Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2+-binding protein, which possesses some characteristics resembling those of regulatory Ca2+-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.

No description available.

Calcium/phospholipid-dependent protein kinase (PKC) was purified from P1798 lymphosarcoma. It was demonstrated that uncomplexed calcium and uncomplexed phosphatidylserine are the activators, that the activation of PKC requires that calcium bind first, that the activation of PKC requires that calcium bind first, that high calcium concentrations inhibit PKC activation, and that calcium inhibition can be overcome by phosphatidylserine.

Nonmuscle myosin from bovine thymus was purified, characterized , and phosphorylated with MLCK, H4PK, and Protein Kinase C. Phosphorylation occured exclusively on the myosin regulatory light chain. Phosphorylation by MLCK and H4PK resulted in the activation of the MgATPase activity as well as filament assembly of nonmuscle myosin.

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is demonstrated to exist in the ß-cell and immunopecipitation. Glucose and potassium significantly stimulate the rapid autophosphorylation of CaM kinase II and proportionally induce autonomous activity of the kinase in a dose-dependent manner that parallels insulin secretion. The activation of CaM kinase II, alloxan, KN-62 and KN-93, suggest that the enzyme is an integral component of insulin secretion and/or related processes in the β-cell.

Nanoparticles (NPs) have become a key tool in medicine and biotechnology; as drug delivery systems, biosensors, and diagnostic devices. However, the mechanism of biocorona formation on nanoparticle surfaces and their impact on drug delivery remains speculative. Nevertheless, functionalized nanoparticles have demonstrated major success in medical applications; having been shown to effectively treat disease. The mechanistic details of protein behavior on nanoparticle surfaces remain poorly understood to date; due to difficulty in determining the orientation and structure of protein on NPs. Furthermore, surface crowding, orientation, and degree of disorder have been shown to perturb the efficacy of protein on NPs; dramatically reducing their benefits. NMR and other biophysical tools can be used to characterize the nanoparticle-protein surface interactions; leading to a better understanding of the biocorona structure. This dissertation investigates the structure, orientation, and function of proteins adsorbed on gold nanoparticles (P-AuNPs). Using hydrogen-deuterium exchange and methylation studies on P-AuNPs, we have elucidated the structure and orientation of proteins on AuNP surfaces. We have also designed fusion proteins that can effectively mitigate structural-, orientation-, and activity-perturbations of P-AuNPs. The benefits of our fusion protein approach have been verified via enzymatic assay; which monitored the enzymatic activity of these P-AuNPs. Biofilms are defined as surface-anchored, multi-cellular, three-dimensional, bacterial communities. Biofilms have a serious impact on public health; because of their role in infectious diseases and medical device-related infections. S. epidermidis is the most common biofilmorming bacteria. Therefore, understanding the mechanisms of biofilm formation could lead to novel therapeutics which prevent biofilm formation. One of the most recognized proteins in the biofilm formation mechanism is the S. epidermidis autolysin domain. Therefore, we have studied the structure and behavior of S. epidermidis autolysin repeat domain R2 (R2ab) via solution NMR and other biophysical techniques. This study has provided a deeper understanding of how R2ab interacts with foreign surfaces and blood proteins; which could lead to future methods of biofilm prevention. Over the course of this dissertation, the characterization of protein-surface interactions was achieved via solution NMR and other biophysical tools; providing insightful information to the fields of medicine and therapeutics.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is involved in Ca2+signaling throughout the body. CaMKII is enriched in the hippocampus and required for learning and memory formation. Four highly conserved genes encode CaMKII in vertebrates: A, B, G, and D. All CaMKII variants are constituted of a kinase domain, regulatory segment, variable linker, and hub domain. These domains comprise an individual subunit which oligomerize together via the hub domain to form multimeric holoenzymes. These four genes are most variable in the linker domain due to extensive alternative splicing. The variable linker significantly impacts the activation of CaMKIIA. Herein, I attempt to develop an in vitro assay which resembles physiological activation of CaMKII via Ca2+ oscillations. I provide preliminary data which indicate that alternative splicing of the variable linker in CaMKIIA modulates the Ca2+ frequency dependent autonomy of these variants. Additionally, neuronal CaMKII variants of CaMKIIA and CaMKIIB decode Ca2+ oscillations into different levels of autonomous activity. Lastly, I assess the impacts of three de novo mutations (Q274P, R275H, and F294S) on Ca2+/CaM sensitivity in CaMKIID by providing data that these 3 mutants increase the sensitivity of CaMKIId to Ca2+/CaM and that Q274P and F294S mutants display Ca2+/CaM independent activity.

The activation of Protein Kinase A (PKA) and of Hyperpolarization-activated and Cyclic Nucleotide-modulated channels (HCN) is controlled by cAMP through cAMP binding domains (CBDs), which serve as cAMP-dependent conformational switches to regulate downstream signaling pathways. The binding of the cAMP allosteric effector removes the auto-inhibition imposed by linkers that are adjacent to the CBDs of PKA and HCN. However, our understanding of how cAMP binding to the structured CBD controls the adjacent inhibitory linkers is currently limited. Herein, we investigate through NMR spectroscopy the interactions between the CBDs of HCN and PKA and the respective adjacent linkers. Chapters 2 and 3 of this thesis focus on the linkers N-terminal to PKA CBD-A and CBD-B, respectively, while Chapter 4 centers on the linker N-terminal to the HCN CBD. We show that in the case of PKA the linker N-terminal to CBD-A is flexible, but is coupled to the CBD-A through state active selective interactions. In the case of the CBD-B of PKA the state selective interactions with the linker N-terminal to it are to a large extent lost and replaced by state-selective inter-CBD interactions, which in turn control the conformational ensemble accessible to the inter-domain linker. Unlike PKA, in the case of HCN, the primary mechanism of cAMP-dependent linker control is through the state-selective destabilization of the structured tetrameric N-terminal linker. Overall, this thesis reports three distinct mechanisms through which linkers in HCN and PKA serve not only as simple covalent threads, but also as integral parts of the allosteric networks underlying auto-inhibition and cAMP dependent activation. / Thesis / Doctor of Philosophy (PhD) / Cyclic adenosine monophosphate or cAMP is a second messenger that is produced by cells to control the internal cellular metabolism in response to external stimuli. The goal of this thesis is to elucidate the structural and dynamical changes that translate the cAMP signal into a specific biological response necessary for the survival of the cell. We used Nuclear Magnetic Resonance (NMR) Spectroscopy to investigate how, under physiological solution conditions, the cAMP interacts with and modifies the cAMP-dependent protein kinase A (PKA) and the hyperpolarization-activated and cyclic nucleotide-gated channels (HCN). Knowledge of both structure and dynamics on both proteins is required in order to fully understand at a molecular level how cAMP works in the human heart. The elucidation of the structural and dynamical changes associated with cAMP-binding is expected to help define general rules applicable to the design of drugs for cardiovascular disorders.

There is a saturable, dose-response relationship between the amount of protein ingested at a meal, the ensuing hyperaminoacidemia, and the subsequent skeletal muscle protein synthesis (MPS) response. Imposition of an external load, usually practiced as resistance exercise, on skeletal muscle is also a potent stimulus for increasing MPS and adds synergistically to the hyperaminoacidemia-induced rise in MPS. The current thesis examined the potential for meal-focused protein/leucine intake strategies, alone and in combination with resistance exercise, to augment MPS in older men. MPS was measured acutely (hours) using the continuous infusion of L-[ring-13C6]phenylalanine (Study 1) or over longer-term, integrated periods via ingestion of deuterated water (Study 2: 2-wk and Study 3: 3-d) while participants were free-living. In Studies 1 and 2 we examined whether a balanced versus a skewed pattern of protein intake across daily meals would enhance MPS during energy restriction (ER) in overweight/obese older men. Study 1 showed that a balanced consumption of protein during ER stimulated acute (%/h) myofibrillar protein synthesis (MyoPS) more effectively than a traditional, skewed distribution. Combining resistance training (RT) with a balanced protein intake pattern restored the lower acute rates of MyoPS during ER to the higher levels observed in energy balance. Study 2 showed no effect of daily protein intake pattern during ER on longer-term integrated MyoPS (%/d). However, the inclusion of RT during ER enhanced integrated MyoPS and the synthesis of numerous individual contractile, sarcoplasmic and mitochondrial skeletal muscle proteins with both protein intake patterns. Study 3 showed that leucine co-ingestion with daily meals enhanced integrated (%/d) MyoPS in healthy older men who were in energy balance and was equally effective among those consuming higher (1.2 g/kg/d) and lower (0.8 g/kg/d) protein intakes. Furthermore, the stimulatory effect of leucine co-ingestion on integrated MyoPS was further potentiated with the performance of resistance exercise. Collectively, these studies support the potential for per-meal recommendations, optimizing the protein dose consumed on a per-meal basis and leucine co-ingestion with meals, to augment MyoPS in older men, especially when combined with RT. These data have implications for recommendations to optimize MyoPS and possibly muscle mass in aging persons. / Thesis / Doctor of Philosophy (PhD)

Diacylglycerol kinases (DGK’s) tightly regulate the intracellular levels of diacylglycerol (DAG) and phosphatidic acid (PA). DAG is an important intermediate in lipid biosynthetic pathways and acts as a lipid second messenger in a number of signaling pathways. Similarly, since PA serves as a potent signaling lipid and is a precursor for lipid biosynthesis, intracellular PA levels must be tightly regulated. There are ten isoforms of DGK in mammals, but we have decided to focus solely on the epsilon form (DGKε) in this work. DGKε is the only isoform that shows specificity for the acyl chains of its DAG substrate; as a consequence, it contributes to the dramatic enrichment of cellular lipids with sn-1 stearoyl and sn-2 arachidonoyl. The most notable example is the highly enriched bioactive lipid 1-stearoyl-2-arachidonoyl phosphatidylinositol. We have purified active human DGKε to near homogeneity and thoroughly characterized its stability as well as examined its secondary structure with CD. We also purified a truncated form (DGKε Δ40) that shows increased stability compared to the full-length protein. Our purified fractions are well suited for a wide range of exciting applications and studies. We have begun incorporating DGKε into liposomes in order to develop a liposome-based assay, which would be a dramatic improvement over the presently used micelle-based assay. This purification also allows for high throughput screens of chemical compounds to test for a specific inhibitor. These studies will reveal valuable information about the structural and functional properties of DGKε and will aid in the development of therapies for DGKε-related diseases. / Thesis / Master of Science (MSc)