Proteins can be labeled with different tags to enable their structural and functional investigations. In addition, labeling proteins at specific sites helps in studying the conformational dynamics of these molecules. A plethora of methods is available to facilitate labeling, choice of which largely depends on the requirements and the anticipated end results. In general, the various labeling methods can be classified into four different classes based on the stage at which labeling is performed, namely post translational labeling, non-ribosomal synthesis, in vitro translation and in vivo translation. Interestingly all these techniques use different unnatural amino acids for this purpose. Protein folding is one among the many applications that requires tailoring proteins with special molecules or labels for deducing the folding pathway. Understanding the protein folding problem is a key for answering questions concerning protein behavior and thus, will provide strategies to solve protein misfolding diseases. Protein folding is one among the unsolved problems in biology and in particular understanding the in vivo behavior of proteins in the complex cytoplasm environment with a cellular density of approximately 350 to 400 mg/ml is more critical. It is evident that there is a difference in the behavior and folding of proteins in vivo and in vitro and to deduce more insights in this aspect the protein of interest is to be labeled with a sensitive probe. The in vivo translation method offers a good method of choice for labeling the protein at a specific position and monitoring its behavior. To study the ultimate goals of acquiring knowledge of the in vivo behavior and folding characteristics of proteins, the first step of establishing an efficient labeling technique is quintessential and as a starting step, this project aims to label a β-clam protein, cellular retinoic acid binding protein I (CRABP I) a 136 amino acid protein, with a sensitive unnatural fluorescent amino acid probe in vivo in E. coli cells.

Proteins—polymers of amino acids—are a major class of biomolecules whose myriad functions facilitate many crucial biological processes. Accordingly, human control over these biological processes depends upon the ability to study, produce, and modify proteins. One innovative tool for accomplishing these aims is cell-free protein synthesis (CFPS). This technique, rather than using living cells to make protein, simply extracts the cells' natural protein-making machinery and then uses it to produce protein in vitro. Because living cells are no longer involved, scientists can freely adapt the protein production environment in ways not otherwise possible. However, improved versatility and yield of CFPS protein production is still the subject of considerable research. This work focuses on two ideas for furthering that research.The first idea is the adaptation of CFPS to make proteins containing unnatural amino acids. Unnatural amino acids are not found in natural biological proteins; they are synthesized artificially to possess useful properties which are then conferred upon any protein made with them. However, current methods for incorporating unnatural amino acids do not allow incorporation of more than one type of unnatural amino acid into a single protein. This work helps lay the groundwork for the incorporation of different unnatural amino acid types into proteins. It does this by using modified aminoacyl-tRNA synthetases (aaRSs), which are key components in CFPS, to be compatible with unnatural amino acids. The second idea is the preservation of DNA templates from enzyme degradation in CFPS. Among the advantages of CFPS is the option of using linear expression templates (LETs) in place of plasmids as the DNA template for protein production. Because LETs can be produced more quickly than plasmids can, using LETs greatly reduces the time required to obtain a DNA template for protein production. This renders CFPS a better candidate for high-throughput testing of proteins. However, LETs are more susceptible to enzyme-mediated degradation than plasmids are, which means that LET-based CFPS protein yields are lower than plasmid-based CFPS yields. This work explores the possibility of increasing the protein yield of LET-based CFPS by addition of sacrificial DNA, DNA which is not used as a protein-making template but which is degraded by the enzymes in place of the LETs.

The cytochrome bc1 complex of the mitochondrial electron transport chain accomplishes the enzymatic reaction known as the modified Q-cycle. In the Q-cycle the bc1 complex transports protons from the matrix to the intermembrane space of the mitochondria, creating the proton gradient used to make ATP. The energy to move these protons is obtained by shuttling electrons from the coenzyme ubiquinol (QH2) to coenzyme ubiquinone (Q) and the mobile cytochrome c. This well studied complex is ideal for examining molecular adaptation because it consists of ten different subunits, it functions as a dimer, and it includes at least five different active sites. The program TreeSAAP was used to characterize molecular adaptation in the bc1 complex and identify specific amino acid sites that experienced positive destabilizing (radical) selection. Using this information and three-dimensional structures of the protein complex, selection was characterized in terms of coevolution and coadaptation. Coevolution is described as reciprocal local biochemical shifts based on phylogenetic location and results in overall maintenance. Coadaptation, on the other hand, is more dynamic and is described as coordinated local biochemical shifts based on phylogenetic location which results in overall adaptation. In this study both coevolution and coadaptation were identified in various locations on the protein complex near the active sites. Sites in the pore region of cyt c1 were shown to exhibit coevolution, in other words maintenance, of many biochemical properties, whereas sites on helix H of cyt b, which flanks the active sites Qo and Qi, were shown to exhibit coadaptation, in other words coordinated shifts in the specific properties equilibrium constant and solvent accessible reduction ratio. Also, different domains of the protein exhibited significant shifts in drastically different amino acid properties: the protein imbedded in the membrane demonstrated shifts in mainly functional properties, while the part of the complex in the intermembrane space demonstrated shifts in conformational, structural, and energetic properties.

[pt] A presença de aminoácidos em cometas e meteoritos levanta questões sobre como estes foram formados em ambientes cósmicos, bem como de que maneira eles foram capazes de sobreviver no espaço sideral; radioresistência é uma informação essencial para prever meias-vidas e avançar os estudos sobre origens da vida. O principal objetivo deste trabalho é determinar, por meio de espectroscopia no infravermelho, as seções de choque de destruição de aminoácidos comuns expostos à radiação de íons e elétrons energéticos. As forças de banda vibracionais (A-values) e a dependência do espectro infravermelho com a temperatura da amostra (10 – 400 K) foram analisadas. Seções de choque de destruição aparente (sigma)d(ap) e rendimentos de sputtering (Y0) para glicina, valina e fenilalanina irradiadas por H+, He+ e Nq+ íons de MeV e elétrons de keV foram medidos. Encontrou-se: i) uma dependência aproximadamente linear entre a seção de choque de destruição aparente e o poder de freamento eletrônico (Se): (sigma)d(ap) = (sigma)d + Y0 /N0 = a Sen (onde n aproximadamente 1) para projéteis de MeV e para amostras à temperatura ambiente; ii) resultados preliminares de σdap para feixes de nitrogênio multi-carregados; e iii) resultados de seção de choque de destruição de valina irradiada por elétrons de keV, bem como sua dependência com a energia de incidência do feixe, e com a espessura e temperatura da amostra. Como contribuição teórica, o modelo CASINO-estendido foi desenvolvido visando descrever a evolução da degradação de matéria orgânica por projéteis carregados, particularmente por feixes de elétrons. Comparadas aos resultados experimentais, as previsões do modelo subestimam o dano causado pelo feixe de elétrons, evidência de que efeitos de sputtering e provavelmente algumas características da amostra (como a estrutura cristalográfica) devem ser incluídos. Como implicações astrofísicas, meias-vidas para valina e fenilalanina irradiadas por raios cósmicos são estimadas em aproximadamente 10 milhões de anos no meio interestelar; da glicina, se irradiadas por vento solar a uma unidade astronômica do Sol, é aproximadamente 3 dias. Visando simular materiais astrofísicos realistas bombardeados por elétrons de keV, a meia-vida de valina envolta por gelos de água e CO2 e depositada sobre silicato é também prevista. / [en] The presence of amino acids in comets and meteorites raises questions about how they have been formed in cosmic environments, as well as how long they can survive in outer space; radioresistance is essential information to predict half-lives and make advances on the origins of life studies. The main objective of the current work is to determine, via infrared spectrometry, destruction cross sections of common amino acids exposed to energetic ion and electron radiation. Before sample irradiation, valine vibrational band strengths and their infrared spectral dependence on temperature (10 – 400 K) were analyzed. Apparent destruction cross sections (sigma)d(ap) and sputtering yields (Y0) for glycine, valine and phenylalanine, irradiated by MeV H+, He+ and Nq+ ions and keV electrons, were measured. From experimental data: i) an approximately linear dependence between the apparent destruction cross section and the electronic stopping power (Se) is found: (sigma)d(ap) = (sigma)d + Y0 /N0 = a Sen (where n approximately 1) for MeV projectiles and for samples at room temperature; ii) (sigma)d(ap) preliminary results relative to multi-charged nitrogen ion beams are discussed; and iii) destruction cross section of valine irradiated by keV electrons, as well as its dependence on incident beam energy, on sample thickness and on sample temperature are presented. As a theoretical contribution, the evolution of organic matter damage by charged projectiles, particularly for electron beams, the CASINO-extended model was developed. When compared to experimental results, the model predictions underestimate the damage caused by electron beams, evidence that sputtering and probably some sample characteristics (as crystallographic structure) are involved. As astrophysical implications, cosmic ray half-lives for valine and phenylalanine are estimated to be about 10 million years in the interstellar medium; solar wind half-life at 1 au from the Sun is approximately 3 days for glycine. Aiming to simulate realistic astrophysical materials bombarded by keV electrons, the half-life of valine embedded into water and CO2 ices over a silicate substrate is also predicted.

The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 µM, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression. To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 … (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.

The degradation of wool keratin under conditions relevant to those of wool dyeing was investigated using the techniques of gel permeation chromatography (GPC), ion exchange gel chromatography, and amino acid analysis. Physical testing of the treated and untreated wool was also carried out to determine the physical changes occurring, parameters used being percentage elongation at the break, and the breaking strain of the fibre. Samples of wool keratin were immersed in various aqueous solutions at 1000C for 24 hours and the filtered, aqueous, oxidised extracts were analysed* The solutions used varied only in the dye, or dye intermediate present in the treatment solution. All treatment baths contained 10% owf 1.02 x 10 -2 MSulphuric VI acid; 10%owf 7.04x 10 -3 MSodium sulphate VI ; A 100 :1 liquor ratio was used in each case. Some of the dye intermediates showed a marked catalytic effect, particularly in their effect on breaking strain, a decrease of 40% in some cases. The GPC profiles of the extracted proteins were examined in detail and compared against previous workers' results. An explanation of the behaviour of the dyes and intermediates was proposed. The amino acid composition data of the extracted and fractionated proteins were compared against various morphological components extracted by other workers, as was the total gelatin obtained from each treatment. / Science Research Council

Over the course of more than ten years, there has been a significant increase in the approach employed to inhibit the function of soluble epoxide hydrolase (sEH). The phenomenon of upregulating soluble epoxide hydrolase (sEH) has been found to result in a decrease in the ratio of epoxyeicosatrienoic acids (EETs) to dihydroeicosatrienoic acids (DHETs) in the body. This has garnered significant attention due to the diverse biological functions attributed to EETs, including the regulation of vasodilation, neuroprotection, increased fibrinolysis, calcium ion influx, and anti-inflammatory effects. Consequently, there has been a growing interest in developing and discovering sEH inhibitors through chemical syntheses and natural extracts, with the aim of increasing the availability of these anti-inflammatory molecules by reducing their hydrolysis. A comprehensive examination of this project was conducted to explore the inhibitory effects of YMSV, a tetrapeptide derived from the castor bean (Ricinus communis), on sEH, as well as to elucidate its underlying mechanism of action. YMSV was determined to function as a mixed-competitive inhibitor of soluble epoxide hydrolase (sEH), and the interaction between the peptide and the protein resulted in the disruption of the secondary structural composition of sEH. Furthermore, the hydrogen bond interactions between YMSV and the Asp 333 residue in the active region of soluble epoxide hydrolase (sEH) were demonstrated using molecular docking investigations. However, quantitative structure-activity relationship (QSAR) research revealed that nonpolar, hydrophobic, and bulky amino acids are favored at the N- and C- terminals of peptides for sEH inhibition. The results of this study indicate that peptides obtained from dietary sources possess unique characteristics as inhibitors of soluble epoxide hydrolase (sEH), displaying significant potency. Consequently, these peptides have promise for further development as therapeutic medicines targeting inflammation and depression in the future.

The traditional treatment for phenylketonuria (PKU) is a phenylalanine (Phe)-restricted diet, supplemented with a Phe-free/low-Phe protein substitute. Pharmaceutical treatment with synthetic tetrahydrobiopterin (BH4), an enzyme cofactor, allows a patient subgroup to relax their diet. However, dietary protocols guiding the adjustments of protein equivalent intake from protein substitute with BH4 treatment are lacking. We systematically reviewed protein substitute usage with long-term BH4 therapy. Electronic databases were searched for articles published between January 2000 and March 2020. Eighteen studies (306 PKU patients) were eligible. Meta-analyses demonstrated a significant increase in Phe and natural protein intakes and a significant decrease in protein equivalent intake from protein substitute with cofactor therapy. Protein substitute could be discontinued in 51% of responsive patients, but was still required in 49%, despite improvement in Phe tolerance. Normal growth was maintained, but micronutrient deficiency was observed with BH4 treatment. A systematic protocol to increase natural protein intake while reducing protein substitute dose should be followed to ensure protein and micronutrient requirements are met and sustained. We propose recommendations to guide healthcare professionals when adjusting dietary prescriptions of PKU patients on BH4. Studies investigating new therapeutic options in PKU should systematically collect data on protein substitute and natural protein intakes, as well as other nutritional factors.

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