All twenty amino acids did not appear simultaneously in nature. Instead some of them appeared early, while others were added into the genetic code later. The amino acids that were formed by Miller (1953) are suggested to have appeared early in evolutionary history, and the amino acids associated with codon capture developed late in the course of evolution. The chronological order of appearance of the amino acids proposed by Trifonov (2000) was G/A, V/D, P, S, E/L, T, R, N, K, Q, I, C, H, F, M, Y, W. According to Romero et al. (1997) amino acids G, D, E, P and S are disorder-promoting residues and C, F, W and Y are order-promoting residues this means that the early or the ancient amino acids were disorder promoting and the order promoting residues came late into the genetic code. These observations led to the hypothesis that the first proteins, which were comprised of the early amino acids only, were disordered, and, furthermore, that the appearance of the late amino acids and the appearance of the structural proteins were concurrent. Software developed by Brooks et al. (2004) to find the amino acid composition of the LUA (Last Universal Ancestor) was used to test this hypothesis. For this work, the Clusters of Orhtologous Groups of proteins (65 COGs) were split into enzymes and non-enzymes. It was found that intrinsic disorder was abundant in both the groups of proteins, with non enzymes being much more disorder than enzymes. Further analysis was done to check for the frequency of the modern amino acids C, F, W, and Y in the Protein data bank (PDB) and Swissprot.

No / Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our consensus domain dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1,695 folds in the CDD as being non-autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predictions suggest that 40% of proteins have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets. / NIH

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein. / October 2007

Most cells within multicellular organisms contain the same genetic information, yet the appropriate tissue-specific expression of genes is required for the proper formation of adult tissues. Genes can either be “turned on” or “turned off” from the initial zygotic state and maintained during subsequent cell divisions. Maintaining the correct expression profiles during cell divisions is accomplished by a number of different nuclear factors. One of the key families of proteins that maintains the repression of target genes during development is the Polycomb group (PcG) of proteins. PcG proteins form a number of different multi-subunit protein complexes that interact with specific regions of chromatin and direct the repression of nearby genes by reducing transcription. One PcG complex, Polycomb repressive complex 1 (PRC1), inhibits transcription and nucleosome remodeling as well as compacts chromatin, both in vivo and in vitro. The in vitro repressive activities map mainly to one subunit of Drosophila PRC1—the Posterior sex combs (PSC) protein. The PRC1 complex is conserved in many other organisms including mammals. To better understand the mechanisms involved in PcG mediated repression we undertook a biochemical structure/function analysis of mouse PRC1. In chapter one, I review the current understanding of PcG biology and a rationale for the dissertation is provided. In chapter two, data are presented that argues that a mouse PRC1 protein, M33/Cbx2, which is non-homologous to PSC, is responsible for chromatin compaction and repression of nucleosome remodeling. Data are presented that suggests these activities are localized to a basic, natively unfolded region of M33/Cbx2. In chapter three, we extend the findings from chapter two in an attempt to predict whether homologous PcG proteins from other species besides fly and mouse have biochemical activity. In agreement with predictions, a panel of recombinant PcG proteins was generated and data are presented that shows the predicted active PcG proteins are capable of both inhibition of nucleosome remodeling and compaction of chromatin. Finally, in chapter four, the implications of the data presented are discussed, and directions for further inquiry are explored.

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.

The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.

This thesis studies the intrinsic disorder effects and the transport and magnetic properties of ring-shaped epitaxial thin films and superconducting tunnel junctions (STJs) of the high temperature superconductor YBa$_2$Cu$_3$O$_{7-delta}$. We used an unconventional contactless technique that allows us to directly measure the persistent current of superconducting rings. In order to study the disorder effects on the persistent current, we slowly increased oxygen vacancies in YBa$_2$Cu$_3$O$_{7-delta}$ by changing $delta$ from 0.03 to 0.55 in steps of $sim$0.021. Monitoring the corresponding changes in the temperature dependence of the persistent current revealed an anomaly in its flow within a certain range of disorder. We found that this anomaly is directly related to the occurrence of a spinodal decomposition of oxygen vacancies in YBCO, which we explain as a competition between two coexisting phases, oxygen rich and oxygen deficient. The analysis of the time dependence of the persistent current revealed that increasing oxygen vacancies transforms the vortex structure from quasi-lattice into a glass and subsequently into a pinned liquid phase. Our results also exhibited the first evidence of self-organization of the vortex structure with increasing disorder. We also performed the first direct measurement of the temperature dependence of the $c$-axis persistent current ($J_c$) that is purely due to tunnelling Cooper-pairs through intrinsic Josephson junctions (IJJs) of YBCO. This is made possible by incorporating IJJs of YBCO into ring-shaped films. Then, we studied the temperature dependence of the persistent current of YBCO nanowires embedded in SrTiO$_3$-barrier integrated between two semi-ring-shaped YBCO thin films and systematically varied the nanowires length. Our observations revealed that $J_c$ has two different temperature dependences: a GL-dependence ($J_c propto (T_c - T)^{3/2}$) at low temperatures which we found the same in all studied samples, and another power law dependence ($J_c propto (T_c - T)^{alpha > 3/2}$) at high temperatures which turned out to depend on the length of the nanowires. We attribute the cross-over between these two temperature dependences to the depinning and the dissipative motion of vortices. These experimental approaches and findings not only provide new information, but more importantly open new avenues of investigating the transport and magnetic properties of superconducting films, junctions, and nanowires.

sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.

No description available.

There are different conformational states of proteins characterized by different Gibbs free energy levels, manifested in folding-unfolding dynamics, for example. Recently, a set of protein states, which require relatively small amount of folding energies, emerged as subjects of intensive research, and proteins or regions characterized by the presence of these states have been termed as ‘Intrinsically Disordered Proteins’ (IDP) and ‘Intrinsically Disordered Protein Regions’ (IDPR), respectively. Predisposition for intrinsic disorder of a query protein is encoded in its amino acid sequence and composition, and can be rather accurately predicted using several intrinsic disorder algorithms. Since pathology of many human diseases can be driven by proteins characterized by high intrinsic disorder scores, research on various disease-associated proteins is often started with the analysis of their intrinsic disorder propensities. In this work, I utilized computational approaches based on the concept of intrinsic disorder to address three health-related issues. To this end, I developed a novel computational platform for disorder-based drug discovery and applied this tool for finding inhibitors of the cancer-related MBD2-NuRD complex, utilized molecular dynamic simulations to explain the effects of mutations on the functionality of the X-linked protoporphyria-related protein ALAS, and used bioinformatics tools to examine the effects ofcardiomyopathy-related mutations in cardiac troponin. Since the complex between the Methyl-CpG-binding domain protein 2 (MBD2) and the Nucleosome Remodeling Deacetylase complex (NuRD) specifically binds to the mCpG-island and blocks tumor suppressor gene expression, finding an inhibitor of this MBD2-NuRD complex is hypothesized to be important for the development of novel anti-cancer drugs. I found that the site, which is responsible for the MBD2 interaction with thetranscriptional repressor p66-α (p66α, which is a part of the NuRD complex), is characterized by a specific disorder-to-order transition pattern, this pattern showed a remarkable similarity to the disorder-to-order pattern of the Myc transcription factor binding site for the Max transcription factor. Importantly, several inhibitors of the Myc-Max interaction targeting the disorder-to-order transition site of Myc were previously described. By applying molecular docking at the disorder-to-order transition site of MBD2, two compounds were identified and further evaluated through molecular dynamics simulations. Anti-leukemia and anti-metastasis effectiveness of these compounds was demonstrated in dedicated in vitro and in vivo experiments conducted by our collaborators. In relation to the defective protein associated with the X-linked protoporphyria (XLPP), the hepta-variant of mouse erythroid 5-aminolevulinate synthase (mALAS2), previously shown to be characterized by a remarkable acceleration of the reaction rate, was investigated through molecular dynamics simulations. In this study, a loop to β-strand transition was observed, and this observation was crucial for a better understanding of the previously described rate-enhancing effects of seven simultaneous variations in the active loop site of this protein. Finally, a wide spectrum of bioinformatics tools was applied to carefully analyze a potential role of intrinsic disorder in a set of cardiomyopathy-related mutations in the components of human cardiac troponin. This analysis revealed that, in comparison with the wild type troponin, chains containing the disease-associated mutations were typically characterized by a local decrease in intrinsic disorder propensity. These mutations affected some disorder-based protein-protein interaction sites and caused remarkable rearrangements of the complex pattern of post-translational modifications. Therefore, this work illustrates that inclusion of the protein intrinsic disorder analysis into the arsenal of techniques used by the biomedical researchers represents an important and promising approach that provides novel inputs for the better understanding of protein behavior in relation to human disease at the molecular level. Techniques and methods developed and utilized in this study will significantly contribute to future biomedical research.