To unravel the mysteries of complex biological processes carried out by biomolecules it is necessary to adopt a multifaceted approach, which involves employing a wide variety of tools both computational and experimental. In order to gain a clear understanding of the function of biomolecules their three dimensional structure is required. X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy are the only two methods capable of providing high-resolution three-dimensional structure of biomolecules. NMR has the advantage of allowing the study of structure of biomolecules in solution and is better equipped to characterize the dynamics of the protein. Protein structure determination by NMR spectroscopy consists of recombinant expression of isotopically labeled proteins, purification, data collection, data processing, resonance assignment, distance restraint and angular restraint generation, structure calculation and structure validation. Apart from 3D structure determination of biomolecules NMR has become the method of choice for studying transient protein-protein interactions, which are notoriously difficult to study at higher resolution by other methods. Mass spectrometry plays an important role in enabling rapid identification of biomolecules and their modifications. The high sensitivity and resolution mass spectrometry offers makes it the method of choice for studying post-transitional modification of proteins. Use of computers in biology has played an essential role in elucidating those structure function relationships in biomolecules that are not possible to study by experimental techniques. The first chapter of this thesis deals with the introduction of methods used in this study. A brief introduction about the theory of Nuclear Magnetic Resonance (NMR) spectroscopy is given. Protein NMR methods used for structure determination of medium sized proteins are discussed. A part of this chapter discusses about the application of mass spectrometry in biochemistry and the use of tandem MS/MS experiments in identification of proteins and peptide fragments. Finally, the last part of this chapter gives an introduction about the theory of molecular dynamics and techniques used in the post processing of MD trajectories to elucidate the dynamics of proteins. The second chapter of this thesis is concerned with NMR characterization of a novel protein-protein interaction between the glycolytic enzyme Triosephosphate isomerase and the redox protein Thioredoxin. Chemical shift perturbation studies have been done to map the binding interfaces of these proteins. The structure of the complex was then modeled using NMR restraints based docking using the known 3D structure of these proteins. The docked complex reveals crucial insights into the glutathione mediated redox regulation of Triosephosphate isomerase and the role of thioredoxin as a deglutathionylating agent. Enzyme activity assays of Triosephosphate isomerase were done to show the inhibitory effects of s-glutathionylation of Cys217 and the role of thioredoxin as a deglutathionylating agent. The third chapter of the thesis is aimed to address some important issues related to the inhibition of Plasmodium falciparum Triosephosphate isomerase by S-glutathionylation. Oxidative stress induces protein glutathionylation which is a reversible post translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione. Mass spectrometric analysis of the kilnetics of glutathionylation along with enzyme activity assays clearly show that gluthionylation of either Cys-13 (situated in the dimmer interface) or Cys-217 (situated in Helix G) can render the enzyme inactive. Molecular dynamics simulations provide a mechanistic basis of inhibition and predict that glutathionylation at Cys217 allosterically induces loop 6 disorder. The fourth chapter of this thesis addresses the stabilizing effect of introduction of a cross-strand disulfide bond across a non-hydrogen bonded position of an antiparallel beta sheet. Multidimensional heteronuclear NMR experiments have been used to get the backbone and side-chain resonance assignments, distance and angular restraints. In addition RDC based restraints have been used to calculate the structure of oxidixed form of L79C, T89C thiroedoxin. The observation of predominantly –RH staple conformation among the NMR ensemble in typical of cross-strand disulfides. The fifth chapter of this thesis deals with the dynamics of thioredoxin using computational methods.In this chapter analysis of known complexes of thiroedoxin was done to determine binding hot spot residues using free energy calculations. The physicochemical basis for the multispecificity of thioredoxin is probed using molecular dynamics simulations. In this chapter it has been shown that conformational selection plays a very important role in thioredoxin target recognition.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science

Thesis advisor: Clare O'Connor / Within cells, reactive oxygen species (ROS) are synthesized naturally and in response to environmental stimuli. However, ROS have deleterious effects on a wide range of cellular molecules. Oxidative stress, caused by the ROS generated by the partial reduction of oxygen, is a major cause of cell damage linked to the initiation and progression of numerous diseases. Thioredoxin peroxidase (Tpx1) plays important roles in cellular defense against ROS. Although homologous genes and their functions have been identified in other eukaryotes, the level of activity as well as the necessity of this protective enzyme in S. pombe exposed to oxidative stress has yet to be fully elucidated. To explore the role of the Tpx1 protein in oxidative stress resistance, novel strains were constructed in which the tpx1 gene was overexpressed. The polymerase chain reaction was used to amplify txp1, and the amplified sequence was cloned into the yeast overexpression plasmid, pNMT41, which allows overexpression under the control of the powerful promoter. DNA sequencing was used to determine that the sequences had been properly inserted into the vector. The plasmids were transformed into two leu- yeast strains: FWP6 and TP108-3C. Production of the Tpx1 protein was ensured using Western Blot techniques. Experimentation to test the responses of the tpx1 strain to oxidative stress will employ a variety of reactive oxygen generators, including hydrogen n peroxide, menadione, tert-butyl hydroperoxide, and paraquat. The results generally supported the proposed role of Tpx1 to confer additional resistance against the oxidative stress. In a complementary line of investigation, knockout strains are being constructed to reduce the levels of the Tpx1 in S. pombe. Gene deletion cassettes were constructed for tpx1. Currently, the strains are being analyzed for the successful replacement of the endogenous tpx1 gene by homologous recombination. If the absence of the protein results in decreased cell viability, the role of Tpx1 indicated by the overexpression experiments could be supported. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

S-nitrosylation (or S-nitrosation) has emerged as an important pathway of non-classical nitric oxide signaling. This post-translational modification involves the transfer of a nitroso group onto a cysteine residue and has been shown to regulate protein function. However, very little is known about the mechanism and structure-dependent factors of the modification. Understanding of S-nitrosothiol chemistry has lagged behind that for the classical nitric oxide signaling pathway due to challenges and limitations of current detection methods of S-nitrosothiols. The S-N bond is typically labile and indirect detection by traditional biotin switch method has low sensitivity and is prone to false positives. In this work, I have explored phosphine derivatization as a new direct approach to labeling protein S-nitrosothiols. Syntheses of aza-ylide derivatives of small organic S-nitrosothiols were successful and the termolecularity of the reaction was overcome by using a bisphosphine. Similarly, S-nitrosated cysteines of thioredoxin were successfully derivatized with the phosphine TCEP and identified by tandem mass spectrometry of the digested protein. Surprisingly, derivatization of S-nitrosoglutathione was found to be unsuccessful and ¹⁸O-labeling of the reaction indicated hydrolysis of the aza-ylide product. We hypothesize that solvent effects are the source of this discrepancy. In addition, x-ray crystallography studies were undertaken to investigate structural rearrangement of a thioredoxin helix to expose residue Cys 62 to S-nitrosation. A new structure of thioredoxin Q63A/C69S/C73S mutant was found to exhibit a highly dynamic N-terminal loop surrounding the pocket of Cys 62 which could have an effect on S-nitrosation of this residue.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Protein import is essential for the biogenesis of mitochondria, as the majority (99%) of mitochondrial proteins are synthesised in the cytosol and thus, have to be imported into mitochondria for their function. The biogenesis of many cysteine-containing proteins of the mitochondrial intermembrane space (IMS), such as members of the small TIM and Cox17 families, is regulated by their thiol-disulphide redox state. Only the Cys-reduced precursors can be imported into mitochondria, whereas oxidised forms cannot. Their import and oxidative folding in the IMS is driven by the IMS disulphide relay system, known as mitochondrial import and assembly (MIA) pathway, whose central components are the oxidoreductase Mia40 and the sulphydryl oxidase Erv1. Currently, little is known about how the MIA precursors are maintained in the cytosol in an import-competent form, and whether they interact with the translocase of the outer membrane (TOM complex) to enter the IMS. In addition, the MIA-mediated protein folding events occurring in the IMS that lead to the generation of fully oxidised substrates are still under investigation. Using Tim9 and yeast as models, studies presented in this thesis showed that Tim9 binding to the mitochondrial outer membrane (OM) does not depend on the receptors of the TOM complex, and occurs without regard to the redox state of the precursor proteins. In addition, it is shown that the oxidised and reduced precursors share the same binding site on the OM, and that this binding site is not important for the translocation process across the OM (Chapter 3). Studies in this thesis investigated the role of the cytosolic thioredoxin and glutaredoxin systems in the biogenesis of mitochondria. Firstly, in vivo studies provided the evidence that the cytosolic thioredoxin system but not the glutaredoxin system is required for growth of yeast cells under respiratory conditions. Secondly, in vivo studies provided the first proof that the Trx system is required for the biogenesis of small Tim proteins. In vitro studies confirmed that the Trx1 system facilitates import of small Tim proteins into isolated mitochondria by maintaining the precursors in a reduced and therefore competent form (Chapter 5). Finally, in vitro studies showed that Mia40 is able to promote the full oxidation of Tim9. Efficient release of Tim9 from Mia40 required the presence of all cysteine residues of Tim9, as effective oxidation and concomitant release from Mia40 failed upon mutation of single cysteine residues. Finally, the study showed that reduced glutathione resolved rapidly the Mia40-Tim9 mixed-disulphide complexes, probably accelerating and/or promoting the Tim9 oxidative (Chapter 4).