Diploma thesis is focused on creation of educational materials supporting the education of biochemistry, specifically amino acids and proteins, at secondary level. At first the analysis of Czech chemistry textbooks concerning the two topics - amino acids and proteins was performed. The major problems found were related to the insufficient graphical representation of biomolecules, unsatisfactory motivational components and insufficient integration of the topic with biology or everyday life experience. The supporting educational materials, presented in this work, supplement the widely used chemistry textbooks. The materials includes a graphic oriented presentation, interactive animations demonstrating the process of denaturation and precipitating of proteins at macroscopic and molecular level, poster presenting the structural formulas of standard amino acids, 3D models of selected proteins, additional texts supporting the current topics concerning amino acids and proteins and the laboratory protocols for students. The presented support materials were evaluated at the secondary school conditions. They were tested in one class and the improvement of student's understanding of the topic was compared to the second class employing only the classical educational methods. The comparison of the results of...

In this thesis, we have advanced a set of distinct bioinformatic and computational tools to address the structure and function of proteins. Using data mining of the protein data bank (PDB), we have collected statistics connecting the propensity between the protein sequence and the secondary structure. This new tool has enabled us to evaluate new structures as well as a family of structures. A comparison of the wild type staphylococcal nuclease to various mutants using the proposed tool has indicated long-range conformational deviations spatially distant from the mutation point. The energetics of protein unfolding has been studied in terms of the forces observed in molecular dynamics simulations. An adaptive integration of the steered molecular dynamics is proposed to reduce ground state dominance by the rare low energy trajectories on the estimated free energy profile. The proposed adaptive algorithm is utilized to reproduce the potential of mean force of the stretching of decaalanine in vacuum at lower computational cost. It is then used to construct the potential of mean force of this transition in solvent for the first time as to observe the hydration effect on the helix-coil transformation. Adaptive steered molecular dynamics is also implemented to obtain the free energy change during the unfolding of neuropeptide Y and to confirm that the monomeric form of neuropeptide Y adopts halical-hairpin like pancreatic-polypeptide fold.

Many biomolecular processes involve mechanical force-induced reactions in the cell, such as translocation, and mechanical stretching of biopolymers. Recent advances in single molecule manipulation techniques make it possible to apply mechanical force to individual biomolecules and study their dynamics. To gain molecular level understanding of these processes and to interpret the single-molecule experiments, we used Langevin dynamics simulations of coarse-grained biopolymer models. Our result show that the mechanism of translocation of proteins through pores depends on the pore diameter, on the magnitude of the pulling force and on whether the force is applied at the N- or the C-terminus of the chain. In addition, the translocation kinetics of peptides varies with their stability. The mechanism of protein translocation is found to be different from that of a structureless polypeptide of the same length. We further showed that unfolding mechanism of translocation process is different from when the same protein is stretched between its C- and N-termini. We also studied the mechanical and chemical/thermal denaturation of proteins. We observed that the free energy profile along the mechanical reaction coordinate and the chemical reaction coordinate are different. In our protein model, the mechanical and chemical/thermal denaturation cannot be simply explained in terms of a simple onedimensional free energy landscape. We further analyzed the spontaneous folding and refolding under a constant force and found that refolding generally occurs via different mechanisms. Similarly, we investigated the protein unfolding/refolding under the applied force that varies with a constant loading rate. This study shows that unfolding/refolding pathways are generally similar for low loading/unloading rates while they become different for high loading/unloading rates. Finally, we studied the dynamics of molecular friction knots formed by a pair of polymer strands. We examined different knot types, and different polymer sequences. Depending on the knot type and the nature of the polymer, we observed two different behaviors when the force F is exerted to separate the polymer strands. The knot between polymer strands can be strong (the time [tau] the knot stays tied increases with the force F applied to separate the strands) or weak ([tau]decreases with increasing F).

Indiana University-Purdue University Indianapolis (IUPUI) / Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.

Indiana University-Purdue University Indianapolis (IUPUI) / Eukaryotic cells rapidly modulate protein synthesis in response to environmental cues through the reversible phosphorylation of eukaryotic initiation factor 2 (eIF2α~P) by a family of eIF2α kinases. The eIF2 delivers initiator Met-tRNAiMet to the translational apparatus, and eIF2α~P transforms its function from a translation initiation factor into a competitive inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which is responsible for the recycling of eIF2-GDP to the translationally-competent eIF2-GTP state. Reduced eIF2-GTP levels lower general protein synthesis, which allows for the conservation of energy and nutrients, and a restructuring of gene expression. Coincident with global translational control, eIF2α~P directs the preferential translation of mRNA encoding ATF4, a transcriptional activator of genes important for stress remediation. The term Integrated Stress Response (ISR) describes this pathway in which multiple stresses converge to phosphorylate eIF2α and enhance synthesis of ATF4 and its downstream effectors. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKα as being subject to both translational and transcriptional induction during eIF2α~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves the stress-induced relief of two inhibitory uORFs in the 5’-leader of the transcript. Also identified as being subject to preferential translation is mRNA encoding the bifunctional aminoacyl tRNA synthetase EPRS. During eIF2α~P, translational regulation of EPRS is suggested to occur through the bypass of a non-canonical upstream ORF encoded by a CUG start codon, highlighting the diversity by which upstream translation initiation events can regulate expression of a downstream coding sequence. This body of work provides for a better understanding of how translational control during stress is modulated genome-wide and for the processes by which this mode of gene regulation in the ISR contributes to cell fate.

Indiana University-Purdue University Indianapolis (IUPUI) / Disruptions of the endoplasmic reticulum (ER) cause perturbations in protein folding and result in a cellular condition known as ER stress. ER stress and the accumulation of unfolded protein activate the unfolded protein response (UPR) which is a cellular attempt to remedy the toxic accumulation of unfolded proteins. The UPR is implemented through three ER stress sensors PERK, ATF6, and IRE1. Phosphorylation of the α-subunit of eIF2 by PERK during ER stress represses protein synthesis and also induces preferential translation of ATF4, a transcriptional activator of stress response genes. Early UPR signaling involves translational and transcriptional changes in gene expression that is geared toward stress remedy. However, prolonged ER stress that is not alleviated can trigger apoptosis. This dual signaling nature of the UPR is proposed to mimic a 'binary switch' and the regulation of this switch is a key topic of this thesis. Adaptive gene expression aimed at balancing protein homeostasis encompasses the first phase of the UPR. In this study we show that the PERK/eIF2~P/ATF4 pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi where ATF6 is activated. Liver-specific depletion of PERK significantly lowers expression of survival genes, leading to reduced expression of protein chaperones. As a consequence, loss of PERK in the liver sensitizes cells to stress which ultimately leads to apoptosis. Despite important roles in survival, PERK signaling is often extended to the vii activation of other downstream transcription factors such as CHOP, a direct target of ATF4-mediated transcription. Accumulation of CHOP is a hallmark of the second phase in the binary switch model where CHOP is shown to be required for full activation of apoptosis. Here the transcription factor ATF5 is found to be induced by CHOP and that loss of ATF5 improves the survival of cells following changes in protein homeostasis. Taken together this study highlights the importance of UPR signaling in determining the balance between cell survival and cell death. A topic that is important for understanding the more complex pathological conditions of diseases such as diabetes, cancer, and neurodegeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins (IDPs) are flexible proteins without defined 3D structures. Studies show that IDPs are abundant in nature and actively involved in numerous biological processes. Two crucial subjects in the study of IDPs lie in analyzing IDPs’ functions and identifying them. We thus carried out three projects to better understand IDPs. In the 1st project, we propose a method that separates IDPs into different function groups. We used the approach of CH-CDF plot, which is based the combined use of two predictors and subclassifies proteins into 4 groups: structured, mixed, disordered, and rare. Studies show different structural biases for each group. The mixed class has more order-promoting residues and more ordered regions than the disordered class. In addition, the disordered class is highly active in mitosis-related processes among others. Meanwhile, the mixed class is highly associated with signaling pathways, where having both ordered and disordered regions could possibly be important. The 2nd project is about identifying if an unknown protein is entirely disordered. One of the earliest predictors for this purpose, the charge-hydropathy plot (C-H plot), exploited the charge and hydropathy features of the protein. Not only is this algorithm simple yet powerful, its input parameters, charge and hydropathy, are informative and readily interpretable. We found that using different hydropathy scales significantly affects the prediction accuracy. Therefore, we sought to identify a new hydropathy scale that optimizes the prediction. This new scale achieves an accuracy of 91%, a significant improvement over the original 79%. In our 3rd project, we developed a per-residue C-H IDP predictor, in which three hydropathy scales are optimized individually. This is to account for the amino acid composition differences in three regions of a protein sequence (N, C terminus and internal). We then combined them into a single per-residue predictor that achieves an accuracy of 74% for per-residue predictions for proteins containing long IDP regions.