Browsing by Author "Huang, Huey W."
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Item 100-microsecond-resolved exafs technique for studying photolyzed hemoproteins(1984) Teng, Tsu-Yi; Huang, Huey W.; Rorschach, Harold E.; Mutchler, Gordon S.This thesis describes a 1-jus-resolved extended x-ray absorption fine structre (EXAFS) technique for studying protein dynamics. Both the time-resolved EXAFS spectrometer and the protein samples are described in detail. About ten years ago synchrotron radiation from electron storage rings began to be used for research in condensed matter. As a result a new technique for studying local structures in non-crystalline system was developed i.e. the extended x-ray absorption fine structure. This technique has now become an important tool for the structural studies of biological systems, particularly metalloproteins. In recent years the development of wiggler and undulator magnets has increased the radiation intensity to a level that, according to photon statistics, it should be possible to measure the EXAFS of hemoproteins in their transient states. Such measurements would provide structural insight to the very difficult but important problem of protein dynamics. However the conventional method of EXAFS measurement is inefficient for time-resolved measurement. We have developed a new spectrometer to take full advantage of the intense radiation. The time resolution of our spectrometer is about 1 us; below that the measurement time would be too long. Myoglobin was chosen as our sample for its importance in protein biophysics. The complex of myoglobin and carbon monoxide (CO) can be dissociated by light (flash photolysis). Its recombination time ranges from microseconds to infinity depending on temperature. The time resolved EXAFS measurement of photolyzed carboxymyoglobin will reveal the structural changes of protein around the CO binding site during the recombination process. We made our sample in the form of a thin film so that it can meet different requirments of x-ray absorption and optical photolysis. A special procedure of sample making was developed, and a transient optical absorption spectrometer was built for testing the samples. All optical absorption properties of our samples are in agreement with known results. An actual time-resolved EXAFS experiment was performed in February, 1983. The result showed no change in EXAFS with time. This might be due to a failure in photolysis (we lacked an on-line transient optical absorption spectrometer). However, these results demonstrated the feasibility and reliability of our spectrometer.Item A 200 MeV proton spin polarimeter(1983) Rice, James Allen; Roberts, Jabus B.; Corcoran, Marjorie D.; Huang, Huey W.The design, construction, and calibration of a 2 MeV proton spin polarimeter are described. This polarimeter monitors the beam at the transfer point between the linear accelerator and the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The polarimeter determines the beam polarization to an accuracy of 1% in less than ten minutes real time, for the anticipated beam current and pulse repetition rate of the polarized beam, with a carbon filament target that intercepts 1% of the beam particles. Scattered beam particles are collected at 12 and 16 degrees to determine left-right and up-down asymmetries and detect beam angle deviations.Item A comparative study of the membrane-active beta-sheet peptide protegrin with the alpha-helical peptide alamethicin(1999) Heller, William Thomas; Huang, Huey W.The interactions of the membrane-active peptides alamethicin and protegrin with lipid bilayers are studied. The influence of the bilayer composition on the orientation transition of alamethicin is studied using oriented circular dichroism. The makeup of the headgroup region of the bilayer is altered while the composition of the hydrocarbon chains remains constant. When a smaller lipid headgroup is added to the bilayer, the insertion transition shifts to higher peptide concentrations. This can be explained as a reduction of the energy cost of adsorbing the peptide in the bilayer due to the smaller lipid headgroup. The interactions of the beta-sheet peptide protegrin with model membranes are studied by oriented circular dichroism and lamellar x-ray diffraction. A transition between two states with distinct oriented circular dichroism spectra is observed which is a function of the peptide concentration and hydration. Lamellar x-ray diffraction indicates that protegrin produces membrane thinning in the same lipid system for peptide concentrations below those needed to bring on the transition. This leads to the conclusion that the low concentration and hydration state has protegrin adsorbed into the headgroup region of the bilayer parallel to the membrane surface. Comparisons between the behaviors of alamethicin and protegrins are drawn which suggests that the two peptides have the same mode of action.Item A feasibility study on neutron reflectivity of lipid bilayers(1996) Heller, William Thomas; Huang, Huey W.The feasibility of studying the mechanical properties of lipid bilayers utilizing neutron reflectivity is examined. The properties of interest are the bulk modulus for compressibility and the membrane rigidity. It is possible to analyze reflectivity measurements of aligned lipid bilayers in terms of theories developed for scattering experiments. Experimental results are presented which indicate that it is possible to study lipid bilayers using neutron reflectivity, but the results point out deficiencies in the particular apparatus used to perform the experiments. Experimental requirements for future work are presented.Item A study of local exchange in continuum processes(1980) O'Connell, James Kevin; Lane, Neal F.; Walters, G. King; Huang, Huey W.The role of one electron orbitals in Many Body Theory and the Generalized Sudden Approximation is reviewed. Various approximations to the exact Hartree Fock static exchange potentials are discussed and a new, screened exchange potential is derived. Phase shifts for the elastic scattering of electrons from Helium, Neon and Argon and Photoionization cross sections for Lithium, Carbon, Oxygen, Neon and Argon were calculated and compared with the exact static exchange results. The screened exchange potential is the best approximation for Photoionization and the Hara exchange potential is the best for Scattering. Ground state eigenvalues, ionization energies and oscillator strengths have been calculated for the 2t2-la1 transition in Methane. The calculations were carried out using the Xalpha Multiple Scattering Method. The ground state eigenvalues were correctly predicted and the oscillator strengths lie within experimental limits.Item Attack on single Escherichia coli spheroplast by antimicrobial peptides(2015-08-13) Sun, Tzu-Lin; Huang, Huey W.; Hafner, Jason H.; McNew, James A.Studies of the molecular mechanisms of antimicrobial peptides (AMPs) have mostly been performed with lipid bilayers, as a substitute for cell membrane. Hence, there is a persistent question as to whether the action of AMPs on bacterial membranes can be reproduced on lipid bilayers. Valuable information was obtained recently from observing the actions of AMPs on E. coli and Bacillus subtilis by time-lapse fluorescence microscopy. The goal of my dissertation is to study the direct action of AMPs on the cytoplasmic membranes by using E. coli spheroplasts, the cell form from which the outer membranes have been removed. The key question is how to reveal the response of the spheroplast to AMPs. In our previous work, the aspiration method on giant unilamellar vesicle (GUV) has been demonstrated as very effective for researching membrane effects induced by peptides. This method is able to measure the membrane expansion due to peptide binding and simultaneously monitor membrane permeability by using dye indicators. In this work, a spray method was developed for introducing AMPs and customized the experimental procedures for performing the experiments of E. coli spheroplasts. The living state of cytoplasmic membrane makes it difficult to deduce the molecular events from the response of live cells. Hence, the physical methods which have been developed for researching peptide activity on lipid bilayers were practiced first. These methods include X-ray diffraction (XRD), oriented circular dichroism (OCD) and GUV aspiration. These methods were practiced by studying the question as to why a hydrocarbon-stapled peptide NYAD-1 drug was reported to have membrane permeating property. Melittin was selected as the representative of AMPs for the E. coli spheroplast experiments. To better understand the characteristics of melittin, the melittin activity on model lipid membrane was examined before advancing to the spehroplast experiment. The melittin transmembrane was proposed by correlating melittin binding on a lipid vesicle (aspirated GUV imaging) with structural studies in multilayers (XRD and OCD). This behavior was further determined by using fluorescence indicator to track the melittin distribution. The toroidal structure of melittin pore was also detected by grazing-angle X-ray anomalous diffraction. In the studies of NAYD-1 and melittin on a model membrane, our discovery of AMP's transmembrane enables us to clarify the pore-formation mechanism which has been disputed for decades. In addition, our past work on the physical property of E. coli spheroplast cytoplasmic membrane has indicated the existence of a lipid reservoir. The lipid reservoir dominates the surface tension by balancing the membrane folds. Finally, we used the aspiration method to hold a spheroplast so as to measure the change of the spheroplast membrane area in response to the AMPs' binding. Further, a fluorescence indicator which is able to associate with AMPs was used to monitor the peptide distribution and another fluorescence dye to monitor the molecule leakage. The spheroplast study shows that there are similarities and differences between the responses of spheroplasts and GUVs. The recent findings on the unique properties of spheroplast membranes are the key for understanding these results. Our work of understanding the AMPs activity on membrane is potentially applicable in improving peptide drug design and delivery for disease treatment.Item Characterization of Structure and Function Relationship between Domains of the ER Membrane Protein Atlastin(2014-02-06) Desai, Tanvi; McNew, James A.; Huang, Huey W.; Braam, Janet; Stern, Michael; Lwigale, Peter YunjuThe endoplasmic Reticulum (ER) is an important site for lipid synthesis, protein synthesis and transport. ER fusion is an essential process for its maintenance and biogenesis. Mutations in genes involved in this process cause Hereditary Spastic Paraplegia (HSP). These mutations are shown to affect intracellular trafficking and localization of membrane compartment. One of the important proteins causing early onset of HSP is Atlastin. Previous work in McNew lab at Rice University (Moss et al., 2011b) has shown that atlastin is involved in the homotypic fusion of the ER and the C-terminal cytoplasmic region of atlastin is essential for atlastin mediated fusion. During my studies presented in this thesis, I was able to demonstrate that the C-terminal cytoplasmic region of atlastin destabilizes lipid bilayers to facilitate fusion. The requirement of C-terminal cytoplasmic region is minimal when fusing two fluid (or unstable) lipid bilayers. The C-terminal cytoplasmic region of atlastin forms an amphipathic helix and mutations on the hydrophobic phase of the helix reduce fusion. These mutations are not dominant, as presence of full length atlastin on even one of the fusing lipid bilayers can significantly improve fusion during a heterotypic fusion reaction. Additionally, domain swaps between human atlastin-1 and drosophila atlastin show that the role of C-terminal cytoplasmic region is highly conserved. Also, during my research presented here in, I found that when the transmembrane region and C-terminal cytoplasmic region of human atlastin-1 were swapped with drosophila atlastin, it showed functional similarity. These results show that although atlastins in organisms play an important role in the ER fusion, there are likely species specific differences in how this is achieved. An understanding of atlastin mediated fusion should help in unraveling mechanisms of HSP pathogenesis and other disorders arising from dysfunctional ER.Item Comparative Study of the Condensing Effects of Ergosterol and Cholesterol(Cell Press, 2016) Hung, Wei-Chin; Lee, Ming-Tao; Chung, Hsien; Sun, Yi-Ting; Chen, Hsiung; Charron, Nicholas E.; Huang, Huey W.Cholesterol, due to its condensing effect, is considered an important regulator of membrane thickness. Other sterols, due to their structural similarities to cholesterol, are often assumed to have a universal effect on membrane properties similar to the condensing effect of cholesterol, albeit possibly to different degrees. We used x-ray diffraction to investigate this assumption. By the combination of lamellar diffraction and grazing-angle scattering, we measured the membrane thickness and the tilt-angle distribution of the lipid’s hydrocarbon chains. This method is sensitive to phase separation, which is important for examining the miscibility of sterols and phospholipids. Mixtures of ergosterol or cholesterol with dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, and dioleoylphosphatidylcholine were systematically studied. We found that mixing ergosterol with phospholipids into a single phase became increasingly difficult with higher sterol concentrations and also with higher concentrations of unsaturated lipid chains. The only condensing effect of ergosterol was found in dimyristoylphosphatidylcholine, although the effect was less than one-third of the effect of cholesterol. Unlike cholesterol, ergosterol could not maintain a fixed electron density profile of the surrounding lipids independent of hydration. In dioleoylphosphatidylcholine and palmitoyloleoylphosphatidylcholine, ergosterol made the membranes thinner, opposite to the effect of cholesterol. In all cases, the tilt-angle variation of the chain diffraction was consistent with the membrane thickness changes measured by lamellar diffraction, i.e., a thickening was always associated with a reduction of chain tilt angles. Our findings do not support the notion that different sterols have a universal behavior that differs only in degree.Item Cooperative phenomena of antimicrobial peptides in membranes: A study by neutron and X-ray diffraction(2001) Yang, Lin; Huang, Huey W.Gene-encoded membrane-active antimicrobial peptides permeablize bacterial plasma membranes without harming the host cells. Furthermore, although most peptides exhibit a broad spectrum of activity against microbes, different peptides preferentially kill different pathogens. Understanding such cell-type specificity is not only fundamental to cell biology but also crucial to potential pharmaceutically applications of antimicrobial peptides. Accumulated evidence indicates that the antimcrobial peptides target the lipid matrix of the plasma membranes. Therefore we focus on the physical states of the peptides bound to lipid bilayers. This thesis describes studies of lipid-peptide systems in the form of aligned multi-lamellae with new neutron and X-ray diffraction techniques developed specifically for such systems, under various conditions with improved temperature and relative humidity control. These technique allow the most detailed structural investigation on the supramolecular assemblies formed by these peptides in model lipid membranes. Interesting phenomena were observed. Peptides form transmembrane pores in fluid lipid bilayers. The sizes of various peptide pores were determined by fitting neutron scattering data with the theory of scattering. By manipulating the temperature and the hydration level of the samples, we observed position correlations developed between the pores located in neighboring bilayers that eventually became long-range and the transmembrane pores were crystallized in lipid membranes for the first time. Diffraction data of the crystallized pores were measured with synchrotron radiation using samples on ultra-thin Si3N4 substrate for transmission X-ray diffraction. A number of different crystalline phases were found. One example is the ABC stacking hexagonal structure, surprisingly also found in pure diphytanoyl phosphatidylcholine samples. Correlating the diffraction data with circular dichroism and other experimental evidence, we separate the pore structures into two categories described by the barrel-stave model and the toroidal model. The implication of these results on the peptide's cell-type specificity is also discussed in terms of the properties of the lipids and environmental variables.Item Effects of membrane inclusions on lipid bilayer structure and dynamics studied by elastic and inelastic x-ray scattering(2003) Weiss, Thomas Michael; Huang, Huey W.The response of the bilayer structure and dynamics to different types of inclusions is investigated using X-ray scattering techniques and the evidence is used to deduce details of their interaction with the membrane. In the case of the antimicrobial peptide RTD-1 we identify, combining the outcome of oriented CD spectroscopy and X-ray diffraction experiments, two different bound states of the peptide that differ in the orientation with respect to the membrane. One of which is shown by lamellar X-rays diffraction to considerably thin the membrane, while the other does not affect the membrane thickness. From this we identify the thinning state to be a surface state in which the peptide is embedded in the headgroup region of the bilayer. Furthermore we investigate the effect of small membrane-spanning helical peptides of different lengths on the bilayer using lamellar diffraction. Contrary to our expectations we did not measure any significant change in membrane thickness upon inclusion of these helices, which leads us review our idea of hydrophobic matching in the case of small single transmembrane peptides. In addition we used inelastic X-ray scattering at high energy resolution to investigate the collective chain dynamics of the membrane and how it is affected by inclusions in the membrane. We measure the inelastic X-ray scattering of DMPC bilayers with and without cholesterol. An analysis of these spectra within a generalized hydrodynamic theory yields the dispersion relation and damping of the high frequency sound modes. We show that this dispersion relation systematically changes with the amount of cholesterol in the sample. Comparing this finding with the situation in the pure lipid above and below the main phase transition we show that under the influence of the cholesterol the dynamics of the lipid becomes more gel-like, a fact that might have important implications for the transport of small molecules across the bilayer.Item Hydrophobic matching and membrane mediated interactions in lipid bilayers(2000) Harroun, Thad Alan; Huang, Huey W.Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its thickness to match the hydrophobic surface of the protein, is a commonly accepted idea in biophysics, but one that until now has not been experimentally tested. One important consequence is that protein interactions will be mediated by the energy cost of deforming the membrane from its protein free state. With X-ray scattering techniques we tested these ideas with the peptide gramicidin embedded in DLPC and DMPC bilayers. Gramicidin pushes the different membranes to a common thickness as expected from hydrophobic matching. Concurrently, gramicidin-gramicidin nearest neighbor distance decreases with increasing mismatch, which confirms that the strain in the lipid bilayer gives rise to an attractive potential between the proteins. We have taken a continuum theory approach to the analysis of the experimental results. This approach treats the energetics of membrane-protein interactions as a function of the material properties of the membrane such as bending rigidity and compressibility. Using numerical methods and a novel simulation technique, we have successfully demonstrated the theoretical relationship between membrane thickness change and protein correlation. By quantitatively reproducing our experimental results, we have shown that the theory of membrane deformation is sufficient to explain the phenomena of hydrophobic matching. We also include a study on the peptide melittin as an example of the type of protein-lipid system we want to understand better. We answer the question of the orientation of the peptide when making membrane pores.Item Improved Biomolecular Crystallography at Low Resolution with the Deformable Complex Network Approach(2013-07-24) Zhang, Chong; Ma, Jianpeng; Huang, Huey W.; Raphael, Robert M.It is often a challenge to atomically determine the structure of large macromolecular assemblies, even if successfully crystallized, due to their weak diffraction of X-rays. Refinement algorithms that work with low-resolution diffraction data are necessary for researchers to obtain a picture of the structure from limited experimental information. Relationship between the structure and function of proteins implies that a refinement approach delivering accurate structures could considerably facilitate further research on their function and other related applications such as drug design. Here a refinement algorithm called the Deformable Complex Network is presented. Computation results revealed that, significant improvement was observed over the conventional refinement and DEN refinement, across a wide range of test systems from the Protein Data Bank, indicated by multiple criteria, including the free R value, the Ramachandran Statistics, the GDT (<1Å) score, TM-score as well as associated electron density map.Item Interaction of a helical peptide with membrane: Study of alamethicin(1994) Wu, Yili; Huang, Huey W.Alamethicin is a transmembrane ion channel at low concentration, and a lytic agent of cell membrane at high concentration. It is a small size polypeptide (20 amino residues), and contains a large section of amphiphilic $\alpha$-helix, which is an often-encountered secondary structure motif in membrane active peptides and proteins. Its membrane-active functions, typical secondary structure, and relatively small size made this peptide be an ideal model for studying the interaction of proteins with membranes. This thesis provides two novel methods to obtain structural information of such a peptide-lipid system. By the method of oriented circular dichroism (OCD), we are able to determine the orientation of the alamethicin with respect to the membrane bilayer. Depending on the alamethicin concentration and the water content in the membrane, alamethicin either perpendicularly inserts into the bilayer or binds parallel to the membrane surface. By the method of lamellar x-ray diffraction, we found the membrane bilayer thickness reduced by increasing concentration of alamethicin and decreasing relative humidity. From these two mutually complementary studies we constructed a consistent picture for the interaction between alamethicin and the membrane. When alamethicin concentration is low, the peptide molecules adsorb near the area of lipid head group, which effectively expands the average cross sectional area of the lipid molecules and makes the hydrocarbon chains more disordered, so that the lipid bilayer becomes thinner than pure lipid membrane. When alamethicin concentration reaches the critical point, where membrane structure becomes favorable for the insertion of alamethicin, alamethicin undergoes a transition from the surface state to the insertion state. The insertion of alamethicin would introduce much water into membrane, so the transition would happen only when relative humidity is high. Our alamethicin-lipid interaction model explains the spontaneous insertion of alamethicin at high aqueous concentration causing the lysis of membrane. It also suggests a possible gating mechanism for alamethicin ion channels.Item Interaction of Daptomycin with Lipid Bilayers Correlated to Its Action on Cell Membranes(2013-05-31) Chen, Yen-Fei; Huang, Huey W.; Toffoletto, Frank R.; Killian, Thomas C.Daptomycin is a lipopeptide antibiotic notably against multidrug-resistant, gram-positive pathogens. Evidence shows that the antibiotic acts neither on DNA nor on the proteins. It has been shown to insert and aggregate in the bacteria membrane; nevertheless, how the molecular interaction leads to cell death is unknown. In this work, the physical properties of interactions between daptomycin and model membranes are studied in order to understand the underlying mechanism of daptomycin. First, daptomycin’s binding affinity to membranes was found to be proportional to Ca++ concentration. The effect of Ca++ cannot be replaced by other divalent ions such as Mg++. After binding, daptomycin was found to form lipid-peptide aggregations on phosphatidylglycerol(PG)-containing vesicles. PG is required for the formation of lipid-peptide aggregates, which eventually lead to membrane ruptures. Cardiolipin, another main component in bacterial membrane, cannot substitute PG to induce the same membrane defect. In addition, with a fixed concentration of Ca++, it requires a minimum concentration of daptomycin to trigger the membrane defect process. The membrane defect results mainly from lipid-peptide aggregations rather than from pores forming in the membrane. Finally, x-ray data imply that daptomycin binds to the headgroup region of the bilayer, which causes membrane thinning. The elastic energy of membrane thinning elevates the energy level of the daptomycin binding state, which explains the transition to the aggregation state.Item Interactions of Amyloid-Forming Peptides with Lipid Bilayer Membranes(2012) Lee, Chang-Chun; Huang, Huey W.Amyloid-proteins are among the most actively researched biological topics today, because they have been associated with many serious human diseases, such as Alzheimer's disease and type II diabetes. In particular the deposition of protein aggregates on cell membranes has been suspected as the causes of the diseases, although the proof is still elusive. Studying the interactions of amyloid-forming peptides with lipid-bilayer membranes may clarify the pathway of the β-aggregate formation and provide new insights into the amyloid hypothesis of diseases. In this thesis, I investigate how three peptides, penetratin, amylin, and LL-37, interact with lipid membranes by using several techniques well-developed in our lab. In the study of penetratin interacting with lipid membranes, we were able to clarify the energy pathway of amyloid formation mediated by membrane-binding. This provides the sole experimental proof for the Jarrett-Lansbury theory of β- amyloid formation. Our investigation on amylin-membrane interaction clarifies how amylin in different forms damage bilayer membranes. Between penetratin and amylin we have clarified the complicated pattern of interactions between amyloid-forming peptides and lipid bilayers. The third peptide LL-37 studied in my thesis turned out to a pore forming peptide. I found the mistake made by previous investigators in several different laboratories that made them erroneously conclude that LL-37 was not a pore forming peptide. The results of these three peptides show that methods we used are a comprehensive set of tools that can reveal a broad range of peptide properties. Both the formation of amyloid aggregates and formation of membrane pores can be explained by a two-state model proposed by Huang describing peptide-membrane interactions. For LL-37, the second state is a pore in membrane. But for penetratin and amylin the second state is an aggregation in the β form. We found that β-aggregates have low affinity within a lipid bilayer, and therefore exit from the bilayer structure. However, this exit process extracts lipid molecules from the bilayer and incorporates them in the peptide aggregates. We suggest that this is the molecular process of how amylin might damage of the membranes of β-cells.Item Interactions of highly charged cationic peptides and large anions with lipid bilayers(2006) Wang, Wangchen; Huang, Huey W.The first chapter is devoted to the highly charged cationic peptides penetratin (pAntp), a 16 residue peptide belonging to the class of CPP (cell penetrating peptides). The translocation of pAntp across cell membranes is believed to occur through a mechanism that is independent of receptors, transporters, and endocytosis. We studied the state of pAntp bound to lipid bilayers by the method of oriented circular dichroism (OCD). In bilayers composed of mixed lipids (DOPC/DOPG) pAntp shows both conformational and orientational changes. At low peptide concentrations (Peptide/Lipid ratio) and high charge densities, the pAntp tends to adopt alpha-helical conformation. At high peptide concentrations and low charge densities, the pAntp tends to adopt beta-sheet and random coil conformations. The alpha-helical pAntp was observed to change its orientation in membrane as the hydration of the bilayers changes. The effect of the peptide termini on its conformation was also examined. The peptides with three different ending forms were compared. The result seems to suggest that the conformation of the peptide is subject to the variation of the peptide termini. The second chapter investigates on the effect of large chaotropic anions on lipid bilayer structure (Hofmeister effect). X-ray diffraction experiments were done on POPC lipid and its mixture with sodium salts (NaI and NaSCN). The result shows that in the present of the salts, the change in the bilayer structure is primarily the thermal motional range of the phosphate headgroup of lipid. The lipid headgroup undergoes a broader motion range in the presence of I- and a narrower range in the presence of SCN-.Item Laser annealing of Ni (001)(1983) McConnell, Robert P.; Walters, G. King; Dunning, F. Barry; Huang, Huey W.Experimental aspects of laser cleaning and annealing of a Ni(1) surface with a pulsed ruby laser are reported. Effects of applying laser energy densities of .4 J/cm to 1.1 J/cm to an Ar-ion sputter-cleaned surface are determined using Auger Electron Spectroscopy (AES), Low Energy Electron Diffraction (LEED), and Polarized LEED (PLEED) as surface diagnostics. AES profiles, LEED Intensity v. Voltage (I-V) curves, and PLEED Polarization v. Voltage (P-V) curves taken on the laser annealed surface are compared to identical information for the thermally annealed surface. Effects of substrate temperature on laser annealing results are investigated. It is concluded that laser-annealing yields clean, well ordered surfaces for elevated substrate temperature, but not at room temperature.Item Membrane Permeability of Hydrocarbon-Cross-Linked Peptides(Biophysical Society, 2013-05) Sun, Tzu-Lin; Sun, Yen; Lee, Chang-Chun; Huang, Huey W.Schafmeister, Po, and Verdine (another study) introduced a method using a hydrocarbon linker (staple) to stabilize a peptide in a helical configuration. One intended goal of this scheme is to facilitate the delivery of peptide drugs into target cells. Here, we investigate whether stapled peptides are intrinsically membrane permeable, by performing a case study on a stapled 12-mer peptide named NYAD-1. We found that the native peptide CAI (an HIV-1 inhibitor) does not bind to lipid bilayers, however NYAD-1 indeed permeates through lipid bilayers even at low solution concentrations. To understand the reason for the membrane permeability, we investigated the physical properties of NYAD-1 as a function of bound peptide/lipid molar ratio P/L. We found that NYAD-1 spontaneously binds to a lipid bilayer. At low P/L, the peptide primarily binds on the polar-apolar interface with its helical axis parallel to the bilayer, which has the effect of stretching the membrane area and thinning the membrane. The membrane thinning reaches its maximum at P/L ~1/15-1/12 in DOPC bilayers. Additional bound peptides have little thinning effect and their helical axes are normal to the plane of bilayers. Thus, the stapled peptide has a membrane interaction behavior similar to helical antimicrobial peptides, such as magainin and melittin. We emphasize that not all peptides that bind to lipid bilayers in the a-helical form behave this way.Item Methods of micro manipulating giant lipid vesicles for the studies of molecular interactions with membranes and membrane-membrane interactions(2011) Sun, Yen; Huang, Huey W.The lipid matrix of cell membranes is a natural binding site for amphipathic molecules. Consequently there are water-soluble, amphipathic peptides and proteins that exert their functions on membranes. Studies also showed that binding of amphipathic molecules (such as drugs) may change the functions of membrane proteins by altering the physical properties of the membrane. Thus, we want to understand how amphipathic molecules interact with membranes and find out the consequences of such membrane-molecule interactions. My thesis consists of development of new methods for studying the kinetics of molecular interactions with membranes and a series of comparative studies on different membrane-active molecules including peptides, proteins and drugs. My contribution to the methods for kinetics is to complement the equilibrium methods already developed in our lab for past twenty years. I established a micropipette aspiration system based on the system developed by Evan Evans in the 80's, but instead of measuring the elastic properties of membranes, we used it to study the dynamic interaction processes between amphipathic molecules and membranes.Item Mode of Action of Antimicrobial Peptides on E. coli Spheroplasts(Cell Press, 2016) Sun, Yen; Sun, Tzu-Lin; Huang, Huey W.We investigated the phenomena of antimicrobial peptides (AMPs) directly attacking the cytoplasmic membranes of Escherichia coli spheroplasts. We developed a procedure for fluorescence recovery after photobleaching to examine dye leakage through bacterial membranes as AMPs in solution bound to the membranes. We found that the AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the response of a lipid-bilayer vesicle, which always showed a membrane area expansion by AMP binding. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of fluorescence recovery after photobleaching showed that the fluxes of dye molecules into and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. We discovered a new, to our knowledge, experimental parameter called the flux rate that characterizes the AMP-induced permeability of dye molecules through bacterial membranes. The phenomena observed in bacterial membranes are consistent with the pore-forming activities of AMPs previously observed in lipid bilayers. The experimental value of the flux rate per pore is much smaller than a theoretical value that assumes no friction for the dye molecule’s permeation through the pore. We believe that experimental studies of the flux rate will be useful for further analysis of AMPs’ permeabilization mechanisms.