Young adults often outperform older adults on tests of associative memory, however, the source of this age-related associative memory deficit is still under debate. There are two main non-mutually exclusive hypotheses: 1) impaired binding processes (i.e. creating and retrieving links between units of information) and; 2) impaired strategic processes (i.e. cognitive control processes that support encoding and retrieval). Although both components are thought to contribute uniquely and interact to support associative memory, they have rarely been studied together. The primary goal of this dissertation is to further characterize associative memory deficits in healthy aging by measuring and controlling binding and strategic processes. Specifically, in this series of three experiments, we studied these two components concurrently by varying the level of demands on binding (i.e. comparing memory for different types of associations) and strategic processes (i.e. varying demands on self-initiated processes). A total N of 97 young adults and 94 older adults studied lists of object-pairs and object-location pairs under intentional encoding conditions. Demands on self-initiated processes were manipulated by increasing the number of foils at test (Experiment 1: 4 alternative forced-choice (AFC), vs. Experiment 2 & 3: 20AFC), and by providing strategy instructions in Experiment 3. We measured the production of strategies with trial-by-trial self-report. In all three experiments, we found that young adults outperformed older adults on object-object memory, but not on object-location memory. Older adults were just as proficient as young adults in generating strategies at study. This remained true even when demands on self-initiated processes increased. However, we found in all three experiments that young adults had greater strategy effectiveness (i.e. accuracy on pairs encoded with a strategy) on the object-object test. In contrast, performance on the object-location task was found to be less related to strategies. Our findings suggest that not all associations are equally affected by aging and that even when strategy production is equivalent between age groups older adults can still be impaired on associative memory. The secondary goal of this dissertation was to explore the contribution of individual variability in age, general cognitive functioning, meta-memory and executive functioning on object-object and object-location memory, strategy production, and strategy effectiveness. Our results highlight the important contribution of executive functioning over and above any effects of age in explaining age-related associative memory decline.

The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membraneous network that, through the cyclical release and re-uptake of Ca²⁺ into and from, respectively, the cytoplasmic space, regulates myofilament shortening and, therefore, muscle contraction. SR derived from the terminal cisternae (HSR) demonstrates the property of Ca²⁺-induced Ca²⁺ release. Upon attainment of a threshold intralumenal Ca²⁺ load, application of a small pulse of extralumenal Ca²⁺ stimulates the release of a pool of intralumenal Ca²⁺ via the ligand gated Ca²⁺ permeable pore of the Ca²⁺ release channel/ryanodine receptor complex. It was hypothesised that intralumenal Ca²⁺ regulates the opening of the release channel. HSR vesicles were purified from skeletal and cardiac muscle by a novel technique. Structural characterisation of these membranes demonstrated an enrichment of harvested fractions in the Ca²⁺ release channel and the intralumenal Ca²⁺ binding protein, calsequestrin. In radiometric studies, skeletal HSR vesicles were shown to bind ryanodine with high capacity at both low and high affinity sites, with 2 fold stimulation of Ca²⁺ accumulation by the polyorganic cation Ca²⁺ channel blocker, ruthenium red. HSR vesicles passively loaded Ca²⁺. Passive loading of HSR vesicles with Ca²⁺ was found to be non-linearly dependent upon the concentration of Ca²⁺ within the loading medium. This suggested the presence of 2 intralumenal Ca²⁺ binding sites with different affinities for Ca²⁺. A spectroscopic dual-wavelength assay of Ca²⁺ release was developed that took advantage of peculiar spectral properties of the metallochromic sensitive dye Antipyrylazo III. In the presence of mM MgATP and mM Mg2+ the initial fast phase of HSR Ca²⁺ was well resolved. Evidence was presented that initial rapid uptake was associated with high affinity binding to an intralumenal compartment. Ca²⁺ -induced Caz+ release was shown to occur with a threshold loading of intralumenal Ca²⁺. The intralumenal Ca²⁺ threshold for Ca²⁺-induced Ca²⁺ release was decreased in the presence of ryanodine. Ryanodine induced Ca²⁺ release was also dependent upon the amount of intralumenal Ca²⁺. Ryanodine was also shown to inhibit sustained Ca²⁺-induced Ca²⁺ release by apparent inhibition of the binding of Ca²⁺ to intralumenal sites. These results suggested that junctional state transitions of the Ca²⁺ channel and calsequestrin were interdependent. Purified mM and mM Ca²⁺ activated neutral protease isoforms selectively cleaved the Ca²⁺ channel into 410 and 150kDa peptides with limited proteolysis. This was demonstrated in both HSR vesicles and the purified Ca²⁺ release channel. A novel 88kDa protein was also shown to be fragmented by both CANP isoforms. The identity of this prominent HSR associated protein remains obscure. CANP fragmentation of HSR protein elevated passive and active 4^Ca²⁺ loading in vesicles. This indicated that selective structural modification of the cytoplasmic portion of the release channel modified the comformational states of a intralumenal Ca²⁺ binding compartment in HSR vesicles. In spectroscopic studies, CANP proteolysis of HSR proteins increased the sensitivity to Ca²⁺ and ryanodine-induced Ca²⁺ release through decreases in the required intralumenal Ca²⁺ threshold for release. These functional alterations coincided with apparent single site cleavage of the release channel. Further proteolysis of the initial 410 and 150kDa peptides was without further significant effect upon function. Based upon the hypothesis that primary sequences rich in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST regions) are recognition sites for CANP binding to substrates, a search for PEST regions within the Ca²⁺ channel was undertaken. It was tentatively proposed that two PEST regions near the N-terminal of the Caz release channel may represent sites close to the CANP cleavage site. The results of this work were discussed in relation to a possible role of Ca²⁺-induced Ca²⁺ release in regulating the patterning of Ca²⁺ cytosolic transients. The frequency and amplitude of cytosolic Ca²⁺ transients appear to be important in regulating protein expression. The requirement of intralumenal Ca²⁺-induced Ca²⁺ release may be a means by which the cyclical uptake and release of Ca²⁺ during muscle relaxation and contraction can be coordinated. This coordination may define the patterning of cytosolic Ca²⁺ transients. The increased sensitivity to Ca²⁺-induced Ca²⁺ release by HSR after CANP treatment may represent a means by which the patterning of cytosolic Ca²⁺ transients can be altered to effect changes in protein synthesis. / Graduate and Postdoctoral Studies / Graduate

The multidrug resistance plasmid TP228 replicates at low copy number in Escherichia coli. Stable partitioning of this plasmid is mediated by three essential components: a ParA homologue, ParF; a centromere binding protein, ParG; and a centromere site, parH. ParF and ParG jointly assemble on the parH centromere forming the segrosome complex, and thereby direct intracellular plasmid transport. ParG belongs to the ribbon-helix-helix (RHH) class of dimeric DNA binding proteins. ParG specifically binds the parH site and also is a transcriptional repressor of the parFG genes. Previous studies demonstrated that unstructured N-terminal tails in ParG are not important for dimerization. Instead the tails are implicated in assembly of higher order nucleoprotein complexes essential for transcriptional repression and segrosome assembly, and also influence ParF nucleotide hydrolysis and polymerization. In this study we defined the role of residues in the RHH folded domain that are crucial for ParG dimerization and function. To achieve our goal the two α-helices, the intervening loop, and two C-terminal residues were analyzed fully by alanine scanning mutagenesis. Initially, ParG mutants were constructed and assessed for effects on normal plasmid partition activity and on dimerization. In vivo segregation assays and bacterial two-hybrid studies revealed mutation of residues F49 in α-helix 1 and W71 and L72 in α-helix 2 of ParG each resulted in defective plasmid partition activity and impaired dimerization. In vitro chemical cross-linking of purified proteins ParG-F49A, ParG-W71A and ParG-L72A demonstrated predominant monomeric species whereas wild-type ParG formed dimeric species as noted previously. Multiangle light scattering and sedimentation equilibrium analysis of the mutant proteins showed shifts in molar mass towards monomeric species with increased Kd values for dimerization. Protein-DNA interactions studied by gel retardation assays showed impaired interactions of ParG-F49A, ParG-W71A and ParG-L72A with parH. Results of conserved substitutions at position 71 showed that aromatic substitutions of W71 to Y71 or F71 are tolerated and have no apparent effects on ParG mediated plasmid segregation, but the non-aromatic W71L mutation blocked the segregation. However, a ParG double mutant bearing the ‘reversed’ amino acid pair (W71L-A52Y) retained plasmid segregation activity and behaved like wild-type ParG in dimerization assays in vitro and in vivo. Thus, substitution of W71 by tyrosine or phenylalanine does not disturb the monomer-monomer interface interactions that pack α-helix 2 from one monomer against residues of α-helix 1 and α-helix 2 of the partner monomer. Moreover, the permissible amino acid combinations at interacting positions 52 and 71 in ParG show significant flexibility and reveal key roles for these residues in function and dimerization of ParG. Overall, our in vivo and in vitro interaction studies provide novel information about the role of hydrophobic residues F49, W71 and L72 in ParG dimerization and activity. In the longer term, interference with dimerization by ParG and other centromere binding proteins using artificial ligands may provide a novel strategy for destabilization of antibiotic multiresistance plasmids.

One common method of improving the nutritional quality of certain food proteins is through fortification with necessary amounts of limiting essential amino acids. This simple and convenient method, however, is not the best. Several disadvantages are associated with the addition of free amino acids to food proteins, such as changes in flavor and color, losses of added amino acids during food processing or cooking, differences in stability and metabolism between free amino acids and amino acids in proteins. Covalent attachment of the limiting amino acids, however, should eliminate these problems, and moreover, could improve the nutritional and functional properties of food proteins. In this study, therefore, an attempt to improve the nutritional value of the soy protein was made by using the carbodiimide condensation reaction to covalently bind methionine and tryptophan to soy protein. In order to confine as much as possible the binding, of amino acid to the protein a-carboxyl groups the soy protein isolate (SPI) was partially hydrolyzed with pepsin to increase the number of a-carboxyls in soy protein. Various conditions of the carbodiimide reaction were analysed by a fractional factorial design in an attempt to determine the factors affecting the amino acid binding to soy protein hydrolysate (SPH). Of the factors investigated, pH, SPH concentration, carbodiimide concentration, activation time and reaction time were found to significantly affect the methionine binding efficiency, whereas pH, SPH concentration, carbodiimide concentration, amino acid concentration and reaction temperature were found to significantly influence the tryptophan binding efficiency to SPH. To determine the best level for each of the selected factors an optimization of the carbodiimide reaction conditions was conducted by carrying out another factorial experiment. Thus, under the best condition found, the methionine and tryptophan contents of methionine - and tryptophan-bound SPH samples were increased 7.7-fold and 18.0-fold, respectively. An in vitro pepsin-pancreatin digestion test demonstrated that the bound amino acids were readily released. In order to improve the low yield of the final product, another analysis of the carbodiimide reaction conditions was carried out. Since the yield could not be markedly improved by this factorial design in which peptic SPH was used, SPI without preliminary hydrolysis was used as the starting material. A product with 95-99% yield was obtained and its methionine or tryptophan content was increased 6.3-fold or 11.3-fold, respectively. High digestibility was still maintained for these products. Gel filtration chromatography demonstrated that the carbodiimide reaction caused an increase in the molecular weights of soy protein fractions. Furthermore, gel filtration chromatography revealed that, there was no selective amino acid binding among the different soy protein fractions during the carbodiimide reaction. / Land and Food Systems, Faculty of / Graduate

acase@tulane.edu / Rational, synthetic design is implemented in a systematic study of the effect of host shape and properties and manifestations of the reverse Hofmeister effect. Hofmeister specific effects were observed at the molecular level wherein it was shown that key to the effectiveness of some “salting-in” anions is their complementarity to hydrophobic cavities and other binding surfaces. A gamut of responses was observed across a range of hosts possessing different structural and functional motifs. These observations were typically manifest at a relatively low (<20 mM) critical precipitation concentration (CPC). Furthermore, it was shown that at low concentrations, typical observations of screening effects are not observed, and binding-site competition is a predominant factor when multiple anions are present in solution. In terms of quantifying the ion recognition sites of different, similarly charged hosts there is little difference in anion affinity, but large differences are observed in 1/CPC values. Thus, subtle changes in the recognition site have dramatic changes in terms of manifestations of the reverse Hofmeister effect. This is (to the authors best knowledge) the first example of a systematic study sequentially modifying small molecular hosts and utilizing them to study reverse Hofmeister trends. In total 12 hosts and 6 host-guest complexes were examined. These studies demonstrate applications of the reverse Hofmeister effect to generate single crystal X-ray structures, with potential applications in protein and small molecule purifications, separations, and crystallizations. / 1 / Jacobs Jordan

Mitogen-activated protein kinases (MAPKs) are an important subfamily of protein kinases that are well conserved in all eukaryotes. MAPKs are the final component of a three-tiered signaling module that regulates the activation of various essential cellular responses. They activate most of their substrates through catalyzing their phosphorylation. However, emerging evidence reveals that some MAPKs also possess non-catalytic functions. In particular, the human MAPK ERK2 can bind to DNA directly and mediate gene expression. The mechanism by which ERK2 binds to DNA is still unclear. In this work, we combined structural, biophysical and biochemical methods to confirm DNA binding by ERK2 and to investigate whether ERK2’s closest plant homolog MPK4 also binds to DNA. First, we identified a possible ERK2-like DNA consensus motif in plant MAPKs. We found that several plant MAPKs, including MPK4, harbor a basic motif (KARK/R or ARR/K) in a region corresponding to the ERK2 KAR motif reported to mediate DNA binding. Next, we determined the DNA binding affinity of ERK2 and MPK4 to different DNA fragments and found that MPK4 associated directly with DNA in vitro, albeit with a significantly lower affinity than did ERK2. Moreover, we observed that ERK2 and MPK4 showed preferred binding to different DNA sequences. Site-directed mutagenesis on the proposed DNA binding region of MPK4 greatly weakened DNA binding, confirming that MPK4 and ERK2 use the same structural elements to associate with DNA. Phosphorylation of the MAPKs through an upstream MKK affected the DNA binding capacity for both ERK2 and MPK4, although the effects differed. Lastly, we observed that a MPK4 mutant with a constitutively increased catalytic affinity displayed a markedly stronger DNA binding affinity compared to wild type MPK4 and phosphorylated MPK4. By demonstrating that the plant MPK4 associated with DNA in vitro, and that this association can be modified by phosphorylation and mutations, we open the possibility of additional kinase-independent functions in plant MAPKs.

Glucans are (1-3)-β-D-linked polymers of glucose that are produced as fungal cell wall constituents and are also released into the extracellular milieu. Glucans modulate immune function via macrophage participation. The first step in macrophage activation by (1-3)-β-D-glucans is thought to be the binding of the polymer to specific macrophage receptors. We examined the binding/uptake of a variety of water soluble (1-3)-β-D-glucans and control polymers with different physicochemical properties to investigate the relationship between polymer structure and receptor binding in the CR3- human promonocytic cell line, U937. We observed that the U937 receptors were specific for (1→3)-β-D-glucan binding, since mannan, dextran, or barley glucan did not bind. Scleroglucan exhibited the highest binding affinity with an IC50 of 23 nM, three orders of magnitude greater than the other (1→3)-β-D-glucan polymers examined. The rank order competitive binding affinities for the glucan polymers were scleroglucan >>> schizophyllan > laminarin > glucan phosphate > glucan sulfate. Scleroglucan also exhibited a triple helical solution structure (ν = 1.82, β = 0.8). There were two different binding/uptake sites on U937 cells. Glucan phosphate and schizophyllan interacted nonselectively with the two sites. Scleroglucan and glucan sulfate interacted preferentially with one site, while laminarin interacted preferentially with the other site. These data indicate that U937 cells have at least two non-CR3 receptor(s) which specifically interact with (1→3)-β-D-glucans and that the triple helical solution conformation, molecular weight and charge of the glucan polymer may be important determinants in receptor ligand interaction.

This work focuses on pigmy sperm whale and horse myoglobins (Mbs), which are distinguished by a single heme pocket residue variant in the CD3 position, when the heme iron is in the +3 oxidation state (i.e. the met form). The strategy employed is as follows: (i) assign heme peripheral protons; (ii) assign the amino acid residues from the heme cavity; (iii) assess the dynamics of ligand binding in the active site by means of hydrogen Iability, solvent isotope effects, and heme-insertion isomer trapping, all by NMR methods. The results of these studies portray dynamic solution structure of the Mb ligand binding site, and provide a set of standard parameters for the studies of larger hemoproteins. The findings are also important for understanding protein-ligand interactions in general. My research investigates the mixed spin metazido and metimidazole complexes of Mbs for the following reasons. First, the allosteric properties of hemoglobin arise mainly from the transition between its two possible quaternary structures. This can be studied by paramagnetic NMR because it is one of the most sensitive tools in terms of changes in the molecular and/or electronic structure of the heme. Second, both the N₃- and imidazole (lm-) complexes are good compromises, in terms of sizes, between the small diatomic oxygen or CN⁻ molecules and the bulky phenyl group. Thus, we can determine the influence of ligand size on structural perturbation of the Heme crevice by comparison among the different size groups. Third, the saturation-transfer phenomenon between metMbIm and metMbH₂0 provides a route to assignments in metMbH₂0 by using assignments of metMbIm. This is crucial because metMbH₂0 is the basis of theoretical calculations of the isotropic shift due to axial ligand field in pure high-spin hemoproteins. Finally, the importance of the metMbIm is underscored by the fact that it is a bis-imidazolium complex, which can then serve as a model other bis-histidyl proteins. Most of the heme peripheral resonances of metEqMbIm and metEqMbN₃ were identified by means of two-dimensional NOESY,COSY, and EXSY spectroscopy. The strongly relaxed upfield protons in metMbIm were assigned based on steady-state 1D NOE and T₁ experiments. Based on the results from metMblm in which saturation transfer of one upfield resonance led to two different free ligand peaks, bound Im equilibration was envisioned and proven by the divergence of broad downfield heme methyl peaks into two peaks each, showing distinctive population preference of each isomer. Dicyanoheme probe, as well as hydrogen Iability comparison studies between pigmy sperm whale Mb and horse Mb in the azido and imidazole states, asserts that single variant pocket residue CD3 is crucial in gating the ligand mobility into and out of the active site. The assignments of heme peripheral and upfield resonances enabled the subsequent assignments of some heme pocket amino acid residues. The facile exchange of bound Im with solvent H₂0 lays the ground work for identification of heme pocket residues in metMbH₂0. Furthermore, while deuterated heme previously allowed only assignment of the non-diastereomeric specific heme 2-vinyl β proton, saturation-transfer from horse imidazole Mb affords the specific identification of 2Hᵦt.

No description available.

The binding energy of the tritium nucleus is calculated by the variational method, using static repulsive core potentials. / Thesis / Master of Science (MSc)