Resolving the backbone tilt of crystalline poly(3-hexylthiophene) with resonant tender X-ray diffraction

The way in which conjugated polymers pack in the solid state strongly affects the performance of polymer-based optoelectronic devices. However, even for the most crystalline conjugated polymers the precise packing of chains within the unit cell is not well established. Here we show that by performing resonant X-ray diffraction experiments at the sulfur K-edge we are able to resolve the tilting of the planar backbones of crystalline poly(3-hexylthiophene) (P3HT) within the unit cell. This approach exploits the anisotropic nature of the X-ray optical properties of conjugated polymers, enabling us to discern between different proposed crystal structures. By comparing our data with simulations based on different orientations, a tilting of the planar conjugated backbone with respect to the side chain stacking direction of 30 ± 5° is determined

Stabilization of Insulin by Adsorption on a Hydrophobic Silane Self-Assembled Monolayer

The interaction between many proteins and hydrophobic functionalized surfaces is known to induce β-sheet and amyloid fibril formation. In particular, insulin has served as a model peptide to understand such fibrillation, but the early stages of insulin misfolding and the influence of the surface have not been followed in detail under the acidic conditions relevant to the synthesis and purification of insulin. Here we compare the adsorption of human insulin on a hydrophobic (−CH3-terminated) silane self-assembled monolayer to a hydrophilic (−NH3+-terminated) layer. We monitor the secondary structure of insulin with Fourier transform infrared attenuated total reflection and side-chain orientation with sum frequency spectroscopy. Adsorbed insulin retains a close-to-native secondary structure on both hydrophobic and hydrophilic surfaces for extended periods at room temperature and converts to a β-sheet-rich structure only at elevated temperature. We propose that the known acid stabilization of human insulin and the protection of the aggregation-prone hydrophobic domains on the insulin monomer by adsorption on the hydrophobic surface work together to inhibit fibril formation at room temperature

Structural Elucidation of the Interaction Between Neurodegenerative Disease-Related Tau Protein with Model Lipid Membranes

A protein\u27s sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into β-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer\u27s disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau\u27s fibrillization mechanism, and what is tau\u27s cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of ta\u27s interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of β-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a \u27hyperphosphorylation\u27 disease mimic helped clarify the role of the microtubule binding domain in anionic lipid affinity and demonstrated even \u27hyperphosphorylation\u27 did not prevent interaction with anionic membranes. Additional studies investigated more complex membrane models to increase physiological relevance. These insights revealed structural changes in tau protein and lipid membranes after interaction. We observed tau\u27s affinity for interfaces, and aggregation and compaction once tau partitions to interfaces. We observed the beginnings of β-sheet formation in tau at anionic lipid membranes. We also examined disruption to the membrane on a molecular scale

Interplay between Mechanics, Electronics, and Energetics in Atomic-Scale Junctions

The physical properties of materials at the nanoscale are controlled to a large extent by their interfaces. While much knowledge has been acquired about the properties of material in the bulk, there are many new and interesting phenomena at the interfaces that remain to be better understood. This is especially true at the scale of their constituent building blocks - atoms and molecules. Studying materials at this intricate level is a necessity at this point in time because electronic devices are rapidly approaching the limits of what was once thought possible, both in terms of their miniaturization as well as our ability to design their behavior. In this thesis I present our explorations of the interplay between mechanical properties, electronic transport and binding energetics of single atomic contacts and single-molecule junctions. Experimentally, we use a customized conducting atomic force microscope (AFM) that simultaneously measures the current and force across atomic-scale junctions. We use this instrument to study single atomic contacts of gold and silver and single-molecule junctions formed in the gap between two gold metallic point contacts, with molecules with a variety of backbones and chemical linker groups. Combined with density functional theory based simulations and analytical modeling, these experiments provide insight into the correlations between mechanics and electronic structure at the atomic level. In carrying out these experimental studies, we repeatedly form and pull apart nanoscale junctions between a metallized AFM cantilever tip and a metal-coated substrate. The force and conductance of the contact are simultaneously measured as each junction evolves through a series of atomic-scale rearrangements and bond rupture events, frequently resulting in single atomic contacts before rupturing completely. The AFM is particularly optimized to achieve high force resolution with stiff probes that are necessary to create and measure forces across atomic-size junctions that are otherwise difficult to fabricate using conventional lithographic techniques. In addition to the instrumentation, we have developed new algorithmic routines to perform statistical analyses of force data, with varying degrees of reliance on the conductance signatures. The key results presented in this thesis include our measurements with gold metallic contacts, through which we are able to rigorously characterize the stiffness and maximum forces sustained by gold single atomic contacts and many different gold-molecule-gold single-molecule junctions. In our experiments with silver metallic contacts we use statistical correlations in conductance to distinguish between pristine and oxygen-contaminated silver single atomic contacts. This allows us to separately obtain mechanical information for each of these structural motifs. The independently measured force data also provides new insights about atomic-scale junctions that are not possible to obtain through conductance measurements alone. Using a systematically designed set of molecules, we are able to demonstrate that quantum interference is not quenched in single-molecule junctions even at room temperature and ambient conditions. We have also been successful in conducting one of the first quantitative measurements of van der Waals forces at the metal-molecule interface at the single-molecule level. Finally, towards the end of this thesis, we present a general analytical framework to quantitatively reconstruct the binding energy curves of atomic-scale junctions directly from experiments, thereby unifying all of our mechanical measurements. I conclude with a summary of the work presented in this thesis, and an outlook for potential future studies that could be guided by this work

Spin diffusion and dynamics studies of the channel forming membrane proteins by solid-state nuclear magnetic resonance

Solid-state nuclear magnetic resonance (SSNMR) is an important tool for the structure, function and dynamics study of many chemical and biological systems, especially powerful in studying membrane proteins, whose structures have been difficult to analyze by traditional x-ray crystallography or solution NMR techniques. In this thesis, various NMR techniques are used to study the structure and dynamics of membrane proteins within lipid bilayers. The main technique applied in this thesis is spin diffusion experiments. We study the structural rearrangement upon membrane binding of colicin Ia by the proton-driven 13C spin diffusion (PDSD) 13C-13C 2D correlation experiment. Membrane bound colicin Ia turns out to have a more extended structure compared to the soluble state. Then a 1D 1H detected 1H spin diffusion experiment is developed to provide the same membrane protein topology information as the 2D 13C detected version, but with significant sensitivity enhancement. We demonstrated this new technique on the colicin Ia channel-forming domain and achieved about 200 fold time saving. Further, the data analysis method is developed to extract the intermolecular distance as long as 12 y from 19F spin diffusion experiment CODEX, where the oligomeric state is obtained at the same time. Demonstrated on the M2 proton channel system, this method is applied to extract the intermolecular distances between a key residue Trp41 in different states of the M2 proton channel. Finally, the water accessibility of the M2 proton channel in different states is studied by the 1H spin diffusion experiment and 3D low resolution models are proposed for this proton channel system by simulating the 1H spin diffusion process between the water and protein. The second focus of this thesis is the dynamics of the M2 peptide in a complex membrane system. Compared to the single component model lipid bilayers, this composite membrane is shown to reduce the rotational rate of the membrane protein by 2 orders of magnitude, which is explained by a rotational diffusion model. The advantage of this immobilization is the ability to acquire high resolution SSNMR spectra at physiological temperatures

Investigation of the peptide-lipid interactions of a beta-hairpin antimicrobial peptide using solid-state NMR

Solid-state NMR (SSNMR) has gained importance as a versatile tool to elucidate the structure and function of many chemical and biological molecules. In this thesis, we have used SSNMR to study the interactions of an antimicrobial peptide (AMP), Protegrin-1 (PG-1) with membranes of varying lipid composition. Based on our studies we have proposed a model for the mechanism of action of PG-1 with two different lipid membranes. PG-1 is a disulfide-linked beta-sheet peptide with broad-spectrum antimicrobial activities. Similar to many AMPs, it selectively disrupts the anionic membranes of microbial cells but leaves the cholesterol-rich zwitterionic mammalian cell membranes intact.;Studying the origin of membrane selectivity and elucidating the quaternary structure of PG-1 and its dependence on the lipid environment is important for understanding its structure-function relation. 31P spectra of oriented lipid bilayers are a very good indicator of the membrane order and we have used this feature to explain the origin of membrane selectivity of PG-1. We have found that the presence of anionic lipids facilitates PG-1 disruption while the presence of cholesterol protects the membrane from PG-1 activity. Rotation-echo double-resonance (REDOR) is a well-established technique to determine heteronuclear distances in solids. We have used 1H{lcub} 13C{rcub} and 13C{lcub}19F{rcub} REDOR to determine the intermolecular packing of PG-1. Measured intermolecular distances show that PG-1 hairpins pack in a parallel fashion in POPC membranes, with the C-terminal strands facing each other.;To investigate whether oligomerization underlies the membrane selectivity, we have determined the oligomeric state of PG-1 and measured its depth of insertion in the anionic and zwitterionic lipid membranes using centerband-only detection of exchange (CODEX) and 1H spin diffusion techniques. 19F CODEX experiments indicate that PG-1 exists as (NCCN)n multimers in both the lipid membranes. 1H spin diffusion experiment shows that these PG-1 multimers are membrane inserted in bacterial-mimetic membranes while in mammalian-mimetic systems they are surface bound. Results obtained from CODEX and spin diffusion experiments suggest that whereas PG-1 forms transmembrane pore in anionic membranes, it exists as beta-sheet on the membrane surface in cholesterol-containing membranes. Thus the oligomeric structure and depth of insertion differ with membrane composition and this helps to explain the basis of selectivity exhibited by PG-1.;Antimicrobial activity of the AMPs depend on various factors such as charge, amphipathicity and conformation of the peptide. Understanding the structure-function relationship of these AMPs is essential to develop a potent antibiotic that is capable of destroying bacterial cells without affecting the host cell membranes. These mutations can change the conformation and other important features which results in altering the membranolytic property of the peptide. The disulfide bonds are known to be important for the antimicrobial activity of PG-1 but the underlying structural reasons are not well understood. We have used SSNMR techniques to study the membrane-bound conformation, dynamics and topology of a disulfide-deleted analog of PG-1 where the Cys residues are replaced by Ala.;Multiple residues were uniformly 13C labeled to measure their chemical shifts, which are excellent indicators of protein conformation. 1D and 2D correlation experiments were conducted to obtain the 13C resonance assignments. The secondary structure of disulfide-deleted PG-1 (Ala-PG1) is a random coil in solution while in the membrane-bound state it exhibits two conformations: a highly mobile random coil and a rigid beta-sheet structure. 1H spin diffusion experiments show that the beta-sheet form of Ala-PG1 inserts into the anionic lipid bilayer while it is surface bound in cholesterol-containing lipid bilayers. The removal of disulfide bonds results in a fraction of the molecule existing as random coils that are loosely bound to the membrane while others retain the beta-sheet form and have similar membrane binding topology as the wild type PG-1. The reduced potency of Ala-PG1 can therefore be attributed to the decrease in the membrane active membrane inserted beta-sheet form of the peptide. Thus using SSNMR we have investigated the peptide-lipid interactions of PG-1 and its mutant and have correlated its structure function abilities

A Bottom-up Computational Approach to Semiconducting Block Copolymers

Conjugated polymers are very attractive materials for the scientists and industry due to low cost of the organic compounds, their lightweight, easy large-area processing from solution at low temperature and mechanical flexibility. Moreover, these materials are multifunctional and advanced technologies require both simultaneous n- and p-type conductance, i.e. ambipolarity. However, there are some hindrances which do not allow the wide spreading of this new generation of semiconductors into the market, first of all, due to their instability to ambient conditions. Moreover, determination of the tunable parameters which are responsible for high efficiency and controlled crystal packing ordering of the devices is rather complicated. A lot of efforts are done in order to improve the performance of the organic electronics as well as to shed light on the relation between the chemical structure and their intrinsic properties. Additionally, the governing factors which define the conductive properties of these materials are still under debate and this remains a great challenge for the researchers. One way to gain insight into the characteristics of polymeric materials is to begin exploring the polymers from their small constitutive units and then step-by-step to construct and characterize every compound up to macromolecular level. In this work, the semiconducting block copolymers, as promising candidates for application in organic transistors, are investigated starting from their small donor and acceptor blocks up to monomers and macromolecules, using computational methods running on different time and length scales. It is found out that the charge transport depends on the symmetry of molecules and the hopping mobilities can be predicted from isolated stacks of dimers, which are defined by minimum energy, without knowledge of the actual crystal structure. Interestingly, the polymers moieties prefer to build up mixed stacks and the flanks form segregated columns if there are no present defects in the samples. At each step of the investigation the results are compared with available experimental data

Charge Transfer in Monomolecular Films and Metal-Organic Frameworks

Characterization and understanding of electronic properties of nanoscale systems is an important issue in modern nanotechnology including molecular and organic electronics. To advance in this topic, charge transfer (CT) properties of two specific nanoscale systems were analyzed in detail in this work. First, electron transfer (ET) dynamics in supported 2D assembles of molecular wires, self-assembled monolayers (SAMs), were studied by resonant Auger electron spectroscopy (RAES) in a combination with a so-called core hole clock (CHC) approach. A variety of suitable SAMs were custom-designed to address specific questions within the general framework of ET dynamics; most of these SAMs were equipped with nitrile tail groups, serving as a predefined site for the resonant excitation of an electron making the ET. The experiments showed a similar electronic coupling efficiency to coinage metal surfaces for the most frequently used S and Se anchors, solving a long-term controversy. Further, an efficient ET was found in acene-based SAM constituents, manifested by a quite low tunneling decay constant (beta) of 0.25 1/Å, similar to that of oligophenyls. In subsequent experiments on an analogous non-benzenoid system, the same ET properties as for its benzenoid isomer were found. As an ultimate proof of the approach, the nitrile groups were attached directly to the substrate, showing an ET time in the sub-fs region, as has been expected. A well-perceptible contribution of the ET process in the RAES [N1s]pi* spectra of pyridyl-substituted molecules revealed that pyridyl is a suitable resonant group for CHC and can be efficiently used as an alternative to nitrile, while NO2-functionalized SAM constitutents exhibited an inverse ET process. Second, static CT properties of surface-anchored metal-organic frameworks (SURMOFs) were studied, taking the basic and well-known HKUST-1 framework as a most suitable reference system. The measurements were performed with the custom-designed two-terminal junction setup and both pristine and guest-molecule loaded SURMOFs were investigated. The pristine SURMOFs showed CT properties similar to hybrid metal-organic molecular wires, as manifested by avery low beta value of 0.0006 1/Å. The CT experiments performed after the incorporation of the guest molecules, viz. ferrocen, TCNQ and its fluorinated analog F4-TCNQ, into the pores of the framework showed a significant increase in the current density. This increase was especially dramatic in the case of TCNQ, achieving up to 6 orders of magnitude. This finding verified a previously reported and highly announced result for this particular guest molecule, obtaining it, however, for the samples of well-controlled thickness, quality and orientation. At the same time, in contrast to the previous report, loading with F4-TCNQ resulted in a similar increase in the current density as for TCNQ, questioning the proposed CT model. These observations were made for several orientations of the SURMOF and different solvents used for the loading. Based on the experimental data, a novel superexchange mechanism for CT in the redox.-molecule-loades SURMOFs was proposed

Insulin unfolding and aggregation: a multi-disciplinary study

This thesis aims at understanding the interaction between insulin and interfaces with a multi-disciplinary approach. We investigate three facets of the interaction. In the first part (chapter 4), we study the interaction of insulin with the air/water interface, for different oligomeric compositions of the solution phase. With the help of Sum Frequency Spectroscopy and calculations of the second order nonlinear susceptibility, we can show that insulin monomers segregate to the hydrophobic air/water interface. Since the insulin monomer is the key species to denature and refold to fibrils, our finding explains for the first time why agitation of insulin solutions and the accompanying increase in air/water interface area accelerates fibril formation. In the second part (chapter 5), we investigate the interaction of insulin monomers at low pH with model hydrophilic and hydrophobic solid surfaces. We use a combination of spectroscopic methods, like ATR FT-IR, XPS, SFG and QCM-D to characterise the silicon functionalised solid surface, to quantify the amount of adsorbed protein and to determine its secondary structure. We show that, contrary to physiological conditions, where insulin monomers are known to change secondary structure upon adsorption, an acidic environment leads to near-native adsorbed insulin, which is stable for at least a day. We further show that heat is needed to restructure the adsorbed insulin monolayer and that this restructured monolayer appears to provide the template for further growth. In the final part (Chapter 6), we apply a comparatively simple experimental method, Reflection Anisotropy Spectroscopy for the first time to the formation of amyloid fibrils at interfaces. In a comparison with FT-IR spectroscopy of our model solid surfaces, we show that a drastic change in the peptide backbone arrangement occurs at a hydrophobic surface, when FT-IR merely detects a thick layer with partial beta-sheet structure. We believe this structural change is the beginning of insulin fibril formation and we use the new tool to explore further changes in the adsorbed layer as it ages over several months

Effect of Solution Shearing Method on Packing and Disorder of Organic Semiconductor Polymers

The solution shearing method has previously been used to tune the molecular packing and crystal thin film morphology of small molecular organic semiconductors (OSCs). Here, we study how the solution shearing method impacts the thin film morphology and causes structural rearrangements of two polymeric OSCs with interdigitated side chain packing, namely P2TDC17FT4 and PBTTT-C16. The conjugated backbone tilt angle and the thin film morphology of the P2TDC17FT4 polymer were changed by the solution shearing conditions, and an accompanying change in the charge carrier mobility was observed. For PBTTT-C16, the out-of-plane lamellar spacing was increased by solution shearing, due to increased disorder of side chains. The ability to induce structural rearrangement of polymers through solution shearing allows for an easy and alternative method to modify OSC charge transport properties