Analysis and Evaluation of the Inhibitory Mechanism of a Novel

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Bioactive Constituents, Metabolites, and Functions

Analysis and evaluation of the inhibitory mechanism of a novel ACE-inhibitory peptide derived from casein hydrolysate Maolin Tu, Hanxiong Liu, Ruyi Zhang, Hui Chen, Fengjiao Mao, Shuzhen Cheng, Weihong Lu, and Ming Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00732 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Journal of Agricultural and Food Chemistry

Analysis and evaluation of the inhibitory mechanism of a novel ACE-inhibitory peptide derived from casein hydrolysate

Maolin Tu a,b, Hanxiong Liu a, Ruyi Zhang a, Hui Chen a, Fengjiao Mao a, Shuzhen Cheng a,c, Weihong Lu b, Ming Du a,b,*

a

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China

b

Department of Food Science and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China

c

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China

Author contributions: Maolin Tu and Hanxiong Liu contributed equally to this study.

*Corresponding author Dr. Ming Du Tel: +86-411-86332275 Fax: +86-411-86323262 E-mail: [email protected].

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ABSTRACT

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Casein hydrolysates exert various biological activities and the responsible

3

functional peptides are being identified from them continuously. In this study, the

4

tryptic casein hydrolysate was fractionated by ultra-filtration membrane (3 kDa), and

5

the peptides were identified by CE-TOF-MS/MS. Meanwhile, in silico methods were

6

used to analyze the toxicity, solubility, stability, and affinity between the peptides and

7

ACE. Finally, a new angiotensin I-converting enzyme-inhibitory (ACEI) peptide,

8

EKVNELSK, derived from the αs1-casein (fragment, 35–42), was screened. The IC50

9

value of the peptide is 5.998 mM, which was determined by an HPLC method. The

10

Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against

11

ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform

12

molecular docking to propose the potential mechanisms underlying the ACEI activity

13

of the peptide. These results indicated that EKVNELSK is a new ACEI peptide

14

identified from casein hydrolysate.

15 16

Keywords: Casein, ACE-inhibitory activity, Peptide, In silico, CE-TOF-MS/MS,

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Molecular docking,

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INTRODUCTION

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Cardiovascular disease (CVD) has been recognized the leading cause of death in

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the worldwide. It is expected that there will be more than 23.6 million deaths in 2030

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caused by CVD 1. Hypertension (blood pressure > 140/90), as one of the independent

23

risk factors for CVD, with characteristics of frequent, chronic, and age-related

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disorders

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can be an effective way to prevent CVD 5. To control the blood-pressure, the

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angiotensin I-converting enzyme (ACE, EC 3.4.15.1) must be taken into consideration,

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which plays a significant role in blood pressure regulation through both the

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renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) in vivo. In the

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RAS, the potent vasopressor angiotensin II (Ang II) can be released by the ACE

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cleaving the C-terminal dipeptide from angiotensin I (Ang I) 6. In the KKS, ACE can

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also inactivate the bradykinin (BK) by removing the two C-terminal dipeptides 7.

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Therefore, more attentions have been gradually paid to find novel ACEI substances

33

for the sake of lowering blood pressure. In the recent years, pharmaceutical drugs

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targeting ACE are shown evident in lowering high blood pressure. However, some

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side effects have appeared in clinical treatments 8. To find safer drugs, increasingly

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scholars have paid attention to food-derived ACE inhibitors, especially the peptides

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which owner the natural advantages of safe and easily to be metabolized in bodies.

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ACEI peptides have been continuously identified from milk, soybean, egg and so on.

39

2-4

, has been paid increasingly attention. Blood-pressure lowering treatment

Casein is an excellent source of bioactive peptides and has been widely used in 9-10

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food industry as a functional ingredient

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been identified from enzymatic hydrolysates of casein, especially ACEI peptides. For

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example, peptide QSLVYPFTGPI (from β-casein) and ARHPHPHLSFM (from

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κ-casein) showed potent ACEI activity in vitro 11.

. To date, lots of bioactive peptides have

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To identify the bioactive peptides more effectively, some in silico methods have

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been employed in recent studies. Quantitative structure-activity relationship (QSAR)

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method, which has been widely applied to elaborate the relationships between the

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peptides’ primary structures and the ACEI activity

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been used to study the active mechanisms of the inhibitors against the receptors, such

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as ACE

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peptides by analyzing the physicochemical characteristics of the peptides. For

51

instance,

52

https://web.expasy.org/compute_pi/, has the function of predicting the molecular

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weight (MW) and Iso-electric point (PI) of the potential bioactive peptides. ProtParam

54

tool can be used to predict instability index of peptides, available at

55

https://web.expasy.org/protparam/. The toxicity of the peptides can be predicted by

56

the online server ToxinPred, available at http://crdd.osdd.net/raghava/toxinpred/.

13

12

. Molecular docking also has

and thrombin Online servers have been used to screen the bioactive

the

online

server

Expasy-Compute

pI/Mw,

available

at

57

The aim of this study is to screen and identify a novel ACEI peptide from casein

58

hydrolysate and elaborate its active mechanisms. The peptides from casein

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hydrolysates were determined by CE-Q-TOF-MS/MS. Toxicity, solubility, and

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stability of the peptides were predicted by in silico methods. A novel ACEI peptide

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was identified and subsequently synthesized. The IC50 value and the inhibition mode

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of the peptide were investigated. Molecular docking analysis also were used to

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elucidate the potential mechanism of ACEI activity of the peptide.

64 65

MATERIALS AND METHODS

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Materials and chemicals

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ACE (from rabbit lung), Hippuryl-histidyl-leucine (HHL), HPLC grade acetonitrile (ACN), and trifluoroacetic acid

(TFA) were purchased from

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Sigma-Aldrich Co. (St. Louis, MO, USA). Casein and trypsin were obtained from

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Solarbio (Beijing, China). All other chemical reagents were of analytical grade.

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Preparation of casein hydrolysates

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Bovine casein solution (50 mg mL-1) was prepared by dissolving bovine casein

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powder in deionized water. Then, 0.1 M NH4HCO3 buffer was used to adjust the pH

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of the casein solution to 8.0. The hydrolysis reaction was started by the adding trypsin

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with 2500 U g-1 of enzyme concentration. Other hydrolysis parameters were set as

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120 min of hydrolysis time and 37 °C of temperature. Heating the samples in boiling

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water (10 min) to quench the reaction before adjusting the pH to 7.0 (after the samples

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cooled to below 25 °C). Subsequently, centrifuged the samples at 8000×g for 20 min

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at 4 °C. Then, the supernatant was fractionated by Amicon Ultra ultrafiltration tube

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(Millipore, [MWCO] = 3 kDa) according to the molecular weight and the fraction

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with peptides’ molecular weight no more than 3 kDa was collected and freeze-dried

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before storage at -80 °C.

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Determination of ACEI activity

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The assay of in vitro ACEI activities of the casein hydrolysate and the peptide

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were conducted by an HPLC method as previously reported 14.

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Identification of peptides by CE-Q-TOF-MS/MS

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CE analysis was conducted on a CESI 8000 Plus System (AB SCIEX, Inc.,

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Redwood City, CA, U.S.A.). The uncoated fused-silica capillary (91 cm of total

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length, i.d. 30 µm) was coupled to Q-TOF-MS/MS (impact II, Bruker Daltonic GmbH,

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Bremen, Germany) through electrospray ionization source (ESI). In the process of

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capillary separation, 10% acetic acid solution and 100 mM ammonium acetate were

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used as the background electrolyte (BGE) buffer and the conductive liquid,

93

respectively.

The

washing

and

injection

of

the

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were

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hydrodynamically. Before loading each sample, the bare fused capillary was

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conditioned by forwarding with 0.1 M NaOH for 3 min, followed by 3 min with 0.1

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M HCl and 4 min with ultrapure water and 3 min with BGE for reversing and

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followed by 4 min forward with BGE. Subsequently, the samples were loaded on

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using under 20 psi pressure (1 min), then followed with BGE (30 s, 20 psi). The

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separation was then conducted electrodynamically (1 h) by applying a voltage of +20

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kV after injection. The CE-ESI-Q-TOF-MS was set as the following parameters:

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150 °C of dry temperature, 3.0 L min-1 of dry gas speed. Moreover, the top 20 intense

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ions were collected in the MS spectra over an m/z range of 50–2200. Finally, Data

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Analysis 4.0 software (Bruker Daltonic GmbH, Bremen, Germany) was used to

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process the MS data. MASCOT searching engine (Matrix Science, London, UK) was

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adopted to identify the sequence of the peptides in the sample.

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Screening of the potential ACEI peptide

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Some properties of the peptides are crucially important for the further studies of

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the peptides especially drug-oriented, such as the toxicity, solubility, and stability.

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Therefore, those factors have been taken into consideration for screening the potential

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ACEI peptides. The ToxinPred server was used to analyze toxicity of the peptides,

111

available at http://crdd.osdd.net/raghava/toxinpred/. The solubility of the peptide was

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evaluated

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www.innovagen.com/proteomics-tools. The instability index of the peptide was

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predicted by using ProtParam Tool (http://web.expasy.org/protparam/). In additional,

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the affinities of the peptides against the ACE were compared by evaluating the

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molecular docking results, which were conducted on the Discovery Studio R2

117

software (Neotrident Technology Ltd., Beijing, China).

118

Chemical synthesis of peptides

by

the

online

Innovagen

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available

at

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Selected peptide was synthesized by a solid-phase method at Cellmano Biotech

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Limited Corporation (Hefei, China), which was with purity of 98.55% and verified by

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HPLC.

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Assay of ACEI kinetics

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The inhibition pattern of the peptide, EKVNELSK, was explored by using

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Lineweaver-Burk plots of 1/V versus 1/HHL. The concentrations of the HHL were set

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as 0.5, 1, 2 and 3.5 mM, meanwhile, the concentrations of the peptide were set as 0,

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4.228 and 8.456 Mm. The Vmax and Km were calculated, respectively, as the Y and

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X-axis intercepts of the primary plot.

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Molecular docking analysis

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Discovery Studio 2017 R2 software has been used to further evaluate the active

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mechanism of the ACEI peptide through molecular docking technology according to

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the reported methods

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PDB ID: 1O8A) was downloaded from RCSB Protein Data Bank. The 3D-structures

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of the peptide was produced by DS 2017 R2, and then minimized it by given the

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CHARMm force field. Meanwhile, ACE molecular was treated by the procedures of

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cleaning, preparing, removing waters and adding hydrogen. Finally, partial flexibility

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program CDOCKER was chosen to perform the docking program with special

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binding sites (coordinates x: 36.189, y: 43.643 and z: 55.175) and receptor radius (16

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Å). The results of molecular docking were evaluated based on the -CDOCKER

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energy scores, interaction site, and interaction force types with ACE.

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Statistical analysis

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15

, with some modifications. ACE crystal structure (Human,

All experiments were conducted in triplicate and the data were presented as the

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mean ± SD in this study. Origin 8.0 (OriginLab Corporation, Northampton, MA, USA)

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software was adopted to process the data. 7

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RESULTS AND DISCUSSION

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ACEI activity and peptide composition of casein hydrolysate

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In this study, the ACEI activity of the casein hydrolysate with the molecular

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weight no more than 3 kDa, has been determined by an HPLC method. As shown in

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Fig.1, the ACEI activity of the mixture became significantly from the concentration of

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0.05 mg mL-1, and the IC50 value of the casein hydrolysate was 0.4424 mg mL-1. To

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analyze the component of the active mixture, CE-ESI-Q-TOF-MS/MS was used to

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identify the peptides (Table 1). There is a total of 32 peptides were identified from the

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casein mixture and 14, 11, 5, and 2 peptides were obtained from αs1-, αs2-, β- and

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κ-CN, respectively. Although the mixture has been fractionated by ultrafiltration tube,

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it’s not hard to find that there are also two peptides with molecular weight more than

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3 kDa, which may attribute to the fact that the ultrafiltration tube can’t separate the

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mixture completely. As casein hydrolysate mixture with potent ACEI activity, the

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identified peptides can be considered as the potential ACE inhibitors.

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Screening of the potential active ACEI peptides

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In the present casein hydrolysate, the ACEI peptides have reported were

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explored

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(http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) and the published literatures.

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As labelled in Table1, there are six peptides (FFVAPFPEVFGK 16, ALNEINQFYQK

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17

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activity, with IC50 values of 18 µM, 264 µM, 600 µM, 11.9 µM, 60 µM, 15 µM,

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respectively. Therefore, those six peptides must be partly of the active factors, which

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made the casein mixture exhibit high ACEI activity.

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, AMKPWIQPK

by

18

searching

, FALPQYLK

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the

, NMAINPSK 17, AVPYPQR

BIOPEP

20

) showed ACEI

The analysis of the human ACE-lisinopril complex has laid a solid foundation 8

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for the related studies of ACE inhibitors. There is a central active site groove that

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extends for about 30 Å in the structure of ACE, which is the active site buried with

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lisinopril

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peptides with large spatial structures are less likely to be bioactive. Moreover, from a

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cost point of view, the peptides with fewer amino acids are more meaningful for

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exploring the drug-derived bioactive peptides. Therefore, the present study was aimed

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to identify a novel ACEI peptide with the number of amino acids less than ten.

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Twelve peptides with the characteristics of the novel and the sequences consist of less

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than ten amino acids (Table 2). Toxicity is one of the fundamental factors should be

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taken into consideration for the various products of foods and drugs. Consequently,

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the identified ACEI peptides in this study were assessed for potential toxicity in silico

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using ToxinPred 22. As shown in Table 2, all of the peptides showed no toxicity.

21

. Consequently, due to the limited space of the ACE active site, the

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Solubility is also an important characteristic of the peptides, which will influence

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the absorption, distribution, and elimination of the peptides in the human body 23. The

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aqueous solubility of the potential ACEI peptides was evaluated by the online tool

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Innovagen, one peptide (VLPVPQK) showed poor water solubility (Table 2).

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Meanwhile, the stability has also been used as an evaluation index to screen

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potential ACEI peptide, which was accessed by the online tool ProtParam. In this

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program, if the instability index is smaller than 40, which is predicted as stable; when

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the instability index value above 40, it is predicted as may be unstable. As shown in

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Table 2, only 5 (EGIHAQQK, EKVNELSK, NRLNFLK, NRLNFLKK, FFSDKIAK)

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out of 12 peptides were predicted as stable. Subsequently, molecular docking analysis

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was adopted to evaluate the affinity of the peptides against ACE. The peptide

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EKVNELSK against ACE acquired highest -CDOCKER ENERGY (kcal mol-1) score

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(185.54), which means this peptide has the highest affinity with ACE (Table 3). 9

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Hence, the peptide EKVNELSK is with higher probability to have the function of

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inhibiting the ACE activity. To confirm the ACEI activity of the peptide,

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EKVNELSK was synthesized for further study.

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IC50 determination of the ACEI peptide activity

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The HPLC method was adopted to evaluate the IC50 value of the peptide

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EKVNELSK. Fig. 2 showed the ACE inhibitory activity of the peptide at various

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concentrations. The regression equation, Y= (1.113E-2) X2+4.57673X+22.14678

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(R2=0.98312), was used to evaluate the IC50 value of the peptide EKVNELSK. The

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results showed that the peptide was a novel ACE inhibitor with an IC50 value of 5.998

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mM. Although it is seemly that the peptide EKVNELSK showed week in vitro ACE

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inhibitory activity. However, as the hypertension pathophysiology is very complicated

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that related to a series of system activities 24, it is hard to judge the in vivo effect of the

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peptide. Some studies showed that the relationship between in vitro ACE inhibition

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and antihypertensive activity is not apparent

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peptide KVLPVPQ with an IC50 value of 1000 µM, which is much higher than

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YPFPGPIPN (IC50 of 15 µM). But, the peptide KVLPVPQ showed significant blood

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pressure-lowering effect in vivo, which is 4.5 times that of YPFPGPIPN 25. Therefore,

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the novel ACEI peptide, EKVNELSK, can be regarded as one of the blood

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pressure-lowering inhibitor candidates and its effect in vivo of this peptide should be

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further studied.

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Inhibition mode of the peptide EKVNELSK

24

. Such as, β-casein derived ACEI

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The ACE inhibition pattern of the peptide EKVNELSK was evaluated by using

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the Lineweaver-Burk plot. As shown in Fig. 3, when the peptide concentration

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increased, the Vm values decreased, which indicated that the existing of the peptide

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EKVNELSK probably hindered the substrate bind to ACE active site. Meanwhile, the 10

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higher of the peptide concentrations, the higher of Km values in the reactions, which

220

implied that the higher concentration of substrate is essential for the ACE catalytic

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reaction. Moreover, the peptide EKVNELSK is a mixed-type ACE inhibitor. Similarly,

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the peptide MLLCS has been identified from the hydrolysates of Styela plicata

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peptides WVYY and WYT derived from the hemp seed proteins

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mixed inhibition pattern against ACE. The mixed-type mode of ACE inhibition of the

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peptide EKVNELSK indicated that the peptide binds to both the active and nonactive

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sites of ACE, which consequently reduces the catalytic activity of ACE 27.

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Interaction of the peptide and ACE

27

26

,

also showed the

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The docking study of the peptide (EKVNELSK) at the ACE active site was

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performed by Discovery Studio 2017 R2 software. As shown in Table 3, the value of

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peptide EKVNELSK from -CDOCKER_Energy indicated that the peptide

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EKVNELSK has the highest strength of the affinity against ACE in the five potential

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ACEI peptides. The most stabilized poses of the peptide EKVNELSK against ACE

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were obtained with the values of electrostatic energy (Eele), Van der Waals energy

234

(Evdw), and potential binding energy (Epot) of -675.211, -14.8202 and -631.721 kcal

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mol-1, respectively. The hydrogen bonds formed between the peptide and ACE played

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a vital role in inhibiting the ACE activity by stabilizing the structure of the

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non-catalytic enzyme-peptide complex 15, 27. Table 4 and Fig. 4 showed that totally of

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15 H-bonds have formed between ACE and peptide EKVNELSK, which contains

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nine (LYS118, ASP121, GLU123, SER219, ASP358, TYR360, VAL399, ARG402,

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ARG522) kinds of ACE residues. The residues GLU123 and ARG522 also formed

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H-bonds with the ACEI peptides (WG and PRY), as reported by Fu et al.

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Meanwhile, three kinds of electrostatic also formed between EKVNELSK and ACE at

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the ACE residues of LYS118, GLU411, and ARG522. Moreover, the electrostatic 11

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interaction of ZN701 and hydrophobic interactions of ALA89, ALA125, PRO519,

245

ARG124, ILE88) with ACE also contributed to the stabilization of the

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EKVNELSK-ACE complex. Therefore, peptide EKVNELSK is a novel and effective

247

casein-derived ACE inhibitor.

248 249

ACKNOWLEDGEMENTS

250

We are grateful to Dr. Fang Luo of the NeoTrident Technology Ltd. (Beijing,

251

China) for the molecular docking instruction and Qin Zhang of the Dalian Polytechnic

252

University for helpful discussion.

253 254 255 256

FUNDING This study was financially supported by the National Natural Science Foundation of China (31371805).

257 258 259

Notes All authors declare that they have no conflicts of interest.

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peptides from food proteins. Trends Food Sci. Technol. 2017, 69, 214−219.

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T.; Park, P. J.; Jung, W. K.; Jeon, Y. J. Effect of angiotensin I-converting enzyme

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(Cannabis sativa L.) peptides. J. Agric. Food Chem. 2014, 62, 4135−4144.

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Figure Captions Fig. 1. Total ion current (TIC) chromatogram and ACEI activity of bovine casein hydrolysate (molecular weight no more than 3 kDa). Hydrolysis was digested by trypsin at 37 °C for 2 h, with 50 mg mL-1 of protein and 2500 U g-1 of the enzyme as the substrate. Fig. 2. The ACEI activity of the peptide EKVNELSK. The ACEI activity of the peptide was determined by the HPLC method. The IC50 value was determined by the regression equation: Y= (1.113E-2) X2+4.57673X+22.14678 (R2=0.98312). Fig. 3. Lineweaver-Burk plot of ACE inhibition by the peptide EKVNELSK. The ACE activities were measured in the absence or presence of the peptide EKVNELSK (■, control; ●, 4.228 mM; ▲, 8.456 mM). 1/[S] and 1/[V] represent the reciprocal substrate concentration and velocity, respectively. Fig. 4. The molecular docking simulation of the peptide EKVNELSK with ACE (PDB: 1O8A). (A), (B) and (C) represent the general overview, local overview, and 2D-diagram of docking pose of peptide EKVNELSK, respectively.

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Table 1 Peptides Profile of Casein Hydrolysate Identified by CE-ESI-Q-TOF-MS/MS. No. 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

Protein αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN

αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN β-CN

m/z meas. 416.1951 455.7382 1070.1953 473.7646 1159.0664 692.8659 669.3396 1118.6130 880.4720 753.8928 460.7463 790.9140 624.9996 634.3531 684.3493 549.8074 490.2843 623.8211 509.7609 817.4429 437.7254 452.7708 344.8805 451.2648 624.3294 415.7288

z 2 2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 3 3 2 2

Mr. calc. 830.3770 909.4668 3206.5859 945.5131 2315.1296 1383.7227 1336.6735 2234.2283 1758.9377 1505.7773 919.4804 1579.8206 1870.9788 1266.6972 1366.6881 1097.6055 978.5538 1245.6353 1017.5090 1632.8835 873.4378 903.5290 1031.6240 1350.7732 1246.6517 829.4446

Scores 53.99 25.78 63.49 47.88 76.75 37.63 63.12 28.98 94.33 67.13 21.25 39.49 40.56 67.12 60.79 32.44 30.38 87.48 49.19 69.10 38.44 27.29 23.17 34.90 51.98 28.95

Start K K K K K R K K K R K K R K K K K K K K K K K K K

AA Sequence EDVPSER EGIHAQQK EGIHAQQKEPMIGVNQELAYFYPELFR EKVNELSK EPMIGVNQELAYFYPELFR FFVAPFPEVFGK HIQKEDVPSER HPIKHQGLPQEVLNENLLR HQGLPQEVLNENLLR LHSMKEGIHAQQK PFPEVFGK VPQLEIVPNSAEER YKVPQLEIVPNSAEER YLGYLEQLLR ALNEINQFYQK AMKPWIQPK FALPQYLK ITVDDKHYQK LTEEEKNR LTEEEKNRLNFLK NMAINPSK NRLNFLK NRLNFLKK RNAVPITPTLNR TKLTEEEKNR AVPYPQR

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End Y E Q D Q E Y F F E L L L F T T A L K E K I E L D

Length 7 8 27 8 19 12 11 19 15 13 8 14 16 10 11 9 8 10 8 13 8 7 8 12 10 7

ACE activity

ACE inhibitor

ACE inhibitor ACE inhibitor ACE inhibitor

ACE inhibitor

ACE inhibitor

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27 28 29 30 31

β-CN β-CN β-CN β-CN κ-CN

507.2634 436.7798 437.2458 390.7523 478.2653

2 2 2 2 2

1012.5164 871.5491 872.4789 779.4905 954.5175

40.11 33.53 33.89 33.29 35.83

K R K K R

32

κ-CN

1052.5454

3

3153.6063

20.63

R

HKEMPFPK INKKIEK VKEAMAPK VLPVPQK FFSDKIAK SPAQILQWQVLSNTVPAKSCQAQPTTM AR

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Y F H A Y

8 7 8 7 8

H

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Table 2 Toxicity, Solubility and Stability of the Potential ACEI Peptides. Number of

Solubility in

residues

water

EDVPSER

7

2

EGIHAQQK

3

No.

Peptide sequence

ToxinPed

Instability index

1

Good

Non-Toxin

unstable (118.63)

8

Good

Non-Toxin

stable (37.64)

EKVNELSK

8

Good

Non-Toxin

stable (-1.86)

4

PFPEVFGK

8

Good

Non-Toxin

unstable (68.01)

5

LTEEEKNR

8

Good

Non-Toxin

unstable (114.33)

6

NRLNFLK

7

Good

Non-Toxin

stable (-25.03)

7

NRLNFLKK

8

Good

Non-Toxin

stable (-20.65)

8

HKEMPFPK

8

Good

Non-Toxin

unstable (141.43)

9

INKKIEK

7

Good

Non-Toxin

unstable (93.04)

10

VKEAMAPK

8

Good

Non-Toxin

unstable (44.65)

11

VLPVPQK

7

Poor

Non-Toxin

unstable (118.63)

12

FFSDKIAK

8

Good

Non-Toxin

stable (-12.48)

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Table 3 The -CDOCKER_Energy Scores of the Potential ACEI Peptides Obtained from Molecular Docking. -CDOCKER_ENERGY

No.

Peptides

1

EGIHAQQK

177.172

2

EKVNELSK

185.540

3

NRLNFLK

154.963

4

NRLNFLKK

159.759

5

FFSDKIAK

169.805

(kcal mol-1)

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Table 4 Hydrogen Bonds, Electrostatic and Hydrophobic Interactions Observed in the Best Peptide Poses Based on the ACE-peptide Complex. Interaction Residues

Distance

Types

EKVNELSK:H132 - A:ASP121:OD2

2.28054

Hydrogen Bond;Electrostatic

EKVNELSK:H133 - A:GLU123:OE2

2.353

Hydrogen Bond;Electrostatic

EKVNELSK:H134 - A:GLU123:OE2

2.16624

Hydrogen Bond;Electrostatic

A:LYS118:NZ - EKVNELSK:O137

4.3831

Electrostatic

A:ARG522:NH1 - EKVNELSK:O15

5.48899

Electrostatic

A:ZN701:ZN - EKVNELSK:O15

2.30831

Electrostatic

EKVNELSK:N1 - A:GLU411:OE2

4.41662

Electrostatic

A:LYS118:HZ1 - EKVNELSK:O111

2.07162

Hydrogen Bond

A:LYS118:HZ3 - EKVNELSK:O114

2.12179

Hydrogen Bond

A:TYR360:HH - EKVNELSK:O64

2.77065

Hydrogen Bond

A:ARG522:HH12 - EKVNELSK:O17

3.02249

Hydrogen Bond

A:ARG522:HH12 - EKVNELSK:O39

2.97451

Hydrogen Bond

EKVNELSK:H66 - A:ARG402:O

2.16968

Hydrogen Bond

EKVNELSK:H67 - A:VAL399:O

3.08084

Hydrogen Bond

EKVNELSK:H86 - A:GLU123:OE1

2.89964

Hydrogen Bond

EKVNELSK:H116 - A:GLU123:OE2

2.05597

Hydrogen Bond

EKVNELSK:H32 - A:ASP358:O

2.658

Hydrogen Bond

EKVNELSK:H88 - A:GLU123:OE1

2.56598

Hydrogen Bond

EKVNELSK:H130 - A:SER219:O

2.79581

Hydrogen Bond

A:ALA89 - EKVNELSK:C98

4.43141

Hydrophobic

A:ALA125 - EKVNELSK:C98

4.26213

Hydrophobic

EKVNELSK:C46 - A:PRO519

4.97062

Hydrophobic

EKVNELSK:C94 - A:ARG124

4.93511

Hydrophobic

EKVNELSK:C98 - A:ILE88

4.68749

Hydrophobic

EKVNELSK:C98 - A:ARG124

4.24887

Hydrophobic

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

A)

B)

C)

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Graphic for table of contents

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