentration

931 932 933 934 935

System

τ1 (ns)

τ2 (ns)

α1

α2

936

With 1.8×10-4 M LYZ + 0 M NaCl

0.139

0.780

0.58

0.41

With 1.8×10-4 M LYZ + 0.05 M NaCl

0.110

0.770

0.77

0.23

With 1.8×10-4 M LYZ + 0.1 M NaCl

0.103

0.744

0.96

0.04


0.099

0.760

0.95

0.05

937 938 939 940 941

#

Error in measuring/fitting lifetime parameters is ~4%.

942 943

Table 5. Anisotropy decay parameters of HPTS collected at 520 nm in different systems.

944

λexc= 375 nm

945 946

τ1rot (ns)

(α1rot)

τ2rot (ns)

In aqueous buffer only

0.154

(1.00)

--

With 1.8×10-4 M LYZ only

0.199

(0.13)

With 1.8×10-4 M LYZ + 0.02 M NaCl

0.205

With 1.8×10 M LYZ + 0.05 M NaCl

System

(ns)

--

0.154

2.17

(0.87)

1.90

(0.66)

1.60

(0.33)

0.663

0.188

(0.75)

1.79

(0.25)

0.580


0.290

(1.00)

--

--

0.290


0.191

(1.00)

--

--

0.191

-4

947

(α2rot)

αirot

th

is the normalized weightage of i component.

948 949



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

950

Table 6. Change in solvent accessible surface area (∆SASA) in Å2 of the concerned residues

951

(residues with % side chain solvent accessibility >10) present around the docked HPTS in

952

LYZ

953 954

Residues

∆SASA (Å)

Trp 34

32.4

Glu 35

39.1

Asn 44

21.23

Asp 53

24.13

Gln 58

13.22

Trp 109

9.05

Val 110

23.99

Ala 111

22.01

960

Asn 114

20.45

961

Arg 115

36.58

955 956 957 958 959

962 963 964 965 966 967 968 969 970 971 972 973 974 975


Page 32 of 33

Page 33 of 33

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


976

TOC Graphic:

977

Detailed scenario of the acid-base behavior of HPTS in the presence of human lysozyme

978 979 980 981 982 983 984


Modulation and Salt-Induced Reverse Modulation ... - ACS Publications

Recommend Documents