Expert answer:I have attached my thesis I need you to do 1/ check the format go over all chapters correct grammar and typo and text make sure there no pilgrims 2/ write a whole chapter about the fundamental of Electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy and different type of spin label radicals 3/ make sure to fix reference by adding For any articles or journals that you found online please add a DOI at the end and remove the hyperlink as well. If you have trouble finding any let me know. They are usually at the top by the title and publishers.3/ add the reference below each figure except the results figures 4/ re write the cancelation and just write that by saying that increasing temperature the spin label incubate more in membrane with further explain based on that idea 5/ make sure to arrange the material and method on one chapter6/ the Result on another individual chapter ( make sure to keep the result on thesis file beside the result I have attached on here ) 7/ the discussion on one individual chapter 8/ make sure to fix index I have attached 3 file the first file include the thesis file the second file include the whole result file the third file include the calculation
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calcultion.pdf
21c_peptide_membrane.pdf
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PROBING THE ACTION OF MAGAININ PEPTIDE IN LARGE
UNILAMELLAR VESICLES VIA SITE-DIRECTED SPIN LABELING
ELECTRON PARAMAGNETIC RESONANCE TECHNIQUE
A Thesis
Presented to
The Faculty of the Department of Chemistry and Biochemistry
California State University, Los Angeles
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
in
Analytical
By
Bayan H ALharbi
2017
© 2017
Bayan H ALharbi
ALL RIGHTS RESERVED
ii
The thesis of Bayan H ALharbi is approved.
Alison McCurdy, Committee Chair
Michael Hayes
Yangyang Liu
Alison McCurdy, Department Chair
California State University, Los Angeles
December 2017
iii
Abstract
Probing the Action of Magainin Peptide in Large Unilamellar Vesicles via Site-Directed
Spin Labeling Electron Paramagnetic Resonance Technique
By
Bayan H ALharbi
Magainin is found in the antimicrobial peptides family and has been proven to have
antibiotic activities in various living things or organisms. Recent researches have also
confirmed an important magainin II antitumor effects against a wide range of cancerous
cells tumor mice models and lines. It is believed that Magainin interacts with the cell
membrane which is usually rich in phospholipids acid. The interaction between Magainin
and cell membranes makes Magainin form channels of ion-permeable in the cell membrane
which kills or denatures the cells that are affected. The peptides of Magainin II exert
antiproliferative and cytotoxic efficacy through forming pores in the cancer cells of the
bladder which do not affect the normal human fibroblasts or normal murine. Furthermore,
Magainin II might provide a strategy of novel therapeutic in the bladder cancer treatment
with probably weak effects of cytotoxic on the healthy cells. The objective of this study is
to identify the mechanism of microbial cell killing peptides; The hypothesize is that
magainin peptides undergo a structural change upon interaction with cell membranes
through, a secondary or tertiary alteration that neutralizes acidic amino acids exposed on
the cell membrane. The structural dynamics, interaction, and topology of a magainin
peptide in a bilayer lipid vesicle will be investigated via spectroscopy of electron
paramagnetic resonance (EPR) using a spin that is site-directed labeled (a method for
examining the structure and local dynamics of peptides) magainin peptide.
iv
The dire needs for new antibiotics and anticancer drugs require the understanding of
multiple mechanisms of potential antimicrobial and anticancer activities.
v
Acknowledgments
My thanks go to both my family and Dr. Ba for their guidance, direction,
assistance, and assistance. I also would like to thank my Country (Saudi Arabia SCAM)
for support me financial during my accommodation in the United States of America.
Without forgetting my lab/classmates for their support they accorded to me in my
research.
vi
TABLE OF CONTENTS
Abstract ……………………………………………………………………………………………………………… iv
Acknowledgments……………………………………………………………………………………………….. vi
List of Tables ……………………………………………………………………………………………………… ix
List of Figures ……………………………………………………………………………………………………….x
Chapter
1. Introduction ……………………………………………………………………………………………….1
Introduction ………………………………………………………………………………………….1
Background Information …………………………………………………………………………3
Objectives ……………………………………………………………………………………………9
Significance…………………………………………………………………………………………11
2. Types of AMPs ………………………………………………………………………………………..12
Introduction ……………………………………………………………………………………….12
2.1 Cecropins ………………………………………………………………………………………14
2.2 Mellitin………………………………………………………………………………………….17
2.3 Cecropin-Mellitin Hybrids ………………………………………………………………18
2.4 Interactions of Peptide-Membrane …………………………………………………….22
2.5 Importance of the Peptide Interactions ………………………………………………29
2.6 Formation of Pores and Osmotic Stress ……………………………………………..31
3. The Models in the AMPs …………………………………………………………………………..33
3.0 Mechanism of the Membrane Disruption …………………………………………..33
3.1 Detergent-Like “Carpet” Model ……………………………………………………….35
3.2 The Barrel-Stave Model …………………………………………………………………..38
vii
3.3 Toroidal Pore Model ……………………………………………………………………….40
4. The Classification of AMPs ……………………………………………………………………………..44
4.0 Introduction ………………………………………………………………………………………..44
4.1 Beta- Sheet Anticancer Peptides …………………………………………………………….44
4.1.1 Defensins ……………………………………………………………………………………….44
4.1.2 Lactoferricin ………………………………………………………………………………………45
4.1.3 Tachyplesin I ……………………………………………………………………………………..46
4.2 Alpha-helical anticancer peptides ……………………………………………………………47
4.2.1 BMAP-27 and BMAP-28 …………………………………………………………………..47
4.2.2 Cecropin A and Cecropin B …………………………………………………………………47
4.2.3 Magainin ………………………………………………………………………………………….48
5. Materials, Methods and Conclusion …………………………………………………………………..52
6. Discussion and Conclusion ……………………………………………………………………………….71
References …………………………………………………………………………………………………………..75
viii
LIST OF TABLES
Table
1. Representative of Amino Acid Sequence …………………………………………………………8
2. Cecropin A, Amino Acids Sequence ……………………………………………………………..11
3. The EPR Simulation Results…………………………………………………………………………48
ix
LIST OF FIGURES
Figure
1. D Structure of Magainin AMP …………………………………………………………………….15
2. Distributions of Non-Polar and Polar Amino Acids ……………………………………….17
3. Association of the Amphipathic…………………………………………………………………..19
4. Single-Cysteine Analogs of CM15 ………………………………………………………………22
5. Cysteine Labeling ……………………………………………………………………………………..22
6. Bilayer Depth ……………………………………………………………………………………………23
7. Circular Dichroism Spectra of CM15 ……………………………………………………………24
8. Localization of the Membrane-bound CM15 …………………………………………………24
9. Models of Transmembrane Channel Formation ……………………………………………..30
10. Model of Membrane Disruption by the Carpet Mechanism …………………………….31
11. Detergent-Like “Carpet” Model ………………………………………………………………….32
12. The Barrel-Stave Model ……………………………………………………………………………..34
13. The Toroidal Pore Model ……………………………………………………………………………36
14. The Structure of the Magainins……………………………………………………………………40
15. MALDI-TOF of Protein K without Radical API ………………………………………….40
16. EPR of Protein 2K (Graphical representation of the data) …………………………….41
17. The Structure of DOPC………………………………………………………………………………42
18 Photomicrograph of the Membrane …………………………………………………………….43
19.
Photomicrograph of the Membrane …………………………………………………………..44
20.
Photomicrograph of the Membrane …………………………………………………………..44
x
21.
Photomicrograph of the Membrane …………………………………………………………..44
22.
Photomicrograph of the Membrane …………………………………………………………..44
23.
Detailed Parameters of the Lipids ……………………………………………………………..45
24. Results for the attachment of the Peptide to the Membrane …………………………..45
25. EPR spectra of magainin with membrane cell at 23 0C………….…………….46
26. EPR spectra of magainin with membrane cell at 25 0C ………………………………….46
27. EPR spectra of magainin with membrane cell at 27 0C ………………………………….47
28. EPR spectra of magainin with membrane cell at 28 0C ………………………………….47
29. EPR spectra of magainin with membrane cell at 29 0C ………………………………….48
30. EPR spectra of magainin with membrane cell at 30 0C ………………………………….48
31. EPR spectra of magainin with membrane cell at 23 0C …………………………………49
32. EPR spectra of magainin with membrane cell at 25 0C ………………………………….49
33. EPR spectra of magainin with membrane cell at 27 0C ………………………………….50
34. EPR spectra of magainin with membrane cell at 28 0C …………………………………..50
35. EPR spectra of magainin with membrane cell at 29 0C ………………………………….51
36. EPR spectra of magainin with membrane cell at 30 0C …………………………………..51
xi
CHAPTER 1
conventional treatment
Introduction
Cancer stills a major mortality and morbidity source worldwide. In the U.S, cancer
is a major source of death for most people who have not attained eighty-five years and
above (Carmieli et al, 2006). Moreover, the numerous cancers rate that includes cancer of
the breast, cancer of the skin cancer of the kidney cancer and cancer of the prostate are in
continuous trend. Nevertheless, each kind of cancer is featured by abnormal development
of cells that result from a fundamentally environmentally-induced mutation of genes or a
modest number of inherited genetic mutation (Milov, et al, 2006). It worth noting that cells
must possess six unique characteristics to be characterized as cancerous; 1) the abilities of
generating their respond signals of growth of less strong growth which the tissues and
responsible for healthy body and life ignore; 2) antiproliferative signs insensitivity; 3)
cellular suicide impedance mechanism which usually causes the death of aberrant cells by
apoptosis; 4) the limit regarding the replication of the boundless; 5) the capability of
fortifying fresh recruits of development vessels that allows for the growth of tumor; 6) the
ability of the tissues cells and tissues of the attack, initially at local level, but later
metastasize or spread all over the bodies (Sato and Feix, 2006). Nevertheless, the treatment
of localized cancerous cells is possible can through radiation therapy, chemotherapy, and
surgery. However, chemotherapy is still the typical choice treatment for metastatic or
advanced cancer.
The development of conventional antibiotics resistance issues has been worldwide
public care issue and the demands for advanced antibiotics have accelerated the
Antimicrobial peptides (AMPs) development as the chemotherapeutic agents of the human
1
therapeutics convectional which develop to target the cancerous cells have impacted
negatively to the healthy cells and are harmful to the cells as well (Carmieli et al, 2006).
Because of changes of the cellular including drug transporters’ increased expression and
drug detoxifying enzymes, alteration of interaction between the drugs and the substrate,
increased abilities to repair DNA defects and damage in the machinery of the cellular which
mediate the cancerous cells of apoptosis develop chemotherapy resistance that causes
antibiotics deactivation.
Antimicrobial peptides (AMPs) therefore, would be a major improvement in the
treatment of cancer because they do not have conventional chemotherapeutic agents’
toxicity and they do not affect the healthy cells and tissues (Milov, et al, 2006). The
antimicrobial peptides (AMPs) are short peptides chains which act as a defensive weapon
against the invasion of pathogens and microorganisms. Various living organisms such as
insects, bacteria, plants as animals, use Antimicrobial peptides (AMPs) to safeguard
themselves against the invasions of the pathogens and microorganisms. In the recent past,
studies on anticancer techniques like chemotherapy have been characterized by various
harmful and negative side effects (Sato and Feix, 2006). The presently used anticancer
drugs pay more attention to the cells that are high proliferated that cause destruction even
to the healthy cell which similarly grows at high rates. Furthermore, the origination of the
Multi-Drug Resistance (MDR) has threatened the cancerous cells features which hinder the
efficiency of the anticancer drug (Gordon-Grossman, et al, 2011). The antimicrobial
peptides (AMPs) can result in the formation of the pores on the surface of the cancerous
cells lipid membranes and therefore triggering their destructions because the cells are not
able to produce the resistance (McMahon, Alfieri, Clark, and Londergan, 2010). The
2
cancerous cells are therefore rendered susceptible to the anticancer drugs as well as the
mechanisms of the body immune. Antimicrobial peptides (AMPs) are not the only peptides
that have the capacity to penetrate the cell membrane, but other peptides like the Cellpenetrating peptides (CCPs) that are as well protein celled, short compounds of
transduction domains that comprise of up to thirty residues of amino acids (AA) that can
enter nuclear and mitochondrial membranes as well as plasmalemmal, without damaging
the membranes (Carmieli et al, 2006). Cell-penetrating peptides (CCPs) and Antimicrobial
peptides (AMPs) consist of various peptide groups that can freely move by the use of
advanced mechanisms across the cell membrane. The cell membranes such as cytoplasm
membrane or plasma membrane are biological membranes which have dynamic
characteristics which separate all the cells’ interior for both eukaryotic and prokaryotic
from the external environment (Milov, et al, 2006). The cell membranes have multilayered
structures with multiplicities of compounds like molecules, lipids, cholesterols, particles
of phosphorous among others. The cell membranes are semi-permeable to certain
molecules like ions and other molecules from accessing to the cell interior.
Background Information
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 microbes (Sato
and Feix, 2006). It is known that chemotherapy results in 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
3
Resistance) that hinder the effectiveness of the anticancer drugs. It was recently reported
that the antimicrobial peptides can create pores in the lipid membranes of the cancerous
cells and hence trigger their destructions since they become unable to form the resistance
(Carmieli et al, 2006). The cancer cells are hence rendered susceptible to the body immune
mechanism as well as the anticancer drugs. AMPs peptides are not only the ones with the
ability to enter the membrane cell, other peptides such as the cell-penetrating peptides
(CCPs) can also 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 (Milov, et al, 2006). The cell membrane (plasma membrane or
cytoplasm membrane) is a biological membrane with a dynamic feature that separates the
interior of all cells including both prokaryotic and eukaryotic from outside environment
(Gordon-Grossman, et al, 2011). The cell membrane has the multilayered structure with a
multiplicity of compounds such as lipids, cholesterol, molecules of phosphorous and many
others. The cell membrane is selectively permeable to ions and other materials to the
interior of a cell.
Cell-penetrating peptide (CPP) refers to the short peptide which is intended to
promote cellular uptake or intake of different molecular equipment. The relation between
cargo and peptides is established either through chemical linkage via covalent bonds or
through non-covalent interactions (McMahon, Alfieri, Clark, and Londergan, 2010). The
primary purpose of the cell-penetrating peptides is to pass the cargo to cells, a process that
usually happens through endocytosis. Nowadays, the use of cell-penetrating peptides is
4
restricted due to the fact of a lack of knowledge and insufficient understanding of the modes
of their uptake.
Antimicrobial peptide (AMP) commonly known as host defense peptide (HPD)
refers to a part of the innate immune response that can be observed in representatives of all
classes of life (Carmieli et al, 2006). Such peptides refer to potent and broad-spectrum
antibiotics. Antimicrobial peptides are able to kill enveloped viruses and cancerous cells.
AMPs may also have the capability to improve immunity by working as
immunomodulators. The antimicrobial peptide is a distinct and unique group of molecules
divided into some subgroup (Milov, et al, 2006). The division is based on the structure and
composition of their amino acid. Antimicrobial peptide includes around fifteen to fifty
amino acids.
5
CHAPTER 2
Types of Antimicrobial Peptides (AMPs)
Introduction
The continuous rise of multi-drug resistant prevalence to pathogens is a worrying
health concern worldwide. Recent studies show that 57 percent of infections of
Staphylococcus aureus as for 2003, the intensive care units in the United States showed
resistant to various antibiotics. Moreover, studies also indicate that there is an emergence
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