Pan Lab

https://pubmed.ncbi.nlm.nih.gov/35247332/
Abstract: Amphiphysin and endophilin are two members of the N-BAR protein family. We have reported membrane interactions of the helix 0 of endophilin (H0-Endo). Here we investigate membrane modulations caused by the helix 0 of amphiphysin (H0-Amph). Electron paramagnetic resonance (EPR) spectroscopy was used to explore membrane properties. H0-Amph was found to reduce lipid mobility, make the membrane interior more polar, and decrease lipid chain orientational order. The EPR data also showed that for anionic membranes, H0-Endo acted as a more potent modulator. For instance, at peptide-to-lipid (P/L) ratio of 1/20, the peak-to-peak splitting was increased by 0.27 G and 1.89 G by H0-Amph and H0-Endo, respectively. Similarly, H0-Endo caused a larger change in the bilayer polarity than H0-Amph (30% versus 12% at P/L=1/20). At P/L=1/50, the chain orientational order was decreased by 26% and 66% by H0-Amph and H0-Endo, respectively. The different capabilities were explained by considering hydrophobicity score distributions. We employed atomic force microscopy to investigate membrane structural changes. Both peptides caused the formation of micron-sized holes. Interestingly, only H0-Amph induced membrane fusion as evidenced by the formation of high-rise regions. Lastly, experiments of giant unilamellar vesicles showed that H0-Amph and H0-Endo generated thin tubules and miniscule vesicles, respectively. Together, our studies showed that both helices are effective in altering membrane properties; the observed changes might be important for membrane curvature induction. Importantly, comparisons between the two peptides revealed that the degree of membrane remodeling is dependent on the sequence of the N-terminal helix of the N-BAR protein family. 

https://pubmed.ncbi.nlm.nih.gov/33095508/
Abstract: Amyloid-b peptides (Ab) assemble into both rigid amyloid fibrils and metastable oligomers termed AbO or protofibrils. In Alzheimer s disease, Ab fibrils constitute the core of senile plaques, but Ab protofibrils may represent the main toxic species. Ab protofibrils accumulate at the exterior of senile plaques, yet the protofibril–fibril interplay is not well understood. Applying chemical kinetics and atomic force microscopy to the assembly of Ab and lysozyme, protofibrils are observed to bind to the lateral surfaces of amyloid fibrils. When utilizing Ab variants with different critical oligomer concentrations, the interaction inhibits the autocatalytic pro- liferation of amyloid fibrils by secondary nucleation on the fibril surface. Thus, metastable oligomers antagonize their replacement by amyloid fibrils both by competing for mono- mers and blocking secondary nucleation sites. The protofi- bril—fibril interaction governs their temporal evolution and potential to exert specific toxic activities.

https://pubmed.ncbi.nlm.nih.gov/32533976/
Abstract: The amphipathic helix 0 of endophilin (i.e., H0-Endo) is important to membrane binding, but its function of curvature generation remains controversial. We used electron paramagnetic resonance (EPR) spectroscopy to study effects of H0-Endo on membrane material properties. We found that H0-Endo reduced lipid chain mobility and increased bilayer polarity, i.e., making the bilayer interior more polar. Lipid-dependent examination revealed that anionic lipids augmented the effect of H0-Endo, while cholesterol had a minimal impact. Our EPR spectroscopy of magnetically aligned bicelles showed that as the peptide-to-lipid ratio increased, the lipid chain orientational order decreased gradually, followed by a sudden loss. We discuss an interfacial-bound model of the amphipathic H0-Endo to account for all EPR data. We used atomic force microscopy and fluorescence microscopy to explore membrane morphological changes. We found that H0-Endo caused the formation of micron-sized holes in mica-supported planar bilayers. Hole formation is likely caused by two competing forces – the adhesion force exerted by the substrate represses bilayer budging, whereas the line tension originating from peptide clustering has a tendency of destabilizing bilayer organization. In the absence of substrate influences, membrane curvature induction was manifested by generating small vesicles surrounding giant unilamellar vesicles. Our results of membrane perforation and vesiculation suggest that the functionality of H0-Endo is more than just coordinating membrane binding of endophilin.

https://pubmed.ncbi.nlm.nih.gov/31919432/
Abstract: The abnormal folding and aggregation of functional proteins into amyloid is a typical feature of many age-related diseases, including type ii diabetes. Growing evidence has revealed that the prevention of aggregate formation in culprit proteins could retard the progression of amyloid diseases. Human Amylin, also known as human islet amyloid polypeptide (hiApp), is the major factor for categorizing Type II diabetes as an amyloid disease. Speci cally, hIAPP has a great aggregation potential, which always results in a lethal situation for the pancreas. Many peptide inhibitors have been constructed from the various segments of the full-length hiApp peptide; however, only a few have their origin from the screening of combinatorial peptidomimetic library. In this study, based on HW-155, which was previously discovered from a one–bead–one compound (oBoc) library to inhibit Aβ40 aggregation, we investigated eight (8) analogues and evaluated their amyloid-prevention capabilities for inhibiting brillization of hIAPP. Characterization studies revealed that all analogues of HW-155, as well as HW- 155, were e ective inhibitors of the bril formation by hIAPP.

https://pubmed.ncbi.nlm.nih.gov/30071193/
Abstract: Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.

https://pubmed.ncbi.nlm.nih.gov/30613815/
Abstract: Polyanionic lipopolysaccharides (LPS) play an important role in regulating the permeability of the outer membrane (OM) of Gram-negative bacteria. Impairment of the LPS-enriched OM is essential in initiating the bactericidal activity of polymyxins. We are interested in how colistin (polymyxin E) affects membrane permeability of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. To describe the vesicle leakage behavior from a structural perspective, we performed liquid atomic force microscopy (AFM) measurements on planar lipid bilayers. We found that colistin caused the formation of nano- and macro-clusters that protruded from the bilayer by ~2 nm. Moreover, cluster development was promoted by increasing the fraction of LPS or colistin concentration but inhibited by magnesium ions. To explain our experimental data, we proposed a lipid clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS-colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria.

https://pubmed.ncbi.nlm.nih.gov/30198000/
Abstract: Membrane interactions play an essential role in physiological and pathological functions of the presynaptic protein α-synuclein (αSyn). Here we used atomic force microscopy (AFM) and electron paramagnetic resonance (EPR) spectroscopy to investigate membrane modulations caused by αSyn. Specifically, we used several lipid bilayers to explore how different lipid species regulate αSyn-membrane interactions. We found that at a protein-to-lipid (P/L) ratio of ~1/9, αSyn perturbed lipid bilayers by generating semi-transmembrane defects that only span one leaflet. In addition, αSyn co-aggregates with lipid molecules to produce ~10-nm-sized lipoprotein nanoparticles. The obtained AFM data are consistent with the apolipoprotein characteristic of αSyn. The role of anionic lipids was elucidated by comparing results from zwitterionic and anionic lipid bilayers. Specifically, our AFM measurements showed that anionic bilayers had a larger tendency of forming bilayer defects; similarly, our EPR measurements revealed that anionic bilayers exhibited more substantial changes in lipid mobility and bilayer polarity. We also studied the effect of cholesterol. We found that cholesterol increased the capability of αSyn in inducing bilayer defects and altering lipid mobility and bilayer polarity. These data can be explained by an increase in the lipid headgroup-headgroup spacing or specific cholesterol-αSyn interactions. Interestingly, we found an inhibitory effect of the cone-shaped phosphatidylethanolamine lipids on αSyn-induced bilayer remodeling. We explained our data by considering interlipid hydrogen-bonding that can stabilize the bilayer organization and suppress lipid extraction. Our results of lipid-dependent membrane interactions are likely relevant to αSyn functioning.

https://pubmed.ncbi.nlm.nih.gov/28459565/
Abstract: A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. Force spectroscopy experiment shows that PrP106-126 reduces Young's modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intra-chain conformation, while the inter-chain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the inter-chain interaction, while the intra-chain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs. 

https://pubmed.ncbi.nlm.nih.gov/28132901/
Abstract: Understanding how antimicrobial peptidomimetics interact with lipid membranes is important in battling multidrug resistant bacterial pathogens. We study the effects of a recently reported peptidomimetic on lipid bilayer structural and mechanical properties. The compound referred to as E107-3 is synthesized based on the acylated reduced amide scaffold and has been shown to exhibit good antimicrobial potency. Our vesicle leakage essay indicates that the compound increases lipid bilayer permeability. We use micropipette aspiration to explore the kinetic response of giant unilamellar vesicles (GUVs). Exposure to the compound causes the GUV protrusion length LP to spontaneously increase and then decrease, followed by GUV rupture. Solution atomic force microscopy (AFM) is used to visualize lipid bilayer structural modulation within a nanoscopic regime. Unlike melittin, which produces pore-like structures, the peptidomimetic compound is found to induce nanoscopic heterogeneous structures. Finally, we use AFM-based force spectroscopy to study the impact of the compound on lipid bilayer mechanical properties. We find that incremental addition of the compound to planar lipid bilayers results in a moderate decrease of the bilayer puncture force FP and a 39% decrease of the bilayer area compressibility modulus KA. To explain our experimental data, we propose a membrane interaction model encompassing disruption of lipid chain packing and extraction of lipid molecules. The later action mode is supported by our observation of a double-bilayer structure in the presence of fusogenic calcium ions.

https://pubmed.ncbi.nlm.nih.gov/27285399/

https://pubmed.ncbi.nlm.nih.gov/27167473/

https://pubmed.ncbi.nlm.nih.gov/26806158/

https://pubmed.ncbi.nlm.nih.gov/26551323/

https://pubmed.ncbi.nlm.nih.gov/26506226/ 

https://pubmed.ncbi.nlm.nih.gov/25992727/