Observations of the position of a microscopic bead attached to a single kinesin protein moving along a microtubule contains detailed information about the position of the kinesin as a function of time, although this information remains obscured because of the fluctuations of the bead. The theory of hidden Markov models suggests a possible theoretical framework to analyze these data with an explicit stochastic model describing the kinesin cycle and the attached bead. We model the mechanical cycle of kinesin using a discrete time Markov chain on a periodic lattice, representing the microtubule, and model the position of the bead using an Ornstein-Uhlenbeck autoregressive process. We adapt the standard machinery of hidden Markov models to derive the likelihood of this model using a reference measure, and use the Expectation-Maximization (EM) algorithm to estimate model parameters. Simulated data sets indicate that the method does have potential to better analyze kinesin-bead experiments. However, analysis of the experimental data of Visscher et al. (1999) indicates that current data sets still lack the time resolution to extract significant information about intermediate states. Considerations for future experimental designs are suggested to allow better hidden Markov model analysis.

A central goal of modern biology is to develop a detailed, predictive understanding of the relationships of three-dimensional structure and biological function. We are attempting build this relationship by combining interdisciplinary work. The dissertation is divided into two parts. In the first part of work, numerous structure-activitiy relationships (SAR) studies of different conformationally constrained peptides and peptide mimetics of human melanotropins have been accomplished and discussed. Through this very tedious hard work, selective agonists and antagonists for each subtype of melanocortin receptors have been obtained. We first started investigating the NMR 3D pharmacophore of agonist and antagonist of human melanotropin based on the NMR structure of AGRP ( PDB: 1HYK) and MTII. After a long struggle with appropriate force fields and calculation methodologies, almost identical structures were obtained for MTII as well as SHU-9119 by employing two different techniques of LLMOD (Large scale Lower Modes of Modeling) and NMR. Combining the existing SAR data with this new modeling approach, a series of linear and cyclized peptides (hybridization of the pharmacophore of AGRP and MTII) have been designed, synthesized and identified. We have been successful in obtaining selective agonists and antagonists of melanocortin receptors and these new discoveries shed new insight into peptide or nonpeptide selective drug design for the future. The second part of the dissertation mainly covers human melanocortin receptor (hMCRs) studies. For the purpose of screening the novel peptides and nonpeptides of melanotropins, a series of human melanocortin receptors have been stably transfected into HEK 293 cell line, and new high throughput screening methodologies have been set up. For the purpose of purification of the melanocortin receptors, hMC4R and hMC1R, -His-tag-flag stably transfected into HEK293 cell lines have been designed and applied in receptor purification. We also further studied the mechanism of selective pathway of melanotropin in the HEK293 cells. It was found that agonist mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin mediated clathrin coated pits, and on the contrary, beta-Arrestin2-GFP recruitment is not dependent on PKA activation. The two-photon fluorescence laser scanning microscopy is a fast, powerful method to study the molecular mechanisms of G protein coupled receptor regulation. In addition, this technique also can serve as a rapid, real-time screening method to differentiate between agonists and antagonists irrespective of any knowledge of their intracellular functional properties (orphan receptors).

Many vertebrate and invertebrate olfactory systems are similar in the organization of their synaptic neuropil into glomeruli, structures surrounded by an incomplete layer of glial processes. Within glomeruli, the axons of olfactory receptor neurons synapse with the dendrites of their target brain neurons. Glomeruli are likely to be odor specific in that each glomerulus processes information from a subset of axons about a particular chemical feature of odorant molecules. Therefore, a large proportion of the neurons within a glomerulus may be excited simultaneously in response to a particular odor. The resulting release of potassium ions from neurons may be sufficient to cause a substantial increase in the extracellular concentration of potassium ions and thus affect the excitability of neighboring neurons. The goal of this study is to develop theoretical models for the diffusion of potassium ions in the extracellular space, and to predict how the glial border affects the spread of potassium ions following the activation of olfactory sensory neurons. Observations of the morphology of the interior and border of the glomerulus were used to estimate the porosity and effective diffusivity of these regions, and the size of the "mouth" region where there is no glial covering. Potassium was assumed to be released into the extracellular space during an initial 0.5 seconds. The time-dependent diffusion equation was solved in spherical coordinates using a finite-difference method. The results indicated that the glial envelope forms a partial barrier to the diffusion of potassium ions, and greatly reduces the spread of potassium ions to neighboring glomeruli following release. According to the model, the decline in potassium concentration within the glomerulus due to the leakage from the mouth and glial boundaries is relatively slow, taking more than 10 seconds to approach its resting level. These findings support the hypothesis that the characteristic distribution of glial cells around glomeruli could play a significant role in olfactory information processing.

How does a biological light sensor convert the energy of a photon through a sequence of structural changes to generate a biological signal? Photoactive Yellow Protein (PYP) isolated from the phototrophic bacterium Ectothiorhodospira halophila, a small water-soluble protein whose three-dimensional X-ray crystallographic structure has been determined to high resolution, serves as a paradigm for structural studies of the interaction of light and proteins. This blue light photosensor has been implicated in the negative phototactic response of these bacteria. PYP undergoes a cyclic series of absorbance changes upon illumination at its λ(max) of 446 nm. In its ground state, the anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becomes protonated in the photobleached state (I₂) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent. To gain an in-depth understanding of these interactions at the molecular level, the active site of the protein and the chromophore structure was modulated via site-directed mutagenesis and incorporation of variant chromophores. The structural, optical, kinetic and thermodynamic properties of several such altered proteins have been investigated and presented in this dissertation. Interestingly, Glu46Asp and Glu46Ala mutations demonstrated dual photoactive species as a result of a pH driven color transition. Met100Ala was the first PYP mutant to exhibit properties of an optical switch. The unique properties of PYP and its mutant forms may eventually permit their use in optical devices for switching, memory, computing and holographic applications. Early stages of the photocycle were characterized using picosecond and femtosecond ultrafast transient absorption spectroscopy. These time-resolved spectroscopic studies have revealed the presence of two new intermediates. The time constants for formation and decay of these intermediates have now been resolved and the structural and mechanistic aspects of these results are discussed. Recently, PYP was proposed as a structural prototype for the PAS domain superfamily. PYP/PAS domains therefore form an important structural motif for biological signaling.

The fingerprint pattern (epidermal ridge pattern) becomes established at about the 10th to 16th week of pregnancy, when the lowest layer of the epidermis, the basal layer, becomes undulated. The pattern established by these undulations becomes visible on the skin surface in subsequent weeks of pregnancy. We argue that the undulation process is initiated by buckling of the basal layer. The necessary compressive stress is generated by differential growth. The instability is investigated using the classic von Karman equations for curved surfaces. The analysis reveals that ridges (rolls) are the most common pattern type and that the local ridge direction of the pattern is perpendicular to the direction of largest stress. For certain parameter regimes dot patterns (hexagons) are a stable solution of the equations. Such dot patterns are, in fact, observed on the palms of certain marsupials. The stress in the basal layer is induced by two mechanisms. First, the basal layer expands faster than the other skin structures. Such expansion is resisted at the major flexion creases and the nail furrow. Second, there is a change in fingertip geometry at the time of pattern generation that provides a different source of growth stress. The combination of the two processes predicts the correct sequence of pattern spread over the fingertip. It also explains the observation that fingerprint configurations are related to the fingertip geometry at the time of ridge formation. Computer simulations for the most important configurations exhibit many features of actual fingerprints and suggest directions for future work.

The ab initio treatments of molecular vibrational motion often invoke only the harmonic oscillator approximation. For vibrational modes whose amplitudes access anharmonic regions of the potential energy surface, the harmonic oscillator approximation fails. Low-frequency large-amplitude vibrations, in particular, can access anharmonic regions in addition to other minima of the potential energy surface. Ab initio harmonic frequencies are often scaled to lower values by empirical factors which presumably account for anharmonicity effects as well as an incomplete basis set and account of electron correlation. However, the scaling of those ab initio harmonic frequencies corresponding to low-frequency large-amplitude vibrations results in theoretical values that are still typically several times larger than the experimental values. It is demonstrated in this dissertation that transforming the nuclear motion Hamiltonian to internal coordinates facilitates construction of ab initio potential energy curves, or surfaces, pertaining to low-frequency large-amplitude molecular vibrational modes. The use of internal coordinates complicates the expression of the kinetic energy in the Hamiltonian, and makes it difficult to obtain. Six different methods for determining the kinetic energy expression in internal coordinates are presented and reviewed. The computational implementation of these six methods was performed to allow their critique. Several example calculations of the presented methodology are given. The solution for the vibrational expectation values of the modes expressed by the developed Hamiltonian was also computationally implemented. The resultant theoretical transition frequencies of the molecular systems of 2-sulpholene and 2-aminopyrimidine are combined with experimental studies, and demonstrate the practical usefulness of the presented methodology.

<p> The energies of protein correlated motions lie in the far infrared or THz frequency range (&lambda; = 1 cm &ndash; 50 mm, f = 0.03 &ndash; 6 THz). The existence of correlated motions has been confirmed by neutron and inelastic x-ray scattering measurements. These techniques require large sample volumes and specialized facilities, limiting their application to systematic studies of changes in correlated motions with functional state and allosteric interactions. Standard terahertz time domain spectroscopy measurements have shown sensitivity to protein-ligand binding, oxidation state, conformation, temperature and hydration. However, the response is broad, in part from the large vibrational density of states and in part from the dielectric response contribution from surface water and side-chains. </p><p> As an overall strategy to measure the correlated structural motions in protein, we use anisotropic and birefringent behavior of molecular crystals to develop a new technique called MOSTS (Modulated Orientation Sensitive Terahertz Spectroscopy). We achieve high sensitivity and mode separation, by using single molecular crystal such as sucrose and oxalic acid, and rapid modulation of the relative alignment of the terahertz polarization and the crystal axes by rotating the sample. By locking into the signal at the rotation frequency, we determine the polarization sensitive signal and map out the optically active vibrational resonances. To illustrate the technique, we compare our measured spectra with the calculated, and find a close agreement. </p><p> We measure dielectric properties of oxalic acid, sucrose and protein crystals and polycarbonate sheet using standard terahertz time domain spectroscopy. We determine the absorbances in oxalic acid and sucrose crystals, using MOSTS technique. We compare the resonances in these two distinct methods. Then, we develop a protein model sample by sticking together two thin plates of sucrose and polycarbonate. We carry out standard THz-TDS and MOSTS measurements on the protein model sample. We show that we are able to isolate the vibrational modes from glassy background in protein model sample by using MOSTS.</p>

<p> Fluids in nanopores with diameters of &lt;100nm exhibit behavior that is not seen at micrometer dimensions and above, such as ion current rectification, ionic selectivity, size exclusion and potential dependent ion concentrations in and near the pore. These properties originate from electrostatic interactions between charges on the nanopore surface and the fluid within the nanopore. The influence of these electrostatic effects is determined from a characteristic screening length for the system known as the Debye length. Typical nanopore systems have diameters on the scale of the Debye length and require the consideration of electrostatic effects which do not need to be considered in micrometer systems. Nanofluidic components may be designed by considering the effect of these surface interactions to control ionic transport and incorporate them in devices.In this study we present single conically shaped polymer nanopores with controlled chemistry of the pore walls and pore opening diameter between 5 nm and 30 nm. Two types of pores were examined. The first group of pores contained a junction between two zones with different surface charges. The first group consists of bipolar diodes, which have a two zones composed of positive and negative surface charges, and unipolar diodes, which have two zones composed of a charged zone and a neutral zone. We find that both bipolar and unipolar diodes show a substantial increase in asymmetrical behavior of current-voltage curves over a conical nanopore with a uniform surface charge. Further is it shown that while both diodes show an increase in current rectification, bipolar diodes in particular have superior rectification abilities. The second group of pores are modified by tethering single-stranded DNA molecules to the pore wall. We find that the DNA occludes the narrow opening of nanopores and that the this occlusion effect decreases with an increase in the concentration of the electrolyte. The results are explained by the persistence length of DNA. At low KCl concentrations (10 mM) the molecules are in an extended configuration, thereby blocking the opening and restricting the flow of ionic current to a greater extent than for high salt concentrations. Attaching DNA creates a system with varying opening diameters that can be used to control neutral and charged species.</p>

To get a comprehensive understanding of protein folding, the structural complexity of many proteins as well as the properties of the cellular milieu must be considered. For oligomeric proteins, not only is there polypeptide folding, but protein-protein interactions are also involved. For all proteins, but perhaps most important for aspherical ones, steric effects due to macromolecular crowding may modulate structure, stability, and folding. This is important as 5--40% of the available volume is occupied by various macromolecules in cells. To address these issues, I have used two model systems. Human mitochondrial co-chaperonin protein 10 (cpn10) is used for folding and assembly studies where the number of monomers is large (i.e., 7) and the fold of each monomer contains mostly beta-structure. In contrast, Borrelia burgdorferi VlsE is a football-shaped, monomeric protein with mostly alpha-helical structure that is employed in studies of how protein biophysical properties are affected by the surroundings in terms of membranes and crowding. Using in vitro biophysical and computational methods, my studies have identified the folding and assembly mechanism of cpn10: whereas heptamer unfolding precedes disassembly, a fraction of unfolded monomers assemble before folding while in the other fraction folding of monomers takes place before assembly. Furthermore, in crowded solutions, the helical structure of VlsE first increases and then, at more extreme conditions, a compact, non-native state with beta-sheet content can be populated that exposes an antigenic region. My results have implications for protein folding in general and for the function of these two proteins in particular ( i.e., chaperonin activity for cpn10 and Lyme disease for VlsE).

All hemoglobins catalyze a reaction in which heme-bound dioxygen reacts directly with nitrogen monoxide (NO) to produce nitrate, resulting in the oxidation of the heme iron. This reaction, NO dioxygenation, has been proposed to proceed through a peroxynitrite intermediate that internally isomerizes to nitrate prior to release from the distal pocket of the globin. The work in this thesis examines the decay of the putative intermediate, using multiple myoglobin mutants to ascertain the role of key stereochemical features in the distal pocket. A reaction scheme is proposed that, we believe, provides a detailed mechanism for the formation and decay of a high-spin, cis-peroxynitrite-Fe(III) intermediate in the NO dioxygenation reaction. In mammalian myoglobins and hemoglobins the rate-limiting step for catalytic NO dioxygenase activity is the slow re-reduction of the heme by auxiliary enzymes, often flavin containing cytochrome b5 reductases. Flavohemoglobins are a family of microbial hemoglobins that contain a FAD-binding reductase domain attached directly to the globin, allowing for rapid re-reduction of the heme iron in the presence of NADH. As a result, these two-domain globins are extremely efficient NO dioxygenases that detoxify NO and are part of a microbial response to host defense mechanisms. Three fungal flavohemoglobins, one from Candida albicans and two from Aspergillus fumigates, have been isolated and preliminarily characterized with respect to ligand binding and NO dioxygenation activity. All three are functional flavohemoglobins as well as active NO dioxygenases. In contrast to other flavohemoglobins characterized thus far, AfFhbA exhibits several peculiar characteristics, the most striking being its unusual stability in the oxy complex. These results suggest the possibility of a different, as of yet undefined, function for this protein.