Glucosyltransferases (GTs) are enzymes which perform glucosylation reactions, which involve attaching a UDP-activated glucose molecule to acceptor molecules specifi c to the enzyme. The enzyme which our lab focuses its research on is a fl avonol-specifi c 3-OGT found in Citrus paradisi, or grapefruit (Cp3GT). This enzyme is part of the class of enzymes known as fl avonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Cp3GT in an attempt to discover the residues important for substrate and regiospecifi city. In this study, we are testing the basis of substrate septicity of Cp3GT. We hypothesize that incorporation of fi ve amino acids specifi c to Citrus sinensis GT (CsGT) into Cp3GT at 308th position may facilitate mCp3GT to use anthocyanidins as one of the substrates. We report our fi ndings thus far concerning the addition of specifi c residues to the Cp3GT’s amino acid sequence based on an alignment with the sequence of a putative fl avonoid GT found in Citrus sinensis.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Running title: Seasonal affective disorder and melanopsin. Typescript (photocopy).

No / Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3alpha and GSK3beta, while a splice variant of GSK3beta (GSK3beta2), containing a 13 amino acid insert, is enriched in neurons. GSK3alpha and GSK3beta deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, beta-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3beta, yet normal in cortex lacking GSK3alpha). Furthermore, the neuron-enriched GSK3beta2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3beta1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3beta isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3beta splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3beta1 and GSK3beta2 are similar.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

This thesis deals with the analysis of natural peptide libraries using mass spectrometry. In the course of the study, both ribosomal and non-ribosomal classes of peptides have been investigated. Microheterogeneity, post-translational modifications (PTM), isobaric amino acids and disulfide crosslinks present critical challenges in routine mass spectral structure determination of natural peptides. These problems form the core of this thesis. Chapter 2 describes an approach where chemical derivatization, in unison with high resolution LC-MSn experiments, resulted in deconvolution of a microheterogenous peptide library of B. subtilis K1. Chapter 3 describes an approach for distinction between isobaric amino acids (Leu/Ile/Hyp), by the use of combined ETD-CID fragmentation, through characteristic side chain losses. Chapters 4-6 address a long standing problem in structure elucidation of peptide toxins; the determination of disulfide connectivity. Through the use of direct mass spectral CID fragmentation, a methodology has been proposed for determination of the S-S pairing schemes in polypeptides. Further, an algorithm DisConnect has been developed for a rapid and robust solution to the problem. This general approach is applicable to both peptides and proteins, irrespective of the size and the number of disulfide bonds present. The method has been successfully applied to a large number of peptide toxins from marine cone snails, conotoxins, synthetic foldamers and proteins. Chapter 7 describes an attempt to integrate next generation sequencing (NGS) data with mass spectrometric analysis of the crude venom. This approach couples rapidly generated cDNA sequences, with high-throughput LC-ESI-MS/MS analysis, which provides mass spectral fragmentation information. An algorithm has been developed that allows the construction of a putative conus peptide database from the NGS data, followed by a protocol that permits rapid annotation of tandem MS data. The approach is exemplified by an analysis of the peptide components present in the venom of Conus amadis, yielding 225 chemically unique sequences, with identification of more than 150 sites of PTMs. In summary, this thesis presents different methodologies that address the existing limitations of de novo mass spectral structure determination of natural peptides and presents new methodologies that permit for rapid and efficient analysis of complex mixtures.

Analyses of protein sequences from diverse genomes have revealed the ubiquitous nature of multi-domain proteins. They form up to 70% of proteomes of most eukaryotic organisms. Yet, our understanding of protein structure, folding and evolution has been dominated by extensive studies on single-domain proteins. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Natural variations (nsSNPs) at these sites alter communication between domains and affect stability leading to disease manifestation. We emphasize this by using explicit all-atom molecular dynamics simulations to study the interface nsSNPs of human Glutathione S-transferase omega 1. We show that domain-domain interface interactions constrain inter-domain geometry (IDG) which is evolutionarily well conserved. The inter-domain linkers modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. These findings led to the development of a method to predict interfacial residues in multi-domain proteins based on difference evolutionary information extracted from at least two diverse domain architectures (single and multi-domain). Our predictions are highly accurate (∼85%) and specific (∼95%). Using predicted residues to constrain domain–domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. Further, we developed and employed an alignment-free approach based on local amino-acid fragment matching to compare sequences of multi-domain proteins. This is especially effective in the absence of proper alignments, which is usually the case for multi-domain proteins. Using this, we were able to recreate the existing Hanks and Hunter classification scheme for protein kinases. We also showed functional relationships among Immunoglobulin sequences. The clusters obtained were functionally distinct and also showed unique domain-architectures. Our analysis provides guidelines toward rational protein and interaction design which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR. These studies enable a deeper understanding of rapport of protein domains in the multi-domain context.

No description available.

No / Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).