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.

Computational simulation techniques, targeted molecular dynamics (TMD) and the quantized elastic deformational model (QEDM, a modified normal mode analysis) in particular, have been employed to determine functionally relevant motions (motions required to perform a specific biological process) of six supermolecular complexes (SMC's): F1F0-ATP synthase, lactose repressor protein, HIV-1 reverse transcriptase, AAA p97, lipid bilayer, and bacterial flagellum. These SMC's are involved in a diverse number of biological processes - production of energy, genetic and allosteric regulation, propagation of viral infection, and cell structure integrity. Understanding the motions of SMC's, as opposed to individual proteins or molecules, is a fundamental step towards determining detailed functional mechanisms. (1) TMD trajectories of F1-ATPase are used to resolve the motions and interactions that occur during the 120° rotation step of the gamma subunit. An ionic track of arginine and lysine residues on the protruding portion of the gamma subunit plays a role in guiding the motions of the beta subunits. (2) The allosteric transition of lactose repressor protein from the repressed (DNA-bound) to the induced (IPTG-bound) state is simulated using TMD. Non-covalent interactions of three interconnected pathways are described. Pathway 2 involves reorganization at the dimer interface and formation of an H74-H74' pi-stacking intermediate. (3) TMD is utilized to investigate the translocation mechanism of HIV-1 reverse transcriptase. The atomic-level interactions between electrostatic (i.e., K263) and hydrophobic (i.e., W266) residues and the DNA primer strand are highlighted. (4) The proposed negative-cooperative ratchet-mechanism between the D1 and D2 rings of p97 is illustrated by means of a QEDM analysis. (5) The intrinsic fluctuations of a DPPC lipid bilayer are investigated via QEDM and elucidate a low-frequency sound mode. (6) QEDM is used to calculate the dimensionless twist-to-bend ratio (EI/GJ) of bacterial flagellar hook and filament. Both ratios are less than one, indicating that within each structure bending is favored over twisting. A theoretical Young's modulus for the hook is proposed, which is orders of magnitude smaller than experimentally determined Young's moduli of the filament. The research results in this thesis are also placed in context of existing experimental data, and in some cases propose future experimental work.

This work describes how new methods for measuring one-bond, two-bond and three-bond heteronuclear and homonuclear residual dipolar couplings (RDCs) and 31P chemical shift anisotropy (31P CSA) values in RNA molecules of average size have been developed, facilitating the efficient, accurate and precise collection of these previously under-exploited but potentially powerful types of NMR structural constraints. These new methods for measuring RDCs and 31P CSA values have been developed using a 24 nucleotide hairpin derived from helix-35 of the 23S rRNA of E. coli, containing the psi746 modification, as a model molecule. A very large body of these types of NMR constraints has been carefully measured for helix-35psi746 using the novel methods. The residual dipolar coupling constraints can be implemented in structure calculations of RNA molecules, although the resultant structures must be interpreted with caution especially when RDCs from dynamic regions are included. The first round of structure calculations for helix-35psi746 has shown that the RDC constraints measured can have a dramatic impact on improving the quality of this RNA structure.

In this thesis I describe two frontier problems in membrane research. The first is about the outer membrane of gram-negative bacteria for which there was little prior research. Using lipopolysaccharide (LPS) bilayer as a model for the outer membrane, the problem of how antimicrobial peptides pass through the outer membrane were studied by oriented circular dichroism (OCD). The second is about non-lamellar structure of lipids that are important for membrane fusion. Two lipid structures, in the inverted HII phase and the distorted HII phase, respectively, are solved by a novel method through neutron scattering. Lipopolysaccharide bilayer was used to mimic the gram-negative bacterial outer membrane. X-ray diffraction patterns show that LPS forms a well aligned multilayer system. Some ions, such as barium (Ba++), were found to be located at the phosphate group in LPS headgroup. OCD spectra show that alpha-helical peptide mellitin and magainin have two different bound states to LPS multilayer that differs in the orientation with the respect to membrane. At high peptide concentration, they are in the active state, i.e. transmembrane state, whereas at low peptide concentration, they are in the inactive state in which they are oriented parallel to the membrane. In contrast, beta-sheet peptide protegrin only showed the transmembrane state. The structures of inverted HII phase of diphytanoylphosphatidylcholine (DPhPC) and a newly discovered distorted HII phase in dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylethanolamine (DOPE) mixture were studied by a new diffraction method to solve the phase problem in neutron scattering experiments. Through model construction, lipid packing patterns were studied. The DPhPC HII Hexgonal structure shows a circular headgroup packing, whereas the DOPC/DOPE mixture distorted HII phase shows that the. DOPC and DOPE are not uniformly distributed, possibly due to their different spontaneous curvatures. The structural information obtained in these studies will shed new light on the research on membrane fusion.

The polyelectrolyte and the hydrophobic effects influence affinity of protein-DNA interactions. Explorations of these phenomena form the focus of this thesis as applied to three biological systems: lactose (LacI) and purine (PurR) repressors from bacterial cells and p53 protein from human cells. First, the presence of guanine or DNA enhanced DNA or guanine binding by PurR, respectively. Compared to the behavior of LacI, a homologous protein, PurR exhibits tighter control of the pur regulon, an effect that derives from both thermodynamic and kinetic differences. In vivo lactose utilization---controlled by LacI---is response to an environmental opportunity to which cells need to respond rapidly. In contrast, purine enzyme biosynthesis---controlled by PurR---is an energetically costly process so that cellular response must integrate signals of purine states over a period of time. Second, the ionic and temperature dependence of DNA binding---reflecting the polyelectrolyte and hydrophobic effects, respectively---for the structurally similar PurR and LacI proteins was examined. Both proteins exhibited a roughly ∼2-fold increase in ion pairs formed in their high affinity form. Both proteins employ basic residues from their N-terminal helix-turn-helix DNA binding motif and core domain; however, PurR employs additional basic residues from its hinge helices and possibly interchain N-terminal to core ion pairs to account for the ∼15 ion pairs when PurR-guanine complexes DNA. The values of Delta Cp determined by van't Hoff analysis, showed greater participation of local folding for PurR than LacI possibly due to more extensive hinge helix folding. These major differences between PurR and LacI, demonstrate that structurally similar proteins can display highly distinct behaviors. Finally, ion concentration dependence of wild type, S392E, and a C-terminal deletion variant (Delta33) p53 proteins showed a remarkably low number of ion pairs formed compared to the number predicted from p53 core domain structural data. CD, temperature dependence, and water release studies of Delta33 p53 are consistent with the notion of a structurally rigid core DNA binding domain. However, the DNA binding properties of Delta33 p53 variant did not substantiate its characterization as "activated" form of p53 and disfavor the popular model of DNA binding regulation by the p53 C-terminal domain.

Phosphatidylcholine multilayers containing $\sim$24% water by total sample weight and gramicidin/lipid molar ratios, were aligned by a mechanical stressing and low temperature annealing ($$80$\mu$ thick x 40mm$\sp2$ area) monodomain defect-free multilayers containing as many as 10$\sp{17}$ uniformly oriented gramicidin channels to be prepared. The alignment of the lipid multilayers was monitored by observing conoscopic interference patterns and orthoscopic images. The smectic defects which appeared during the alignment process were identified and dissolved. The incorporation of gramicidin in the multilayers in the form of the transmembrane channels was proven by its circular dichroism spectra (CD). The well defined CD spectrum of uniformly oriented gramicidin channels was obtained. With proof that gramicidin is in the transmembrane conducting state, it is hoped that these oriented samples will permit spectroscopic studies of the ion channel in its conducting state and diffraction studies of the channel-channel organization in the membrane. (Abstract shortened with permission of author.)

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No description available.

The partial differential cross section for the scattering of thermal energy neutrons from liquids and solids contains information about the diffusive motion of the nuclei. This experimental method is known as quasielastic neutron scattering (QNS). We have used QNS to study the effect of macromolecules and supramolecular structures on the diffusive motion of water in four systems: pure water, agarose gels, poly(ethylene oxide) (polyox) solutions, and the cyst stage of the brine shrimp (Artemia salina). The QNS spectra were studied using 0.95THz neutrons for values of the momentum transfer in the range 0.7(ANGSTROM)('-1) to 1.9(ANGSTROM)('-1), and energy transfers in the range -0.2THz to 0.2THz. Agarose gels were studied using a gel of 20% agarose in water by weight, and 20% agarose in deuterium oxide. Polyox solutions were prepared consisting of 20% polyox in water and 18.5% polyox in deuterium oxide (equivalent to 20% polyox in water). Brine shrimp were studied at hydrations of 0.10, 0.31, 0.78, and 1.20 grams of water per gram of dry brine shrimp. We have interpreted our data using the simplest heterogeous model for water in complex systems, the "bound-free" model. Analysis of QNS line shapes permits separation of the contribution of the tightly bound protons in the agarose and polyox from protons in the water. This analysis has allowed us to estimate the number of grams of water bound per gram of dry solid (c). For agarose c = 0.64 (+OR-) 0.26. The agarose results are in good agreement with previous nuclear magnetic resonance (NMR) studies of the diffusion coefficient. For polyox c = 1.46 (+OR-) 0.64. For the two highest brine shrimp hydrations c = 0.11 (+OR-) 0.07, but c decreased as the hydration decreased for the two lowest hydrations. The latter result is consistent with the results obtained by other workers with NMR. The free component in all of these systems showed deviations from the behavior of pure water. These deviations were all toward more "solid-like" behavior.

Steric interactions between ligand molecules and the valine E11 methyl group of human hemoglobin and sperm whale myoglobin have been examined by high resolution NMR. The methyl proton resonances of this amino acid are shifted markedly upfield by the heme ring current. In all the proteins the position of the E11 valine resonance showed little change in going from bound CO to bound methyl and ethyl isocyanide. In alpha chains and myoglobin, the binding of n-propyl and n-butyl isocyanide produced marked downfield shifts of the valine resonance, indicating that the valine residue was forced away from the center of the heme ring. In beta subunits only tert-butyl isocyanide produced a marked decrease in the ring current shift of the valine methyl protons. New peaks observed in the isonitrile protein spectra were identified as ligand proton resonances by comparing the spectra of normal and deuterated isonitrile complexes. The magnitudes of the ring current shifts for the terminal methyl protons of ethyl isocyanide suggest a linear geometry for the Fe = C = N - Cl bonds in beta chains and a bent geometry for alpha chains. Myoglobin ethyl isocyanide complexes exhibit ligand ring current shifts intermediate to those observed for the hemoglobin subunits. Kinetic and equilibrium constants for the reactions of CO and the isonitriles with a large group of proteins and a model heme compound have been determined. Results were analyzed in terms of a 3 barrier model for ligand binding. The observed rate and equilibrium constants for the protein reactions showed a complex dependence on each individual step in the binding process. Pentacoordinate heme dissolved in soap served as a simple model for the proteins. When the protein equilibrium data are converted into free energy terms and the heme-soap results subtracted, the resulting value, (DELTA)G(,prot), describes the distal protein steric barrier. The results indicate that CO binds easily to all of the heme proteins while methyl isocyanide shows a large increase in (DELTA)G(,prot). This increase in steric hindrance has been attributed to the distal histidine residue. In most of the proteins, bound ethyl isocyanide exhibits about the same steric barrier as methyl isocyanide and the longer isonitriles show increasingly larger steric hindrance.