<p> Site-directed mutagenesis provides a powerful tool in the study of enzyme function. Residues suggested to be important for catalysis can be readily mutated and the energetic effects measured. However, the limited repertoire of naturally occurring amino acids constrains the substitutions that can be made. To obtain a deeper understanding of how enzymes work requires using unnatural amino acids to systematically perturb enzymatic residues. For example, hydrogen bonds in an &lsquo;oxyanion hole&rsquo; are a common feature of enzyme active sites and often suggested to be important for catalysis. However, water can form hydrogen bonds, so for enzyme-mediated hydrogen bonds to be catalytic the energetics of these hydrogen bonds must be different than those made with water. A previous study in the enzyme ketosteroid isomerase (KSI) used a series of fluorotyrosine analogs to perturb the p<i>K_a</i> of the tyrosine hydrogen bond donor and results suggested a modest catalytic contribution of oxyanion hole hydrogen bonds. However, challenges in synthesis limited the set of fluorotyrosine analogs used. To overcome these challenges and extend the series of fluorotyrosines used in enzymatic studies, we developed an approach to selectively incorporate fluorotyrosines in peptides using silyl-based protecting groups. The fluorotyrosine must be protected on the amino and phenol groups and then incorporated in a peptide using solid phase synthesis. More so, the protection chemistry must be friendly in such a way that it does not have drastic side effects on any other part of the system. We tested the stability of silyl groups including TBDMS-Cl, TIPS-Cl, and TBDPS-Cl for their use in peptide synthesis. With the doubly protected fluorotyrosines, solid phase peptide synthesis will occur in order to place the new tyrosine into the protein of interest where these analogs will be used to investigate the energetics of enzymatic hydrogen bonds.</p>

<p> Recently, it has been shown that a decrease in activity of acetylcholinesterase (AChE) in AD patients is compensated by an increase in butyrylcholinesterase (BuChE) activity. Therefore, BuChE also becomes a significant target in the treatment of cognitive loss associated with Alzheimer&rsquo;s disease. While the majority of drug development so far has centered on AChE inhibitors in order to increase the acetylcholine level in AD patients, the development of specific and potent BuChE inhibitors have also begun to attract attention. We recently synthesized and assayed a library of bisthiophosphates as potential BuChE inhibitors. To evaluate if the analogs were selective for BuChE, their inhibitory properties against both BuChE and AChE were determined. </p><p>

Gold nanoparticles hold promise as both a stable drug delivery system as well as a targeted inhibitor of protein:protein interactions. The flexibility and ease of modifying the surface of nanoparticles enables them to be tailored to specific cellular environments and tasks. Due to these characteristics, I have been able to effectively modify gold. To further investigate gold nanoparticles as drug delivery systems, it is important to understand where they localize once inside the cell. One possible way of determining the cellular fate of the nanoparticles is conduction cell fractionization studies coupled with ICP-MS. A protocol using sucrose gradients along with high-speed ultracentrifugation allows for the various cellular organelles to be separated and digested. Once each fraction is digested, I can monitor where the gold nanoparticles localize inside the cell.

Surface functionized nanoparticles provide versatile tools for biotechnology. Their unique nano-scale size and large surface-volume ratio make them ideal for recognition of small molecules, DNA and proteins. Here, we are using such nanoparticles as DNA/drug delivery agents in mammalian cells. The release processes were mediated inside cells using glutathione (GSH) or near-UV light. Both in vitro and in vivo release of drug/DNA payload from nanoparticle surface were established. Additionally, a systemic study of nanoparticles surface charge relationship of various aspects of drug delivery (e.g. intracellular payload release, cellular uptake, and tissue penetration) has been performed using fluorescein-conjugated model systems. Furthermore, the GSH- or light- mediated release, combining with the controlled interactions of biomolecules with surface functionalized nanoparticle scaffolds, can be further utilized to realize targeted delivery into cancer cells.

Proteins and nanoparticles (NPs) provide a promising platform for supramolecular interaction. We are currently exploring both fundamental and applied aspects of this interaction. On the fundamental side, we have fabricated a series of water-soluble anionic and cationic NPs to interact with cationic and anionic proteins respectively. A Varity of studies such as, activity assay, fluorescence titration, isothermal titration calorimetry etc. were carried out to quantify the binding properties of these functional NPs with those proteins. Those studies reveal the prospect of tuning the affinity between the nanoparticles and proteins by the surface modification. On the application side, we have used this protein-nanoparticle interaction in protein refolding where we successfully refolded the thermally denatured proteins toward its native structure. We have also applied this particle-protein recognition to create a biocompatible protein sensor using a protein-NP conjugate. Green fluorescent protein and a series of cationic NPs were used for a protein sensor for the identification of protein analytes through displacement process. We have extended this application even in sensing the proteins in human serum.

Analytical methods of separation are the focus of this dissertation. Several of the studies are of a clinical nature and concern the effects of various medication and materials additives on low birth weight infants. In all cases, the chemicals under consideration are federally approved species, but must be reevaluated because of the underdevelopment of the infants. The first study investigates butylated hydroxy toluene (BHT), a common food and drug antioxidant, which is a component of the nutrition supplement administered to children. A high performance liquid chromatographic determination for the BHT and metabolites was required. Reverse phase chromatography was employed using photodiode array detection. This allowed for post-run optimization of detection wavelengths which was critical due to the limited sample size available. Sample preparation utilized solid phase extraction methodology to reduce the biological matrix. Results which highlight the differences between adult and infant samples are given. The second study incorporates element specific detection with a microwave induced plasma for gas chromatographic analysis to determine isothiazolinones in infant blood. This feasibility study illustrates the ability of the technique to discriminate the analytes from a complex matrix. Lysing of red blood cells was found to be necessary in order to quantitatively determine the analytes at the part per billion level necessary. The final clinical study exploits the features of photodiode array detection for liquid chromatography. A chromatographic peak is shown to contain two components and the source of the contaminant has been traced to drug infusion tubing. This peak has not been positively identified but spectral and retention characteristics suggest that it is a polymeric stabilizer used in the manufacture of polyvinylchloride tubing. Next, capillary isotachophoresis is employed to separate the conformers of calmodulin, a metallo-protein which undergoes changes in its tertiary structure as it binds up to four calcium ions. In isotachophoresis analytes travel with constant velocity in an electric field. Differences in electrophoretic mobilities allow analytes to migrate into discrete zones. Conductivity detection was used in this analysis. A description of the process and instrumentation is followed by a section of troubleshooting analysis. The separation of the calmodulin conformers is shown and experimental considerations for working with this and other metallo-proteins are discussed.

The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38&alpha;. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38&alpha;-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38&alpha;-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38&alpha;-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38&alpha; was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.