Graduation Year

2020

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Physics

Major Professor

Jianjun Pan, Ph.D.

Committee Member

Jianfeng Cai, Ph.D.

Committee Member

Sagar Pandit, Ph.D.

Committee Member

Martin Muschol, Ph.D.

Committee Member

Ghanim Ullah, Ph.D.

Keywords

AFM, Colistin, ULV, SUV, GUV, Lipid Bilayer, Matrix Protein and Endophilin

Abstract

Peptide-membrane interactions depict the cell’s response to an external molecule. This is a critical event to evaluate the peptide’s function and effect as well as the response of target molecule. The understanding of the mechanism of action of peptide in a molecular level is important, for example, this may be useful in developing the therapeutic strategy. Peptides are the functional macromolecules which are actively researched among bio-related fields. Model membrane systems that mimic the real cell have been useful platform to test the perturbation upon peptide addition. In this thesis, we investigate the membrane modulation behavior of three peptides (modulators) such as colistin, matrix protein, and N-endophilin on the model as well as real membrane by using several experimental techniques.

Polymyxin-E or Colistin; first peptide we used in my thesis turns out to increase the permeability of bilayer and impair the membrane of Gram negative (G (-)) bacteria is also known as a class of an antimicrobial peptide (AMP). Understanding how AMP interacts with pathogens is important in combating with the multidrug resistant bacteria. We are interested in the molecular mode of action of colistin on lipopolysaccharides (LPS) containing model membrane and the G (-) bacteria in native state. LPS is a structural molecule presents in the outer leaflet of outer membrane of G (-) bacteria which stands for their structural integrity. At the meantime, its inherent property of bearing negative charge and hydrophobic center lead the central target for antibiotics. Colistin being an AMP has become a last line of resort combating pathogens. Our ULV leakage experiment showed the permeabilization of membrane in a concentration as well as LPS dependent manner. Supported bilayer measurement by liquid atomic fore microscopy (AFM) reveled the structural change in the form of nano to macro cluster formation on similar dependence. To address the modulations caused by colistin binding to LPS; a lipid clustering model was proposed. We also reported the morphological modulation on E. Coli upon in vivo exposure to the increasing concentration of colistin by using dry AFM. The reaction of colistin against the bacteria was examined considering two variables which play the role i.e. incubation period and the concentration of peptide and elucidated the extent and the degree of bacterial membrane modification. Finally, determination of physical parameter such as roughness of bacterial outer surface and the measurement of their dimension using AFM images support the mode of action. This provides an insight in understanding the mode of impairing the outer membrane and hence mechanism of killing of the bacteria.

The second peptide we used in my study is a segment of matrix protein which turns out to cause the lipid assemblage and membrane fusion. This protein has been known as the critical component of structural poly-protein gag of HIV-1 virus. The matrix domain (MA) with myristoylation motif of gag of human immunodeficiency virus type 1 is believe to be required in general for assemble process of env protein during the viral particle production after the infection of host cell. Here, we studied the broad spectral effect of 14-31 segment of MA alone i.e. MA14-31 on lipids which contains the highly basic region to play a critical role in targeting the lipids at plasma membrane. We used fluorescence microscopy (FM) to observe the real time effect of peptide upon its exposure to the model membranes in a vesicular form (i.e. Giant Unilamellar Vesicle (GUV)). This test revealed the evolution of up to the micron sized vesicles around the parent vesicles either leading to a breakage event or remain intact. Interestingly, this effect was obtained to be specific to the lipids head and suppressed upon the involvement of cholesterol. Atomic force microscopy (AFM) employed on the supported lipid bilayer (SLB) indicates the perturbation of the lipid by forming the transmembrane hole or rupture of depth ~5 nm and the planar raised structure of height ~1 nm at their proximity. In addition to these features, the bilayer which contains the anionic lipids showed an extra feature which appear to be the flake/bead or bleb like structure of height ~5 nm on the top of bilayer structure. With the involvement of cholesterol, the effect was suppressed quantitatively. Forster Resonance Energy Transfer (FRET) revealed the fusion of nano sized anionic small unilamellar vesicles (SUV) which was further quantified by Dynamic Light Scattering (DLS) and complemented by FM. The effect of MA14-31 in each measurement appeared to be consistent in general. With this observation, we purposed a model of interaction to depict the membrane modulation and evolution of new features due to the peptide. As this study suggests not only the independent potency of peptide without myristoylation requirement for interaction, the specificity of target lipid’s head and the membrane stabilizing role of cholesterol but also comprises the broad spectrum of the abilities of MA14-31 on model membrane which may be helpful in assessing the overall function of matrix protein in viral research.

The third peptide we used in my work is the helix 0 of N-endophilin which turns out to change the membrane’s material property and create the local curvature on membrane. N-endophilin has been recognized as a functional protein enriched in the nerve terminal. N-Endophilin belongs to BAR (BIN/Amphiphysin/Rvs) superfamily. Its N-terminal helix called as helix 0 being the amphipathic in nature known to play a critical role in sensing and generating membrane curvature for synaptic vesicles endocytosis by interacting with other proteins and lipids. However, its actual role remain elusive and controversial. We used electron paramagnetic resonance (EPR) spectroscopy to study the impact on H0-Endo on the material property of lipid membranes such as mobility, bilayer polarity and acyl chain order. Upon addition of the H0-Endo on lipid liposome; we found that the lipid chain mobility is reduced whereas the bilayer polarity of interior increased. This changes were explicit on anionic lipids as compare to the zwitterionic. Interestingly, the involvement of cholesterol has in-between impact. Furthermore, we obtained the decrease in order of lipid upon the measurement of lipid acyl chain orientation on magnetically aligned bicelles. Specifically, the decrease fall in two linear trends i.e. gradual for low peptide to lipid (P/L) ratio followed by that of dramatic (for high P/L). We also used atomic force microscopy (AFM) and fluorescence microscopy to examine the perforation of supported bilayer and remodeling of giant unilammelar vesicles respectively. The AFM reveled the formation of transmembrane hole of up to the micron sized extension. The fluorescence microscopy on the other hand showed the curvature generation in the form of mini vesicles as a cloudy stuff around the parent vesicles. Thus our overall results present the helix 0 as a functional segment of N-endophilin that can actively modulate the material property as depicted by proposed interfacial-bound model, topographical disruption caused by competing adhesive force and line tension and the morphological change in agreement with established wedge-type insertion theory.

The results of three peptides interacting with membrane show their functional ability for membrane modulation which become useful to understand their mechanism of action and hence this knowledge may be applied to the other peptides of similar nature.

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