Please use EndNote program to organize your references.

Probing Aaction of the Magainin Peptideprotein in Large Unilamellar Vesicles via Site-

Directed Spin Labeling and Electron Paramagnetic Resonance TechniqueSpectroscopy.

Bayan Hassan

Mentor: Dr. Ba

California University, Los Angeles

Department of Chemistry and Biochemistry

1
Abstract:

Magainins belong toare among the developing class of antimicrobial peptides, being

membrane dynamic peptides with antimicrobial action. It was believed that magainins interact

kills cell through interaction with cell membranes which are cell rich in acidic phospholipids. As

a result, mMagainins form ion-permeable channels in the membrane that lead to cell death.

Furthermore, Magainins acts via self-association within a membrane rather than via interaction

with a chiral center which usually contains at least one carbon atom and four non identical

substituents such as bilayer phospholipids. The structural dynamic, interaction and topology of a

magainin in bilayer lipid vesicles will be investigated using a site-directed spin labeled

magainining and electron paramagnetic resonance spectroscopy (EPR). Furthermore, Magainin

acts via self-association within a membrane rather than via interaction with a chiral center.

Moreover, the natural D-amino acid enantiomers contained in the peptide are not sensitive to

proteolytic cleavage, which is favorable for the development of these peptides as therapeutic

agents.

2
Objective:

To study the mechanism of microbial cell killing peptides, by magainins, which are active

inconnects phospholipid biwith layers rich in acidic phospholipids.? InteractionConnection with

the lipid bilayers prompts this class of peptides to shape a secondary structure, an amphiphilic a-

helix described by a cationic and hydrophobic face, in the bilayer. Describe by a cationic and

hydrophobic face. Previous experimental resultsConfirm recommends that a bunch ofthe peptide

molecules assemble together to form ion-permeable channels within the membrane that

ultimately leading to cell death.

Complete this section with the following items:

(1) Hypothesis: (What is your opinion that magainins can cause the cell death of the

microbial?)

(2) Method to prove the hypothesis: (Design of experiment to prove your hypothesis.)

(3) Impact of your research in the field of developing antimicrobial agents:

3
Background:

AMPs (antimicrobial peptides) are chains of short peptides that function as defensive

weapons against the invading microorganisms. Different animals, plants, insects, as well as

bacteria, use AMPs to protect themselves from the invasions by microbesial (Lin Yang). It is

known that In the recent past, research on anticancer approaches such as the chemotherapy result

inis met by several side effects. The currently applied anticancer drugs focus on the high

proliferated cells; these drugs do not spare even the healthy cells that grow at similarly high or

even lower rates. Moreover, there has originated another yet threatening character of the

cancerous cells; the MDR (Multi Drug Resistance) that hinder the efficient of the anticancer

drugs. It was recently reported that tThe antimicrobial peptides can createause the pores to form

4
on the lipid membranes of the cancerous cells and hence trigger their destructions since they

become unable to form the resistance (Tomas, 2003). (references?) The cancer cells are hence

rendered susceptible to the body immune mechanism as well as the anticancer drugs (Xuan Liu).

AMPs peptides are not the only the ones with the ability to enter the membrane cell, but other

peptides such as the Cellcell-penetrating peptides (CCPs) can also penetrate the plasmalemmal,

as well as mitochondrial and nuclear membranes without causing any damage to the membranes.

which also celled protein transduction domains are short compounds, comprising up to 30 amino

acid (AA) residues, which can penetrate the plasmalemmal as well as mitochondrial and nuclear

membranes, without causing any damage to the membranes. AMPs and CCPs are part of several

peptides groups with the ability to move freely using advanced mechanism across the cell

membrane (Becher and Sagan). The cell membrane (plasma membrane or cytoplasm membrane)

is a biological membrane with a dynamic feature that separates the interior of all cells

includingfor both prokaryotic and eukaryotic from outside environment. Cell membraneIt hasis

multilayered structure with a multiplicity of compounds such as lipids, cholesterol, molecules of

phosphorous and many others (Sahu). The cell membrane is selectively permeable to such as

ions and other materials to have access to the interior of a cell (Mitra)

AMPs are currently classified in several ways, depending on their individual properties.

AMPs can be classified by the functions of their original proteins, their uptake mechanisms,

intracellular evoked reactions, and their chemical properties. These groups are described

below:including

 Beta-sheet anticancer peptides

5
1. Defensins

Defensins are a part of closely- related Cys-Arg-Rich, cationic AMPs consist of 29 to 45 amino

acid residues. Defensins can form three intermolecular disulfide bonds between the NH2-terminal

and COOH- terminal regions of the peptide, which gives cyclic, triple-stranded amphiphilic beta

sheet structure that gives the feature of defensins - like. Defensins are isolated from different

sources. Tthe best known examples related to alpha helix and beta sheets areones are alpha and

beta defenses from human.

The best known are Human neutrophil peptides such as

(HNPs)-1 (ACYCRIPACIAGRRYGTCIYQGCC),

(HNPs)-2(CYCRIPACIAGERRYGTCIYQGRLWAFCC) and

(HNPs)-3(DCYCRIPACIAGERRYGTCIYQGRLWAFCC) of which HNPs – 3 are belong to

alpha defensines.

Hypothesis:

At high concentration (> 25 mg/ml) of alpha defensins suppress DNA synthesis in renal cell

carcinoma lines, as well as reducing cancer cell viability.

Method:

Tumor cells killeding by alpha defensins throughincluding membrane binding event which is,

through mediated by electrostatic interaction followed by rapid collapse of the membrane

potential and loss of membrane integrity. Membrane permeabilization by alpha defensins has

6
been attributed to the channel- forming ability of these peptides because HNP-1 has been shown

to form voltage- dependent, ion -permeable channels in planar phospholipids bilayer membranes.

2. Lactoferricin

It is a cationic AMP with 25 amion acid residues ( FKCRRWQWRMKLGAPSITCVRRAF).

Lactoferricin isolated from cows milk consist of two cys that creat dislified bond linking between

NH2- terminal region and COOH-terminal region of the peptide.

Hypothesis:

The cytotoxic activity of the BOVINE LACTOFERRICIN (LfcinB (this name has not been

defined) against cancer cell depend on both amphipatic structure and high net positive charge of

the peptide .

Method:

LifcinB binds to cell membrane , causing membrane intergrity to be lost as a result the

formation of transmembrane pores which allow the peptide to enter the cytoplasmic

compartment of cancer cell and co localize with negatively -charged mitochondria.

3. Tachyplesin I

This peptide has 17 Amino Acid Residues (KWCFRVCYRGICYRRCR).

Hypothesis:

7
Interaction between tachyplesin I and hyaluronan are believed to contribute to selective killing of

cancer cells by tachyplesin I since many tumor cells tend to express hyaluronan at levels that are

much higher than those found on normal tissue.

Method:

Tachyplesin I have a unique method toof killing cancer cells., Rrecent study shows that,

tachyplesin I was shown to bind to hyaluronan on hyaluronan-over expressing human TSU

prostate carcinoma cells, as well as to Complementary Component C I q in human serum,

leading to activation of the classical complement pathway and complement-mediated lysis of

tachyplesin I-coated cancer cells.

 Alpha- Helical anticancer peptides

1.BMAP-27 and BMAP-28

BMAP-27 ( GRFKRFRKKFKKLFKKLSPVIPLLHL) and

BMAP-28 ( GGLRSlGILRAWKKYGPIIVPIIRI) wereit predicted to form alpha helicesx due to

the cationic NH2- terminal that is followed by a hydrophopic tail at COOH- terminal.

Hypothesis:

Both BMAP-27 and BMAP-28 are showed awere found to cause dramatic reduction in

membrane permeablizing activity.

Method:

BMAP peptides caused a rapid reduction in mitochondria membrane potential in situ that was

induced opeing of the mitochondria permeability transition pore that result in cytochrom C

release and initiation of cell death by apoptosis.

8
2.Cercropin A and B

Ceropin A ( KWKLFKKEKVGQNIRDGIIKAGPAVAVVGQATQIAK) and

Ceropin B (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL) were derived from insect

sources . They consist of two alpha helices that are characterized by the secondary structurres of

the peptides.

Hypothesis:

Cercropin A and B have selective and cytotoxic efficancy by taracting nonpolar lipid cell

membrane and forming ion-permeable channels.

Method :

Cercropin A and B can lys cancer cells at concentraation below that which damage normal cells

Cecropin B and analogs have IC50 values which ranged from 3-17 mm against different cell

lines. Ccecropin B also showed in vitro killing activity gainst multi drug resistance humman

breast cancer cell lines and also in vivo anticncer effects in mice with colon adenocarcinomas

The analysis of the effect of cecropin B on Ags humman stomach carcinoma cell line revealed

that peptide treatment caused short outward currents that were consistent with the formation of

transient channel like pore while the cecropin A analogue faild to form pore.

3.Magainins

The wild type magainin is anAn α-helical peptide isolated from the African clawed frog Xenopus

laevis.

The original sequence is:

9
GIKKFLHIIWKFIKAFVGEIMNSNS-NH2;

Our modifiedThe modify sequence is:

GIKKFLHIIWKFIKAFVGEIMNCNC-NH2.

In the latter one, Replaced sSerine residue is replaced withwith cysteine residue as shown in the

diagram, R- Residue (Indicated as R).

Cytosine which is located on the core of the protein forms a disulfide bond and is almost similar

to cysteineAlmost the same but the main different is the cytosine is located on the core of protein

and form disulfide bond. While, serine can form hydrogen bond and found on the surface

interacting with water.

Hypothesis

10
Magainin acts through self-association within a membrane rather than through interaction with a

chiral center. Furthermore, the α- o-amino acid enantiomers commonly used in the biosynthesis

of proteins particularly in some methanogenic archaea as well as Bacteria are not sensitive to

proteolytic cleavage a property that has been suggested to be favorable for the development of

these peptides as therapeutic agents. It was suggested that

Method:

mMagainin suggested forming a toroidal pore with a diameter of 2–3 nm, inducing lipid flip-flop

and the translocation of peptides into the inner leaflet of the bilayer coupled to membrane

permeabilization. However, the interactions of magainin AMPs with bacterial membranes are not

well-characterized. For instance, membrane potential assays using bacterial cells reveal the

kinetics of the permeabilization, but do not give information on pore size, which is essential for

discriminating between various proposed models of membrane permeabilization. Electron

microscopic observations failed to capture the dynamic process of membrane permeabilization.

However, this process can be improved by using electroporation where temporary pores are

introduced by the assistance of high external direct current (DC) electrical field or the use of an

exponentially decaying pulses. When this is done, the dynamic process of membrane

permeabilization can be observed.

 AMPs and the cell penetration peptides, CPPS, are very similarly. CPPs are basically

short peptides facilitating either the cellular intake or uptake of several forms of

molecular equipment. However, there are a few differences that distinguish between even

though they have comparable characteristics. Bboth are made of short amino acid chains

11
 with the ability to pandurate the cell membrane using more than onedifferent

mechanisms, and, both are cationic in nature (Ganz). These peptides have drawn much

attention due to their ability to cross cell membrane with low toxicity making them

promisingthe perfect to detect a new class of therapeutics. techniqu The study of these

peptides can also help toe and for understanding the transportation materials acrossinside

and outside the cell through the cell membrane. (Epand and Vogel).

 CPPs and AMPs are alsoThey distinguished in some aspectsbetween CPPs and AMPs.

First, is the fact that CPPs transport various

 macro-molecules across the cell membrane while the AMPs fight invading

microorganisms. Second, for CPPs to access inside the cell it used endocytosis

pathway but AMPs depends totally on their self-induced action to pandurate the cell

membrane.

 Third, by considering the field of research they are useful in. CPPs are mostly useful

for gene therapies and cellular drugs delivery methods. While the AMPs are useful

in researching a new way to control bacteria cell (Henriques, Melo and castanho).

AMPs are also known also as multifunctional molecules due to the fact that they stimulate a

myriad of activities in the cells and can be applied in various useful activities in the human life

(Dennison and Harris)

 Some of the actions toin triggers in the cellular activities ofbody of an organism include

stimulation of the production of cytokines, prevention of the making of the protein C that

is responsible for reducing chances of cell death and increasing cell permeability in

animals, spurring tumor cell lysis stimulating adhesion of molecules expression and many

12
 other activities in the cell pointing to the vitality of these peptides (Balandin, Emelianova

).

 AMP peptides use various means to pass through the plasma membrane when AMPs are

characterized according to the methods they use to access the cytosol, theywhich are

classified into two categories includingthat are lytic and non-lytic. T AMPs these two

distinct mechanisms can also be termed as either pore dependent or pore independent.

The pore dependent mechanism is the once in which these peptides induce the

permeability throughof the cell membrane.

 This cell membrane is made more porous through the carpet model in which AMPs

assemble on the surface of the membrane to disrupt the membrane through barrel-

stave, toroidal -pore. Most of the AMPs have more than 40 amino acids in length and

they also possess a 3D structure having a kink at the middle.

3D Model of AMP structure (Source: "File:Adenosine-monophosphate-anion-3D-balls.png -

Wikimedia Commons", 2017)

13
(Please have the AMP’s 3D structure in the prospectus. The 3D structure could be obtained from

a literature, or you may make one using computational programs. Describe the structural

properties.)

 Carpet Model

In this model, the peptides disrupt the membrane by orienting parallel to the surface of

the lipid bilayer and forming an extensive layer or carpet. Hydrophilic regions of the

peptide are shown red, hydrophobic regions of the peptide are shown blue.

 The barrel-stave model

In this model, the attached peptides aggregate and insert into the membrane bilayer so

that the hydrophobic peptide regions align with the lipid core region and the hydrophilic

peptide regions form the interior region of the pore.

14
  Toroidal pore model

In this model, the attached peptides aggregate and induce the lipid monolayer to bend

continuously through the pore so that the water core is lined by both the inserted peptides

and the lipid head groups.

15
Method

Spin labeling technique is useful in determining the structural organization of proteins. It

involves investigation of the structure and local dynamics of proteins and peptide units using

electron spin resonance.

Step 1: investigating the structure and local dynamics of proteins using spin labeled

AMPsing

1) .5 mg of protein was weighed,t then, dissolved in 500 micro liter buffer (AFP) pH=7.4...

2) . 5.09 mg of Inhibitor of apoptosis (APIAIP) was weight then, dissolve in 0.25 ml ethanol

3). Transfer the spin label ethanol solution to the AFP AMP buffer solution. Close the cap and

shake to mixture.

4. Shake with vortex and allow the reaction for 2-4 hours in athe cold room at 4oC.

5). Lyophizle theyour sample.

6). Dialysis in deionized water at 4oC. Three changes of water at 1 h, 2 h, 4 h and overnight.

7). Lyophilize the sample and store it at -20oC.

Step 2: prepare large unilamellar vesicles.

Generally, unilamellar vesicles are spherical liposome chambers bound by a single bilayer of an

amphiphilic fluid contained within the chamber. They are used as transportation vehicle for the

artificial administration of either pharmaceutical drugs or nutrients or both. zwitterionic

phospholipids form the major phospholipid composition in unilamellar vesicles and are the key

16
vehicular agents. These lipids form lamellar phases having an equilibrium water gap of 2 to 3 nm

due the dominating van der Waals forces of attraction between dipolar bilayers. The description

below shows how these vesicles can be formed.

1. A lipid of 3mg is dissolved in 1 ml chloroform.

2. The above solution is then heated till it evaporates. It should be done in a vacuum,

within a cylindrical reaction vessel. As the process takes place, the lipid separates

onto the walls of the reaction vessel and a lipid film of homogenous appearance is

formed on the walls (Avanti Lipids Polar, Inc 1).

3. Salt solution (≤ 1Mm KCL) or distilled water (5-10 ml) is added.

4. The lipid film is lyophilized for 1-4h. It is done under a 70℃ water bath without

disturbance. The lipid film separates from the walls of the vessel, and forms lipid

liposomes of ¿1 μm in diameter. In the period of 4h, the liposomes form a large

sphere of approximately 5-10 mm in diameter (Avanti Lipids Polar, Inc 1).

5. Gently shake the vessel for 5 s, which will result in disintegration of the lipid

sphere. It will, however, reform vesicles which have 1-50 μm in

diameter. Shaking with a greater force may also result in vesicle formation

(Avanti Lipids Polar, Inc 1).

6. Clean the apparatus after use.

Note: It is important to note that the size and amount of vesicles relies on the time used in

incubation, and the time taken to shake the sample at 70℃. The smaller sizes allow for fast

movement within the cells hence faster transportation of the nutrients. The unilamellar vesicles

17
were prepared by hydrating the lipid films so as to establish the large sized molecules. These can

then be used as control set up to monitor any other changes.

Significance

Magainin is a cationic alpha helical anticancer and antimicrobial peptide,, lacking the toxicity of

conventional chemotherapeutic agent without excessive damage to t without harming the normal

cell. Unlike the conventional antibiotic that based on targeting specific target protein, ,

magaininand can cause membrane permeabillizezation membrane leading to cell death, unlike

the conventional antibiotic that based on targeting specific target protein. Therefore, Magainin is

excellent antibiotics for human therapeutics. (Hoskin, Ramamoorthy). The significance of the

research findings are that it can establish the optimum vesicles to be used as vehicles for

transportation of nutrients and pharmaceutical drugs into the cell. This is vital because only the

targeted cells require the nutrients or pharmaceutical drugs and because of this, there will be

higher efficiencies in treatment with very minimal side effects since only the right parts are

targeted.

18
Result:

Figure 1: MALDI-TOF of protein 2K that functions as a signal peptide without radical API

19
Figure 2: MALDI-TOF of protein 2K with addition of radical Inhibitor of apoptosis protein

(IAP)API

20
Figure 3: Electron Paramagnetic ResonanceEPR of protein 2K.

Figure 4: Large unilamellar vesicles seen after incubateing at 70 0C.

21
22
 References

1. Bahar, Ali Adem and Dacheng Ren. "Antimicrobial peptides." Pharmaceutical (2013):33

Journal

2. Balandin, S V, et al. "Molecular mechanism of antitumor effect of natural antimicrobial

peptides." Russia Journal of Bioorganic Chemistry (2016):15. Journal

3. Bechara, Cherine and Sandrine Sagan. "Cell Penetrating-peptides: 20 Years Later where

do we stand." Elsevier B.V (2013): 10. Journal.

4. Bechingar, Burk Hard, and Karl Lohner. "Detergents-like actions of linear amphipathic

cationic antimicrobial Peptides." Elsevier Science Direct (2006):10. Journal.

5. Dennison, Sarah R, and Fredrick Harris. "Antimicrobial peptides: Their

Histroty,Evaluation, and Functional promiscuity." (2013): 38. Journal

6. Epand, Richard M and Hans J Vogel. "Diversity of Antimicrobial peptides and their

mechanism of action." Biochimic a et Biopphysica (1999): 17. Journal.

7. Ganz, Tomas "Defensins Antimicrobial peptides of innate immunity." Nature Reviews

8. Immunology (2003): 11. Journal.

9. Henriques, Sonia Troeira. Manuel Melo and Miguel a Castanho. "Cell- Penetrating

peptides and microbial peptides: How different they are." Biochemical Journal (2006):7.

Journal.

10. Fuminori Yoneyama, Yuichi Imura, Kanako Ohno, Takeshi Zendo, Jiro Nakayama,

Katsumi Matsuzaki, and Kenji Sonomoto. "Peptide-Lipid Huge Toroidal Pore, a New

Antimicrobial Mechanism Mediated by a Lactococcal Bacteriocin, Lacticin Q."

American Society for Microbiology (2009): 3211–3217. Print.

11. Hardy, Jay. Small, But Potent Killers. Santa Maria: Hardy Diagnostics, 2015. Print.ont

23
Lin Yang, Thomas M. Weiss, Robert I. Lehrer, and Huey W. Huang. "Crystallization of

Antimicrobial Pores in Membranes: Magainin and Protegrin." Biophysical Journal

(2009): 2002-2003. Print.

12. Mohammad Abu Sayem Karal, Jahangir Md. Alam, Tomoki Takahashi, Victor Levadny

and Masahito Yamazaki. "Stretch-Activated Pore of the Antimicrobial Peptide, Magainin

2." American Chemical Society (2015): 1-2. Print.

13.Shai, Yechiel. "Mechanism of the binding, insertion and destabilization of phospholipid."

Elsevier (1999): 55-70. Print.

14.Tomàs Pérez, Víctor. Antimicrobial Peptides (AMPs). Barcelona: universitat autónoma de

Barcelona, 2015. Print.

15.. Xuan Liu, Yi Li, Zhi Li, Xiqian Lan, Polly Hang-Mei Leung, Jiashen Li, Mo Yang, Frank

Ko, Ling Qin. "Mechanism of Anticancer Effects of." Journal of Fiber Bioengineering

and Informatics (2015): 25-36. Print.

16. Yamazaki, Masahito. Kinetic Pathway of Antimicrobial Peptide. Workshop. Shizuoka :

Shizuoka University, 2010. Print.

17. Avanti Lipids Polar, Inc. “Liposome Preparation”. Avanti Lipids Polar, Inc., 2017,

https://avantilipids.com/tech-support/liposome-preparation/.

18. Lu, Jun-Xia, et al "Solid State Nuclear Magnetic Resonance Relaxation Studies of Interaction

Mechanism of Antimicrobial peptides with phospholipid Bilayer Membranes." American

Chemical Society (2005): 10.

19. Mitra, Abhijit. "Structural transitions in membranes: Mechanisms for Cell- Penetrating

peptides." Ahmedabad, n. d. Book.

24
20. Sahu, Indra D. " Probing Structural Dynamic and Topology of the KCNEC 1 membrane

protein in lipid Bilayers via site-Directed Spin Labeling and Electron Paramagnetic Resonance

Spectroscopy." American Chemistry Society (2015): 11. Journal.

22. Hiroaki, Ramamourthy.“Studies on Anticancer Activities of Antimicrobial Peptides”.

Biochim Biophys Acta. 2008 February: 1778(2): 357-375.

23. File:Adenosine-monophosphate-anion-3D-balls.png - Wikimedia Commons. (2017).

Commons.wikimedia.org. Retrieved 19 July 2017, from

https://commons.wikimedia.org/wiki/File:Adenosine-monophosphate-anion-3D-balls.png

25