The work presented in this thesis describes the determination of structures of peptides and proteins at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. The thesis is divided into three parts. Part I of the thesis consists of chapters 1 to 4, and deals with structural studies of two novel conotoxins. Part II of the thesis consists of chapter 5 and deals with structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2. Part III of the thesis consists of Appendices A to D. Appendix A describes implementation of a novel pulse sequence for determination of disulfide connectivity using long-range 13 C–13 C scalar couplings across disulfide bonds. Appendices B, C and D contain supplementary infor- mation (acquisition parameters and chemical shifts) for the structural studies presented in parts I and II of the thesis. Part I: Structural studies of novel conotoxins from Conus monile Chapter 1 gives a brief overview of the conotoxins and their structural studies. The first half of the chapter describes biosynthesis, classification schemes, nomenclature, com- monly observed post-translational modifications and applications of conotoxins. The latter half of this chapter summarizes the challenges involved in the structural studies of conotoxins in light of the recent developments in integrated transcriptomic and venomic studies of conotoxins. The key homonuclear and heteronuclear NMR experiments that are employed for structural studies of conotoxins are summarized. Emphasis was laid on describing the spectral features and the structural information that can be gleaned from these experiments. Finally, the current mass spectrometric and NMR methods available for determination of disulfide connectivity are discussed Chapter 2 describes sample preparation and preliminary biophysical characteriza- tion of a conotoxin Mo3964 that contains a hitherto uncharacterized cysteine framework (C–CC–C–C–C). The sequence of Mo3964 was identified at the nucleic acid level as a cDNA clone. Analysis of the signal sequence revealed that the toxin belongs to the M-superfamily, while the cysteine framework bears more resemblance to O- and K- super- family of conotoxins. Structural studies were initiated to determine the disulfide connec- tivity, tertiary structure and biological activity. The gene corresponding to the mature toxin sequence was cloned in a bacterial expression vector pET21a(+) as a C-terminal tag to the cytochrome b5 fusion protein host system. The fusion protein was obtained by recombinant expression using the bacterial expression host E. coli BL21(DE3) and the mature toxin was obtained by either enzymatic or chemical cleavage of the fusion protein followed by size exclusion chromatography and reverse phase HPLC. Proton 1D NMR spectra of the purified peptide exhibited sharp lines and good spec- tral dispersion indicating that molecule was well folded. Formation of disulfide bonds in the mature toxin was ascertained by high resolution mass spectra of intact and chemically modified Mo3964. The peptide toxin exhibited remarkable stability to chemical denatu- ration and proteolytic digestion. Spectroscopic studies clearly showed that Mo3964 pos- sesses a very stable and well defined structure as long as its disulfide bonds are intact. Analytical size exclusion chromatography and Multi Angle Light Scattering (MALS) studies showed that Mo3964 exists in solution as monomer albeit with a non-globular structure. Electrophysiological studies showed that Mo3964 inhibits outward potassium currents in rat Dorsal Root Ganglion (DRG) neurons and increases the reversal potential of rat voltage gated sodium channel rNav 1.2 stably expressed on Chinese Hamster Ovary (CHO) cells at peptide concentrations as low as 10 nM. Chapter 3 describes the determination of disulfide connectivity and tertiary stricture of Mo3964. Initial attempts to determine disulfide connectivity using direct fragmenta- tion of the intact peptide in the mass spectrometer failed due to the relatively large size of the molecule and its resistance to endoproteases. Partial reduction alkylation based methods failed as the first stage of partial reduction gave rise to a mixture of various single disulfide bond reduced species which could not be separated from each other. Subsequently, information about the disulfide connectivity was obtained using a method that does not necessitate separation of such a mixture of single disulfide bond reduced species. This method involves partial reduction, cyanylation of the reduced cysteines and alkali mediated cleavage of the peptide backbone on the N-terminus of cyanylated cysteines. Structural studies were carried out using homonuclear and heteronuclear NMR meth- ods. The hydrogen bond network and hence topology of the molecule was determined with high accuracy using the long-range HNCO-COSY experiment that correlates hydrogen- bond donor-acceptor pairs. This experiment utilizes the three bond heteronuclear scalar coupling, i.e., the h3JN C O′ coupling across the hydrogen bonds. All these restraints proved crucial to the assignment of the disulfide connectivity in Mo3964, given its novel cysteine framework. The structure of Mo3964 was calculated using a total of 549 NOE distance restraints, 84 dihedral angle restraints and 28 hydrogen bond distance restraints. The tertiary structure was constructed from the disulfide connectivity pattern 1–3, 2–5 and 4–6, that is hitherto undescribed for the M–superfamily conotoxins. The ensemble of structures showed a backbone Root Mean Square Deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (φ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The remarkable stability and anomalous spectral properties exhibited by Mo3964 could be rationalized using the disulfide connectivity and the tertiary structure. The tertiary structural fold has not been described for any of the known Conus peptides. Further, a search for structures similar to that of Mo3964 using the web server DALI returned no hits indicating that the peptide scaffold of Mo3964 has no structural homologues. Hence, the conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules. The structure of Mo3964 was submitted to the Protein Data Bank (PDB ID: 2MW7)[1]. Chapter 4 describes the structural studies of a 17 residue, single disulfide containing conopeptide Mo1853. The samples for structural studies were obtained either by chemical synthesis or by recombinant expression methods. Structural studies using homonuclear solution NMR methods revealed that Mo1853 exists as two equally populated cis and trans X–Pro conformers which are in slow exchange regime, compared to the chemical shift timescale. Sequence specific assignments were obtained for both the conformers by analysis of homonuclear 2D 1 H,1H–DQF–COSY,1H,1 H–TOCSY, 1H,1 H–NOESY and 1H,1 H–ROESY spectra. Temperature dependence of chemical shifts was measured and coalescence was observed for two amide protons at 318 K. At this temperature, the rate of exchange and the free energy of activation were determined to be 59 Hz and ≈ 67.2 kJ mol−1 respectively. The evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D- NOESY and ROESY spectra. Tertiary structures of both the cis and trans conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. Tertiary structures of both the conformers consist of a 29-membered macro-cyclic ring formed by 9 amino acid residues which are cyclized by side chain to side chain disulfide bond. The conformation of the X–Pro peptide bond which is located within this macro-cyclic ring causes the cis structure to be compact and the trans structure to be in an extended form. Analysis of the tertiary structures indicated that the trans conformer is stabilized by hydrogen bonds while the cis conformer is likely to be stabilized by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. Preliminary electrophysiological studies carried out on rat DRG neurons indicate that Mo1853 transiently reduces late outward potassium currents. Part II: Structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2 Chapter 5 presents the solution NMR studies of the Smr domain from MutS2 of H. pylori , henceforth called as HpSmr. In H. pylori , MutS2 is involved in suppression of homologous recombination and its Smr domain was shown to be necessary for this activity. As of date, in spite of the availability of structural information for the Smr domain, unambiguous identification of the residues involved in metal binding, DNA binding and catalysis remains elusive. Structural studies were carried out on two different constructs of HpSmr viz., HpSmr– (His)6 and GSHM–HpSmr, with and without the hexahistidine tag respectively. Se- quence specific assignments of HpSmr–(His)6 were obtained at two different sample pH conditions viz., pH 8.0 and pH 5.35 using the standard suite of triple resonance NMR experiments. Since, valines and leucines constitute about 25% of the total number of amino acid residues in HpSmr–(His)6 , stereospecific assignments were obtained for di- astereotopic methyl groups of these residues by preparing a fractionally 13C labeled sample of HpSmr–(His)6 . Solution structure of HpSmr–(His)6 at pH 8.0 was determined using 766 NOE restraints, 170 backbone dihedral angle restraints and 70 hydrogen bond distance restraints. The tertiary structure exhibits the canonical α/β sandwich fold ex- hibited by all the other known structures of Smr domains. Further, NMR studies and analytical gel filtration studies indicated the presence of pH dependent conformational exchange in HpSmr that involves strand to coil transition in the C-terminal β-strand. In order ascertain that the conformational equilibrium is not at an artifact caused by the C-terminal hexa-histidine-tag, HpSmr protein construct GSHM–HpSmr, which does not have the hexa-histidine-tag, was prepared. Conformational exchange was observed in this construct as well. The preliminary NMR evidence suggests that the conformational exchange is caused by pH dependent cis–trans isomerization of a semi-conserved Proline residue Pro66 . We have hypothesized that the pH dependent modulation of the activity of Smr domain of MutS2 can be advantageous to H. pylori . Such a regulation could help the bacteria to achieve optimal rate of homologous recombination in response to changes in pH, which is necessary for maintaining homeostasis and tiding over stress conditions. Part III: Appendix Appendix A describes an NMR pulse program LRCC_CH2 that was designed with the aim of determining disulfide connectivity using long-range 13C–13 C (C β –C β ′ ) couplings across the disulfide bond. This experiment is a modification of an earlier experiment pub- lished by Bax and co-workers designed to measure the side-chain χ3 dihedral angle in me- thionines. The experiment described here is optimized for the detection of 3 bond scalar coupled methylene carbons. The details of modifications introduced in LRCC_CH2, its product operator analysis, a representative spectrum acquired on [U-13C,15 N]–Mo3964, short-comings and future directions are described. The C programming code that was used to implement the pulse program is also included in the appendix. Appendices B, C and D contain the supplementary information (acquisition pa- rameters for the NMR experiments and chemical shifts) for the structural studies carried out on Mo3964, Mo1853 and HpSmr.

Ces travaux de thèse présentent une étude des propriétés optiques et de la photofragmentation de protéines en phase gazeuse. Les expériences ont eét effectuées sur un montage couplant la spectroscopie optique (un LASER UV/Vis) à la spectroscopie de masse (un piège linéaire LTQ). Le photodétachement d'électron est utilisé comme sonde d'action pour l'étude des propriétés optiques des biomolécules ou comme initiateur de radicaux pour les expériences d'a-EPD. L'efficacité de la fragmentation a-EPD a été démontrée sur des petites protéines et son application aété étendue aux polymères synthétiques. Une étude comparative de l'a-EPD avec l'EDD a permis de mieux comprendre les mécanismes liés à la fragmentation d'anions radicalaires. Cestravaux de thèse ont également porté sur l'apport de la spectroscopie d'action par photodétachement sur des protéines entières dans le visible et le VUV. Le domaine du visible a été exploré au sein de notre laboratoire sur une métalloprotéine pour montrer l'importance de l'environnement protéique sur les propriétés optique du groupe porphyrine (hème). L'accès au rayonnement synchrotron SOEIL nous a pemis d'enregistrer les spectres d'action de protéines entières dans le VUV, encore inexplorés dans ce domaine de longeur d'onde. Pour la premièère fois sur une protéine anionique, un double détachement direct d'électrons corrélés a pu être observé. Enfin, des expériences de spectroscopie de photoélectrons (PES ont été réalisées en collaborations avec M. Kappes (Karlsruhe) afin d'enregistrer des spectres de photoélectron résolu en conformères et d'étudier ainsi la stabilité de protéines anioniques fortement chargées. / These thesis present a study of the optical properties and photofragmentation of gas phase protein . The experiments were performed on a mounting EET coupling optical spectroscopy ( UV / Vis LASER ) with mass spectroscopy ( linear trap LTQ ) . Photodetachment electron is used as a probe of Action for the study of optical properties of biomolecules or as radical initiator for experiments a- EPD . The efficiency of the fragmentation a-EPD has been demonstrated on small proteins and extended to synthetic polymers. A comparative study was to ESD -EPD has a better understanding of the mechanisms associated with the fragmentation of radical anions . Cestravaux thesis also focused on the contribution of action photodetachment spectroscopy on whole proteins in the visible and VUV . The visible range has been explored in our laboratory on a metalloprotein to show the importance of the protein environment on the optical properties of porphyrin group ( heme ) . Access to synchrotron radiation Soeil a management permit us to record the action spectra of whole proteins in the VUV , unexplored in the field of wave length . For premièère both an anionic protein , a direct double correlated electron detachment was observed . Finally , experiments with photoelectron spectroscopy (PES were carried out in collaboration with M. Kappes (Karlsruhe ) to record photoelectron spectra resolved conformers and thus to study the stability of highly charged anionic proteins .

This dissertation describes the purification and partial characterization of CBP from the epidermis of the monarch butterfly larvae (Danaus plexippus). A yellow protein-carotenoid complex was extracted from the yellow pigmented epidermal tissue from monarch butterfly larvae by homogenization. Additional steps in the purification process included differential precipitation with ammonium sulfate, cation and anion chromatography, and lastly size exclusion chromatography. Polyacrylamide gel electrophoresis demonstrates that a single protein was isolated (M-LBP) having a ~60 kDa molecular weight, the value has subsequently been confirmed by HR-tandem MS. Lutein is the sole carotenoid bound by M-LBP with a stoichiometry of the binding of 2: 1. Immunohistochemistry results show that M-LBP has no cross-reactivity to antibodies for silk worm CBP (Bombix mori) but does have cross-reactivity with antibodies for horn worm epidermal CBP (Agrius convolvuli). Binding affinities were determined using surface plasmon resonance for the carotenoids lutein (KD = 18.6 ± 0.7), R,R-zeaxanthin (KD = 990 ± 60), R,S-zeaxanthin (KD = 60 ± 2). Tryptophyphan fluorescence lifetimes were determined for the apoprotein and compared to those of the native M-LBP. Tryptophan fluorescence lifetimes were found to be 3.9 ns and 3.0 ns, respectively for these two forms of the protein, indicating that upon dissociation of the carotenoid from the protein the tryptophan fluorophore adopts a position where it has less interaction with the polar surface environment.

One of the main components underlying drug addiction is the emergence of tolerance. Although its development is a complex issue, and is believed to have both psychological and physiological connotations, it is clear that some physiological change must occur that would enable an organism to withstand drug concentrations lethal to a naïve system. The purpose of this thesis was to identify and study a physiological mechanism, whose characteristics were altered due to chronic exposure to ethanol. Vasopressin (AVP), whose primary function is to control water balance, release from the neurohypophysis is suppressed by an acute ethanol challenge. Therefore, I hypothesized; 1) that chronic ethanol exposure would reduce the normal suppression of AVP release during an acute ethanol challenge and 2) that the ion channels that are acutely sensitive to ethanol, involved in the control of AVP release, would exhibit a change in their ethanol sensitivity and characteristics. To study the hypothesis, I utilized the neurohypophysis from rats chronically exposed to ethanol and yoked controls to determine whether chronic exposure would modify the acute ethanol sensitivity of the neurohypophysial vasopressin release mechanism. I examined whether the long-term ethanol exposure affected the suppression of vasopressin release from either or both the intact neurohypophysis and the isolated neurohypophysial terminals. In addition, I investigated how chronic exposure affected two types of potassium channels, the ethanol sensitive large conductance Ca+2-activated (BK) channel and the fast inactivating (IA) channel known to be insensitive to physiologically relevant concentrations of ethanol. I was able to establish that chronic ethanol exposure reduced the suppression of vasopressin release by an acute ethanol challenge from both the intact neurohypophysis and the isolated neurohypophysial terminals. In addition, I discovered that oxytocin release was affected similarly. I concluded from this data that chronic exposure to ethanol affected a general mechanism, which controlled hormone release from the neurohypophysis, and that this mechanism could be isolated to the neurohypophysial terminals. I also used electrophysiological techniques to study ion channel characteristics of both the BK and IA potassium channels. I found that in naïve rats, BK channels were potentiated and IA channels insensitive to physiological relevant concentrations of ethanol. But in chronic ethanol-exposed rats the BK channels exhibited a reduced sensitivity to ethanol while IA channels were inhibited. In addition, the current density of the BK channel was significantly reduced. These results show that at least one characteristic of each potassium channel has been modified. This suggests that chronic exposure can not only modify the ethanol sensitivity of ion channels known to be ethanol-sensitive, but also those believed to be relatively insensitive. Therefore, since modifications in these channels have previously been shown to alter the duration and frequency of action potentials, I conclude that these ethanol-induced modifications play a role in the modified hormone release patterns observed in the chronically exposed rats.

Chromatin remodeling is crucial to transcriptional regulation in vivo and a number of protein complexes capable of altering genomic architecture in the budding yeast Saccaromyces cerevisiaehave been identified. Among these, the SWI/SNF complex, a 2 MDa, eleven subunit protein assembly, has been the most extensively characterized. The SWI/SNF complex is required for the proper expression of a number of genes in yeast, although it is completely dispensable for the expression of others. Likewise, some, but not all, transcriptional activator proteins require SWI/SNF activity in order to function in vivo. The goal of this thesis work was to identify those components of the transcription process which dictate this dependence on SWI/SNF activity. Using the well characterized UASGALsystem, we have determined that one of these components is the nucleosome state of activator binding sites within a promoter. We find that while SWI/SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind and function at low affinity, nucleosomal binding sites in vivo. The SWI/SNF -dependence of these nucleosomal binding sites can be overcome by 1) replacing the low affinity sites with higher affinity, consensus GAL4 binding sequences, or 2) placing the low affinity sites into a nucleosme-free region. These results provide the first in vivo evidence that the SWI/SNF complex can regulate gene expression by modulating the DNA binding of a transcriptional activator protein. To determine whether specific components of the GAL4 protein are necessary in order for the SWI/SNF complex to modulate binding to nucleosomal sites in our model system, we tested the SWI/SNF-dependent DNA binding of various derivative GAL4 proteins. We find that a functional activation domain is not required for SWI/SNF to modulate GAL4 binding in vivo. Interestingly, like the full length protein, GAL4 derivatives in which the activation domain has been mutated are able to partially occupy nucleosomal sites in the absence of SWI/SNF (binding in the absence of SWI/SNF is at least forty percent lower than in the presence of SWI/SNF), indicating the activation domain is also not required for SWI/SNF-independent DNA binding. These results support a model in which the SWI/SNF-dependence of a gene reflects the nucleosomal context of its important regulatory sequences, e.g. binding sites for transcriptional regulatory proteins. Although nucleosomal promoter regions have been correlated with SWI/SNF-dependence in the past, there has of yet been no gene at which nucleosome location has correlated with a specific genetic function. In the final part of this thesis work, we initiated a search for an endogenous SWI/SNF-dependent gene for which the nucleosome state of activator binding sites could be determined.

Transcriptional regulation of cell cycle controlled genes is fundamental to cell division in eukaryotes and a broad spectrum of physiological processes directly related to cell proliferation. Expression of the cell cycle dependent human H4, H3 and H1 histone genes is coordinately regulated at both the transcriptional and posttranscriptional levels. We have systematically analyzed the protein/DNA interactions of the immediate 5'regions of three prototypical cell cycle controlled histone genes, designated H4-F0108, H3-ST519 and H1-FNC16, to define components of the cellular mechanisms mediating transcriptional regulation. Multiple biochemically distinct protein/DNA interactions were characterized for each of these genes, and the binding sites of several promoter-specific nuclear DNA binding activities were delineated at single nucleotide resolution using a variety of techniques. These findings were integrated with results obtained by others and revealed that the in vitro factor binding sites in H4, H3 and H1 histone promoters coincide with genomic protein/DNA interaction sites defined in vivofor the H4-F0108 and H3-STS19 genes, and with evolutionarily conserved cis-acting sequences shown to affect the efficiency of histone gene transcription. Specifically, we have defined binding sites for Sp1, ATF, CP1/NF-Y, HiNF-D, HiNF-M, HiNF-P and HMG-I related factors. Based on sequence-similarities and cross-competition experiments, we postulate that most of these protein/DNA interaction elements are associated with more than one class of histone genes. Thus, the protein/DNA interactions characterized in this study may represent components of a cellular mechanism that couples transcription rates of the various histone gene classes. Regulation of the protein/DNA interactions involved in transcriptional control of these H4, H3 and H1 histone genes was investigated in a spectrum of cell types using several distinct in vitro cell culture models for the onset of differentiation and quiescence, as well as cell cycle progression. Moreover, we studied control of histone gene associated DNA binding activities during hepatic development from fetus to adult in transgenic mice reflecting the onset of differentiation and quiescence in vivo. We show that the H4 histone promoter protein/DNA interaction mediated by factor HiNF-D is selectivelymodulated, and directly at the level of DNA binding activity, during the entry into, progress through and exit from the cell cycle in normal diploid cells, as well as during hepatic development. The regulation of this protein/DNA interaction occurs in parallel with analogous interactions occurring in H3 and H1 histone genes. Moreover, these proliferation-specific protein/DNA interactions are collectively deregulated during the cell cycle in four distinct cell types displaying properties of the transformed phenotype. Hence, the cellular competency to coordinately transcribe distinct classes of histone genes during the cell cycle may be mediated by the intricate interplay of constitutively expressed general transcription factors and temporally regulated, cell growth controlled nuclear factors interacting specifically with cell cycle dependent histone genes. Finally, we show that HiNF-D is represented by two electrophoretically distinct species. The ratio of these forms of HiNF-D fluctuates dramatically during the cell cycle of normal diploid cells, but remains relatively constant in tumor cells. Total HiNF-D binding activity embodied by both HiNF-D species is negatively influenced in vitro by incubation with exogenous phosphatase activity. These observations provide a first indication for the hypothesis that HiNF-D may exist in distinct post-translationally modified forms that are subject to a stringent cell growth control mechanism involving protein kinases and phosphatases. Such a cellular post-translational modification mechanism, which directly impinges on (or activates) the DNA binding activity of a key factor controlling histone genes, would provide a highly efficient means by which to influence the rate of transcription in rapid response to intra-cellular requirements for histone mRNA and extra-cellular cues signalling the onset and cessation of cell proliferation.

Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway. An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.

Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. HIV-1 expresses multiple genes from a single provirus by alternative splicing. Early in viral expression, fully spliced 2-kb viral RNA is exported from the nucleus and encodes the viral regulatory protein, Rev, which is essential for nuclear transport of partially spliced and unspliced genomic-length RNA. Rev binds to an RNA structural element called the Rev response element (RRE) and mediates nuclear export through the leucine-rich nuclear export signal (NES) pathway. The human Rev Interacting Protein (hRIP) interacts specifically with the Rev NES. Rev NES mutants that are unable to export Rev-dependent RNAs are also unable to bind to hRIP. The hRIP cDNA encodes a 562 amino acid protein containing an N-terminal zinc finger with homology to Arf GAP domains, a central serine and threonine rich region, and C-terminal phenylalanine-glycine (FG) repeats characteristic of nucleoporins. To identify an hRIP ortholog in a genetically tractable organism, we performed database searches using the N-terminal zinc finger of hRIP. Using this approach, we identified a novel gene in Schizosaccharomyces pombe. Alignment of the entire reading frame of the putative ortholog with hRIP indicates similarity with the serine/threonine rich region and with the FG repeats, suggesting that S. pombecould be a good model system to study the cellular function of hRIP. We find that the S. pombe ORF is an essential gene, which encodes a 483 amino acid protein that is also able to interact with the NES of HIV-1 Rev. Based on being an essential gene, and the presence of a putative Arf GAP domain, the ORF was named an Arf GAP essential for viability, agv1+. We show that Agv1 is not directly involved in the nuclear export of poly(A+) RNA or 5S rRNA, nuclear export of leucine-rich NES-containing proteins, or nuclear import of nuclear localization signal (NLS)-containing proteins. However, Agv1 does appear to play a role in the cytoplasmic localization of 5S rRNA. We demonstrate that loss of Agv1 alters the localization of endoplasmic reticulum (ER) membrane and Golgi membrane resident proteins, accumulates intracellular membrane, and blocks processing of carboxypeptidase Y. Furthermore, the S. cerevisiae ADP-ribosylation factor (Arf) GTPase activating protein (GAP) Glo3, but not a catalytically inactive Glo3 mutant [R59K], is able to partially compensate for the loss of Agv1 function in temperature sensitive strains, indicating that Agv1 is an S. pombe Arf GAP with some functional features similar to S. cerevisiae Glo3.

The aim of the thesis is to utilize Raman optical activity (ROA) to get unique information on peptide/protein conformation, which is otherwise difficult or even impossible to obtain. We have focused on investigation of amide and disulfide groups. Utilizing tailor-made model structures (rigid tricyclic spirodilactams with two interacting nonplanar amide groups), special model peptides and even biologically active molecules (neurohypophyseal hormones and their agonistic and antagonistic analogs, antimicrobial peptide lasiocepsin and its analogs having different disulfide pattern) we have traced specific spectral manifestation of nonplanar amides and disulfides. ROA results were supplemented by data obtained by complementary chiroptical methods - electronic (including vacuum UV - SRCD) and vibrational circular dichroism. When used in a concerted fashion, these techniques provide complex information on peptide/protein secondary structure. Where possible, experimental chiroptical data were compared to ab initio calculations. In chiroptical spectra we have found and interpreted signals reflecting nonplanarity of the amide group. Moreover, in ROA spectra we have identified signals due to S-S stretching vibrations which seem to reflect sense of the disulfide group torsion.

All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-­‐messenger systems. In bacteria, metabolites also affect post-­‐transcriptional regulatory mechanisms, but there are only a few isolated examples of this regulation in eukaryotes. Here, I present evidence that RNA-­‐binding by the stem cell translation regulator Musashi-­‐1 (MSI1) is allosterically inhibited by 18-­‐22 carbon ω-­‐9 monounsaturated fatty acids. The fatty acid binds to the N-­‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. I identify stearoyl-­‐CoA desaturase-­‐1 as a MSI1 target, revealing a feedback loop between ω-­‐9 fatty acid biosynthesis and MSI1 activity. To my knowledge, this is the first example of an RNA-­‐binding protein directly regulated by fatty acid. This finding may represent one of the first examples of a potentially broad network connecting metabolism with post-­‐transcriptional regulation.