Browsing by Subject "Atomic force microscope"
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Item Open Access Nanomechanics of Ankyrin Repeat Proteins(2011) Lee, WhasilAnkyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.
Item Open Access Nanomechanics of Nucleic Acid Structures Investigated with AFM Based Force Spectroscopy(2010) Rabbi, Mahir HaroonNucleic acids are subjected to many different mechanical loadings inside. These loadings could cause large deformations and conformational changes to these molecules. This is why the mechanical properties of nucleic acids are so important to their functions. Here we use a newly designed and built high-performance AFM force spectrometer, supplemented with molecular dynamics simulations and NMR spectroscopy to investigate the relationship between mechanical properties and structure of different nucleic acids.
To test the mechanical properties of nucleic acids, we successfully designed and purpose-built a single molecule puller, an instrument to physically stretch single molecules, at a fraction of the cost of a commercial AFM instrument. This instrument has similar force noise to hybrid instruments, while also exhibiting significantly lower drift, on the order of five times lower. This instrument allows the measurement of subtle transitions as a molecule is stretched. With the addition of a lock-in amplifier, we possibly could obtain better force resolution, the order of femtonewtons.
We find that helical structure does indeed have an effect on the mechanical properties of double-stranded DNA. As the A-form double helix has a shorter, wider structure compared to the B-form helix, its force spectra exhibit a shorter initial length before the overstretching force plateau, compared to B-form DNA. Contrarily, the Z-form double helix has a narrower, more extended helical structure than B-form DNA, and we see this fact manifest in the force spectra of Z-DNA, which has a longer initial length before the overstretching force plateau. Also, interestingly, we find that neither A, nor Z-DNA force spectra display the second melting force plateau. Indicating this plateau is not necessarily cause by melting of strands apart, but rather a feature of B-DNA.
To better understand the forces that stabilized these different structures, specifically base stacking, we also mechanically characterize different single-stranded helical polynucleotides using AFM based force spectroscopy. We expand on previous studies by confirming that single helical polynucleotides undergo a force transition at a force of ~20 pN as they are uncoiled, and also demonstrating, that when stretched beyond this force transition, the molecules behave differently depending on base sequence and backbone sugar. Specifically, the force spectra of poly-adenylic acid possess a linear force region, which persists to ~300 pN, after the force plateau. We also observe that poly-deoxyadenylic acid is comparatively stiffer than other polynucleotides after undergoing two force transitions. By supplementing our force spectroscopic data with MD simulations and NMR spectroscopy, we find that base stacking in adenine is quite strong, persisting above 100 pN. We find that initial helical structure, which is defined by base stacking and backbone sugar, guides the stretching pathway of the polynucleotides. This finding can possibly be extrapolated to the elasticity of double-stranded DNA.
Item Open Access New Approaches To Studying Non-Covalent Molecular Interactions In Nano-Confined Environments(2010) Carlson, David AndrewThe goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.
In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.
In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.
In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.
Item Open Access STIFFNESS CALIBRATION OF ATOMIC FORCE MICROSCOPY PROBES UNDER HEAVY FLUID LOADING(2010) Kennedy, Scott JosephThis research presents new calibration techniques for the characterization of atomic force microscopy cantilevers. Atomic force microscopy cantilevers are sensors that detect forces on the order of pico- to nanonewtons and displacements on the order of nano- to micrometers. Several calibration techniques exist with a variety of strengths and weaknesses. This research presents techniques that enable the noncontact calibration of the output sensor voltage-to-displacement sensitivity and the cantilever stiffness through the analysis of the unscaled thermal vibration of a cantilever in a liquid environment.
A noncontact stiffness calibration method is presented that identifies cantilever characteristics by fitting a dynamic model of the cantilever reaction to a thermal bath according to the fluctuation-dissipation theorem. The fitting algorithm incorporates an assumption of heavy fluid loading, which is present in liquid environments.
The use of the Lorentzian line function and a variable-slope noise model as an alternate approach to the thermal noise method was found to reduce the difference between calibrations preformed on the same cantilever in air and in water relative to existing techniques. This alternate approach was used in combination with the new stiffness calibration technique to determine the voltage-to-displacement sensitivity without requiring contact loading of the cantilever.
Additionally, computational techniques are presented in the investigation of alternate cantilever geometries, including V-shaped cantilevers and warped cantilevers. These techniques offer opportunities for future research to further reduce the uncertainty of atomic force microscopy calibration.
Item Open Access The Design Of A Nanolithographic Process(2007-07-02) Johannes, Matthew StevenThis research delineates the design of a nanolithographic process for nanometer scale surface patterning. The process involves the combination of serial atomic force microscope (AFM) based nanolithography with the parallel patterning capabilities of soft lithography. The union of these two techniques provides for a unique approach to nanoscale patterning that establishes a research knowledge base and tools for future research and prototyping.To successfully design this process a number of separate research investigations were undertaken. A custom 3-axis AFM with feedback control on three positioning axes of nanometer precision was designed in order to execute nanolithographic research. This AFM system integrates a computer aided design/computer aided manufacturing (CAD/CAM) environment to allow for the direct synthesis of nanostructures and patterns using a virtual design interface. This AFM instrument was leveraged primarily to study anodization nanolithography (ANL), a nanoscale patterning technique used to generate local surface oxide layers on metals and semiconductors. Defining research focused on the automated generation of complex oxide nanoscale patterns as directed by CAD/CAM design as well as the implementation of tip-sample current feedback control during ANL to increase oxide uniformity. Concurrently, research was conducted concerning soft lithography, primarily in microcontact printing (µCP), and pertinent experimental and analytic techniques and procedures were investigated.Due to the masking abilities of the resulting oxide patterns from ANL, the results of AFM based patterning experiments are coupled with micromachining techniques to create higher aspect ratio structures at the nanoscale. These relief structures are used as master pattern molds for polymeric stamp formation to reproduce the original in a parallel fashion using µCP stamp formation and patterning. This new method of master fabrication provides for a useful alternative to conventional techniques for soft lithographic stamp formation and patterning.