The successful application of bio/nano materials in medicine, materials science, and molecular electronics is dependent on the development of functional, well-defined hybrid materials. In this thesis, I show that the overall structure and function of protein-gold nanocrystal conjugates is influenced by protein surface charge, stoichiometry, and orientation on the nanostructure. By comparing the non-specific conjugation behavior of the lac Repressor (LacI), lysozyme, and alpha-lactalbumin, we establish that basic regions are significantly involved in the assembly of bio/nano conjugates. Super structures, such as controlled nanocrystal aggregates, can result from non-specific protein conjugation depending on the number of basic regions on the protein surface. Moreover, proteins with basic functional domains, like the DNA binding domain of LacI, pose a challenge because non-specific conjugation through these regions adversely affects biofunction. This obstacle can be avoided by specifically conjugating proteins through regions not significantly involved in function. In order to prevent conjugation through the LacI DNA binding domain, we developed a mutant with solvent exposed cysteine residues to direct conjugation to gold nanocrystals through a gold-sulfur bond. The formation and stoichiometry of LacI- and T334C-gold nanocrystal conjugates was followed by protein radiolabeling and analytical ultracentrifugation, two solution techniques that circumvent the challenges associated with spectroscopic characterization of bio/nano conjugates. These techniques provided additional confirmation that LacI conjugates through a weaker, reversible electrostatic interaction, whereas T334C conjugates are more robust, in agreement with the prediction that T334C conjugates through a non-reversible gold-sulfur bond. Lastly, the operator DNA binding function of these conjugates was assessed with nitrocellulose filter binding, analytical ultracentrifugation, and electrophoretic mobility shift. Interestingly, the order of DNA-repressor-nanocrystal complex formation had an impact on operator binding. Regardless of the order of complex formation, LacI conjugates retain little to no DNA binding function. T334C, however, retains significant operator binding if the operator-repressor complex is formed prior to conjugation. These results demonstrate that specific conjugation through regions of low functional significance can greatly improve the biofunction of conjugated proteins. This thesis provides an improved understanding of the interaction between biomolecules and nanostructures, which will benefit the design of materials that are structurally and functionally sound.

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-.

We have developed a mathematical model of the L-type Ca2+ current and cytosolic Ca2+ transient, which is based on data from whole-cell voltage clamp experiments on rat ventricular myocytes. Modified Goldman-Hodgkin-Katz (GHK) equations are provided to account for the different ion selectivity of the DHP-sensitive Ca2+ current channel. The decay of whole cell currents obtained by maintained depolarization is characterized by means of voltage and Ca2+-dependent inactivation embedded in a 5-state dynamic DHP channel model. To characterize a reduced amount of steady-state inactivation of DHP channel in the presence of [Ca 2+]o, a mechanism is used in the model whereby Ca 2+ also inhibits the voltage-dependent inactivation pathway. The 5-state DHP model is also used to simulate single-channel activity. Cytosolic Ca 2+ transients are studied as well. They derive mainly from secondary Calcium-Induced-Calcium-Release (CICR) from the Sarcoplasmic Reticulum (SR). We have developed a 4-state RyR-sensitive Ca2+ model that describes the kinetics of the release channel. This model provides close fitting of cytosolic Ca2+-transient data and mimics the high gain, graded Ca2+ release behavior of the channel. Overall, the model provides a quantitative description of the Ca2+ subsystem in the mammalian heart.

The RecA protein of Escherichia coli plays essential roles in homologous recombination. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo process. During this process, a key intermediate comprised of three DNA strands simultaneously bound to a RecA filament (RecA·tsDNA complex) forms. To date, the questions about the high-resolution structure of this intermediate and how the it forms have not been addressed. We present a systematic characterization of the helical geometry of the three DNA strands of the RecA·tsDNA complex using fluorescence resonance energy transfer (FRET). Measurements of the helical parameters for the RecA·tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We then monitored the formation of the RecA·tsDNA complex in real time using a fluorescent base analog. Time-dependent changes of polarized emission from the fluorophore demonstrated this process involves at least three phases. The first phase is strongly dependent on substrate concentration and reaction temperature. Kinetic simulation revealed a sequential four-steps mechanism as the best description for the reaction. The association rate constant approaches the magnitude of 107 M-1s -1. Thermodynamic analysis indicates the reaction is entropy favorable and a large activation energy is necessary for the association step. A comparison of the energy diagrams between a homologous, partially homologous and a completely heterologous dsDNA substrates suggests that the RecA protein discriminates homology both kinetically and thermodynamically. Based on these data, a structural and mechanistic model for RecAmediated strand exchange was constructed.

A mathematical model of midbrain dopamine neurons has been developed in order to understand the mechanisms underlying two types of calcium-dependent firing patterns that these cells exhibit in vitro. The first is the regular, pacemaker-like firing exhibited in a slice preparation, and the second is a burst firing pattern sometimes exhibited in the presence of apamin. Since both types of oscillations are blocked by nifedipine, we have focused on the slow calcium dynamics underlying these firing modes. The underlying oscillations in membrane potential are best observed when action potentials are blocked by the application of TTX. This converts the regular single-spike firing mode to a slow oscillatory potentials (SOP) and apamin-induced bursting to a slow square-wave oscillation. We hypothesize that the SOP results from the interplay between the L-type calcium current ($I\sb{Ca,L}$) and the apamin-sensitive calcium-activated potassium current ($I\sb{K,Ca,SK}$). We further hypothesize that the square-wave oscillation results from the alternating voltage activation and calcium inactivation of $I\sb{Ca,L}$. Our model consists of two components: (a) a Hodgkin-Huxley-type membrane model, and (b) a fluid compartment model. A material balance on $Ca\sp{2+}$ is provided in the cytosolic fluid compartment, while calcium concentration is considered constant in the extracellular compartment. Model parameters were determined using both voltage-clamp and calcium imaging data from the literature. In addition to modeling the SOP and square-wave oscillations in DA neurons, the model provides reasonable mimicry of the experimentally observed graded modification of the amplitude and frequency of the SOP in response to injected current, as well as the elongation of the plateau duration of the square wave oscillations in response to calcium chelation.

The heme-based aerotaxis transducer (HemAT) from B. subtilis is a heme-containing protein and functions as an oxygen sensor. It can detect oxygen and transmit the signal generated from oxygen binding to regulatory proteins through its putative methyl-accepting chemotactic domain. Through other components, the signaling information is transferred to motor proteins, which control the direction of rotation of flagella and in turn lead to changes in the swimming behavior of bacteria. There is a great deal of information known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium. However, the detailed molecular mechanism of chemotaxis of Bacillus subtilis is in a sense reversed, because attractant binding to chemotactic receptors strengthens the activity of the downstream histidine kinase, instead of inhibiting reaction in Escherichia coli and Salmonella typhimurium. Multiple-wavelength anomalous dispersion (MAD) data were collected from crystals of HemAT using the intrinsic anomalous scatterer, iron, with synchrotron radiation. Three wavelength iron MAD data were collected to 2.8A resolution. The native data set was collected to 2.15A resolution. The crystallographic analysis reveals that the crystal belongs to P21212 1 space group with the cell dimension a = 50.00A, b = 80.12A, c = 85.95A. There are two molecules in one asymmetric unit with 40% solvent content. I have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at resolutions of 2.15A and 2.7A. The structures show that the HemAT sensor domain is a dimeric protein with one heme group in each subunit. The structure of liganded form of HemAT sensor domain reveals a more symmetrical organization than that of the unliganded form. Tyrosine70 in one subunit shows distinct conformations in the liganded and unliganded structures. Our study suggests that disruption of HemAT symmetry plays an important role in initiating the chemotaxis signaling transduction pathway. Our kinetic and thermodynamic studies of ligand binding suggest that HemAT may employ negative cooperativity for detecting external ligand in the signal transduction. The sensor domain provides the structural evidence for such a molecular mechanism.

The neuronal hemoglobin from Cerebratulus lacteus (CerHb) has an O2 affinity (P50 ≈ 1 muM) similar to mammalian myoglobin (Mb), which are both optimized for oxygen storage and release. Mb has a histidine at the E7 helical position, which provides a strong, stabilizing hydrogen bond to bound O2 and a leucine and valine at the B10 and E11 positions, respectively. In contrast, CerHb has three potential hydrogen bonding donors, tyrosine at B10 and glutamine at E7, called the YQ motif, and an unusual Thr at the E11 position. Invertebrate hemoglobins displaying the YQ motif typically have much higher oxygen affinities (P50 ≤ 0.1 muM) and significantly lower rates of O2 dissociation (≤5 s-1) than Mbs. Using mutagenesis and IR spectroscopy, we have been able to show that the beta-hydroxyl of ThrE11 accepts a proton from the TyrB10 side chain, causing the non-bonded electrons of the phenoxyl group to point toward bound O2. The resultant partial negative charge destabilizes the FeO2 complex, causing the high oxygen dissociation rate constant, kO2, and moderate affinity of the wild-type protein.

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.

The high-resolution structures of two separate RNA molecules were determined using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. Both RNA molecules, in their respective systems, perform critical functions of regulating gene expression. To explore the dynamic properties of one molecule, an extensive investigation into its fast (<5ns) intra-molecular motions was carried out. The dynamics investigation was performed using a novel application of heteronuclear relaxation measurements from several base and ribose sites within the molecule. The results of this work were then interpreted using various forms of the Lipari and Szabo motional model. The significance of this research is two fold: (1) it provides detailed structures of two RNA molecules and a basis for their roles in protein recognition and binding and (2) augments the structural characterization with direct measurements of fast internal motions using a novel application of heteronuclear NMR relaxation analysis.

This work shows how to decrease the complexity of modeling flexibility in proteins by reducing the number of dimensions necessary to model important macromolecular motions such as the induced fit process. Induced fit occurs during the binding of a protein to other proteins, nucleic acids or small molecules (ligands) and is a critical part of protein function. It is now widely accepted that conformational changes of proteins can affect their ability to bind other molecules and that any progress in modeling protein motion and flexibility will contribute to the understanding of key biological functions. However, modeling protein flexibility has proven a very difficult task. Experimental laboratory methods such as X-ray crystallography produce rather limited information, while computational methods such as molecular dynamics are too slow for routine use with large systems. In this work we show how to use the Principal Component Analysis method, a dimensionality reduction technique, to transform the original high-dimensional representation of protein motion into a lower dimensional representation that captures the dominant modes of motions of proteins. For a medium-sized protein this corresponds to reducing a problem with a few thousand degrees of freedom to one with less than fifty. Although there is inevitably some loss in accuracy, we show that we can approximate conformations that have been observed in laboratory experiments, starting from different initial conformations and working in a drastically reduced search space. As shown in this work, the accuracy of protein approximations using this method is similar to the tolerance of current rigid protein docking programs to structural variations in receptor models.