The feasibility of studying the mechanical properties of lipid bilayers utilizing neutron reflectivity is examined. The properties of interest are the bulk modulus for compressibility and the membrane rigidity. It is possible to analyze reflectivity measurements of aligned lipid bilayers in terms of theories developed for scattering experiments. Experimental results are presented which indicate that it is possible to study lipid bilayers using neutron reflectivity, but the results point out deficiencies in the particular apparatus used to perform the experiments. Experimental requirements for future work are presented.

A method has been developed to measure the spin-lattice and spin-spin relaxation times of the various components of a multi-component spectrum. By Fourier transforming the free induction decay after the inversion recovery or spin echo pulse sequences, the different frequency components can be resolved and their individual relaxation times can be measured. This method has great advantages over the conventional method of analysis for multiple component decays which are very common in chemical and biological system. Based on this method, many interesting studies can now be done with NMR. The details of this method are presented with a comparison between our method and the conventional method and results and discussion of some applications.

Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37$\sp\circ$C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, His$\sp{64}$ (E7) was replaced with Gln, Gly, Val, Leu, and Phe; Val$\sp{68}$ (E11) was replaced with Ala, Ile, and Phe; Leu$\sp{29}$ (B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His$\sp{64}$ (E7) was replaced with Val; Val$\sp{68}$ (E11) was replaced with Thr and Ser; Thr$\sp{67}$ (E10) was replaced with Ala, Val, Glu, and Arg; Lys$\sp{45}$ (CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo first order rate constants varied 5 orders of magnitude, from 44 hr$\sp{-1}$ (H64G) to 0.055 hr$\sp{-1}$ (native) to 0.005 hr$\sp{-1}$ (L29F) at 37$\sp\circ$C, pH 7 in air. The dependence of autooxidation on oxygen and pH were measured for selected proteins. In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high oxygen levels, autooxidation proceeds by direct dissociation of the neutral superoxide radical (HO$\sb2$) from oxymyoglobin, and this process is accelerated by decreasing pH. At low oxygen levels, autooxidation also occurs by a bimolecular reaction between molecular oxygen and a very weakly bound complex between water and ferrous deoxymyoglobin. The neutral side chain of the distal histidine (His$\sp{64}$) inhibits autooxidation by hydrogen bonding to bound oxygen. Replacement of His$\sp{64}$ by amino acids incapable of hydrogen bonding to the bound ligand causes a change in the mechanism of autooxidation and marked increases in the rate of autooxidation. Increasing the polarity of the distal pocket by substitution of Val$\sp{68}$ with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II)-O$\sb2$ complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 (E11) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.

Biological macromolecules do not function as static molecules. A variety of biological functions are the result of fluctuations in protein and nucleic acid structure. The method of X-ray diffuse scattering analysis has been extended to provide information about intermolecular and intramolecular disorder in protein and nucleic acid crystals. Yeast initiator transfer ribonucleic acid (tRNA) carries the initial methionine to the ribosome during translation initiation. Results from analysis of the yeast initiator tRNA crystal diffuse scattering indicate that anisotropic, lattice-coupled motions contribute to the overall disorder in the crystals. The lattice-coupled disorder implies a flexing motion between the anti-codon and acceptor arms. This type of flexing has been implicated in tRNA's role in protein synthesis on the ribosome. Diffuse scattering analysis also shows that the distal half of the anti-codon arm of tRNA undergoes isotropic short-range motion correlated over a distance of approximately 3 A. This distance corresponds well with the base-pair stacking distance in RNA helices. These results are consistent with nearest-neighbor base pairs moving isotropically and as coherent units in the tRNA anti-codon arm. Aspartate aminotransferase (AspAT) is an important enzyme in the catabolism of amino acids and catalyzes the removal of their amino groups. Crystallographic evidence suggests that the small domain of one of the subunits in the biologically active dimer of AspAT in acetate buffer is disordered. Analysis of the X-ray diffuse scattering from AspAT crystals shows that the small domain moves isotropically and as a rigid body with an amplitude consistent with the crystallographically determined average B-value for the small domain. A preliminary attempt has been made to map the three-dimensional diffuse scattering from a set of simulated still diffraction photographs. This technique has been applied to actual diffraction data from AspAT crystals. The results show that an important diffuse scattering feature is weak but discernible in the reconstructed 3-D volume. X-ray diffuse scattering analysis provides unique experimental evidence for motions in biological macromolecules in crystals. Information obtained about global as well as local intramolecular motion is useful in understanding the role of molecular flexibility in biological function.

The structure of the liganded form of phosphate-binding protein, an essential component of the Pst inorganic phosphate active transport system of E. coli has been determined and refined at 1.7 A resolution to an R-factor of 14.7% for 32,222 reflections. The initial model was derived by fitting the amino acid sequence to a 2.5 A resolution electron-density map computed with isomorphous replacement phases from a single iodo derivative with anomalous dispersion data. The mean figure of merit is 0.69 for 11,477 reflections. The model consists of all 321 amino acid residues (2,439 non-hydrogen atoms), the phosphate substrate and 259 ordered water molecules. The molecule is ellipsoid with overall dimensions of 35 A $\times$ 40 A $\times$ 70 A. Phosphate-binding protein structurally resembles six other periplasmic binding proteins (specific for sc L-arabinose, sc D-galactose/ sc D-glucose, leucine/isoleucine/valine, leucine, sulfate and maltose) solved in our laboratory. The molecule consists of two domains, each with a central $\beta$-sheet flanked by $\alpha$-helices. The domains are connected by a hinge which is composed of three strands and one helix. The substrate is bound in the cleft formed between the domains. The general folding pattern is parallel $\alpha$/$\beta$ with the exception of one antiparallel strand in each $\beta$-sheet as a result of the crossover between the domains. The bound phosphate anion is inaccessible to the bulk solvent. It interacts with the protein through twelve hydrogen bonds. Molecular dynamics simulations using free energy perturbation techniques reproduce the experimentally observed specificity of phosphate-binding protein. A decreased affinity is almost entirely due to differences in electrostatic interactions. Electrostatic field calculations based on the finite difference method compute a strong field gradient for the binding site in the absence of the substrate, caused by the proximity of several ionized side chains as well as a number of peptide amides and polar side chains. The gradient is complementary to the net dipole moment of the bound HPO$\sb4\sp{=}$. The electrostatic potential at the binding site is positive averaging about 100 kT/e despite the uncompensated charge of Asp56. (Abstract shortened with permission of author.)

Myoglobin is the subject of continuing investigations because of its ability to bind oxygen reversibly. This physiological role depends on the modulation of iron reactivity and heme affinity by the globin. The hypothesis that both of these factors are affected by residues in the proximal heme pocket is tested by site-directed mutagenesis of four proximal residues: Leu89(F4), His97(FG3), His99(FG5), and Leu104(G5). The structures of several mutant myoglobins have been determined by X-ray crystallography and then used to interpret the results of functional studies. These four proximal pocket residues support and maintain the structure of the porphyrin ring via steric and/or electrostatic interactions with the heme prosthetic group. In turn, stereochemical changes in the heme group induced by a proximal pocket mutation modulate heme-iron reactivity via changes in the displacement of the iron from the heme plane: a more accessible heme iron results in greater affinity for carbon monoxide and dioxygen. Leu89 and His97 are important surface residues that protect the hydrophobic heme pocket from hydration through steric and/or electrostatic interactions with the heme. Substitutions at either position with a small or polar residue expose the heme pocket to solvent and accelerate heme loss. Ile99 and Leu104 are located in the interior region of the heme pocket beneath the heme prosthetic group. Substitution with smaller or polar residues at positions 99 and 104 also results in water penetration of the heme pocket and promotion of heme loss. Furthermore, Leu104 is located between two sites previously found to bind xenon. Crystal structures of xenon-containing Leu104 mutant myoglobins support the hypothesis that changes in the volume accessible to photodissociated ligands affects the rates of primary and secondary recombination.

Gene-encoded membrane-active antimicrobial peptides permeablize bacterial plasma membranes without harming the host cells. Furthermore, although most peptides exhibit a broad spectrum of activity against microbes, different peptides preferentially kill different pathogens. Understanding such cell-type specificity is not only fundamental to cell biology but also crucial to potential pharmaceutically applications of antimicrobial peptides. Accumulated evidence indicates that the antimcrobial peptides target the lipid matrix of the plasma membranes. Therefore we focus on the physical states of the peptides bound to lipid bilayers. This thesis describes studies of lipid-peptide systems in the form of aligned multi-lamellae with new neutron and X-ray diffraction techniques developed specifically for such systems, under various conditions with improved temperature and relative humidity control. These technique allow the most detailed structural investigation on the supramolecular assemblies formed by these peptides in model lipid membranes. Interesting phenomena were observed. Peptides form transmembrane pores in fluid lipid bilayers. The sizes of various peptide pores were determined by fitting neutron scattering data with the theory of scattering. By manipulating the temperature and the hydration level of the samples, we observed position correlations developed between the pores located in neighboring bilayers that eventually became long-range and the transmembrane pores were crystallized in lipid membranes for the first time. Diffraction data of the crystallized pores were measured with synchrotron radiation using samples on ultra-thin Si3N4 substrate for transmission X-ray diffraction. A number of different crystalline phases were found. One example is the ABC stacking hexagonal structure, surprisingly also found in pure diphytanoyl phosphatidylcholine samples. Correlating the diffraction data with circular dichroism and other experimental evidence, we separate the pore structures into two categories described by the barrel-stave model and the toroidal model. The implication of these results on the peptide's cell-type specificity is also discussed in terms of the properties of the lipids and environmental variables.

Allosteric transition, the basis of signal transduction and central to the function of regulatory proteins (e.g., transcriptional factors), is widely involved in biological systems with conformational change as a key characteristic. Although end-state structures are known for many proteins, less is known about the underlying detailed mechanism of the allosteric transition. We have used LacI as a model system to investigate this process at the atomic level. The work in this thesis focuses on three regions of LacI: the core pivot region, the N-subdomain monomer-monomer interface, and the hinge region. Characterization of representative mutants (L148F, S151P, P320A, and Q60G/L148F) demonstrated that the core pivot region exerts long-range effects on LacI function. For L148F and S151P, operator and inducer binding are altered in an inverse fashion with binding for one ligand strengthened, and binding for the other ligand weakened. Further characterization of L148F and S 151P has indicated that the conformational equilibrium is shifted towards the induced state in L148F and towards the repressed end in S151P. This conclusion is supported by detailed thermodynamic ligand binding assays and UV difference spectra. Detailed unfolding/refolding studies further suggest that the intrinsic ligand-binding properties of L148F and S151P are altered. Global fitting of all ligand-binding data is underway to further characterize these shifts. Our data for K84 hydrophobic variants (K84A/L) disclose impeded allosteric response to inducer, a state that is supported by a unique pattern in UV difference spectra. Operator release kinetics for K84A/L in response to IPTG suggest that two inducer molecules are required to release operator DNA. Characterization of 13 substitutions at V52, including binding to operator sequence variants, indicates a dominant role of the protein-operator interaction in LacI allostery and high affinity operator binding. Moreover, subsets of mutants that decouple inducer binding and conformational change were identified. In summary, this thesis work emphasizes the key role of several regions in LacI allostery, identifies several LacI allosteric intermediates, and discloses intermediates trapped along the allosteric pathway by mutation that correlate with points along the TMD simulation.

Electric force microscopy has been adapted for fast, high-resolution imaging of the charge density of biological systems. The electric double layer interaction between the tip and sample provides a contrast mechanism that is sensitive only to surface charge density, as other parameters are held constant. This contrast is well described by an expression for the tip-sample double layer interaction in electrolyte solutions. The resultant charge maps are acquired at typical atomic force microscopy scan rates. Fluid electric force microscopy has proven highly sensitive and non-destructive, as demonstrated with charge density maps of fluid-phase supported bilayer membranes and single DNA molecules.

The method of X-ray multi-wavelength anomalous dispersion (MAD) is a phasing method routinely used in protein crystallography, but the same method is difficult for lipid systems for the practical reason that the commonly used lipid samples for diffraction do not have a well-defined thickness. The first chapter is devoted to a practical approach to use the MAD method for lipid structures. The procedure is demonstrated with the lamellar phase of a brominated lipid. In the second chapter, we apply this new MAD method to an inverted hexagonal structure. The bromine distribution obtained from the MAD analysis provides the information for the chain packing in the hexagonal unit cell. The intensity of the bromine distribution is undulated around the unit cell. The analysis shows that the lipid chains pack the hexagonal unit cell at constant volume per chain, with no detectable effect from a high-energy interstitial region.