No / D-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of L- and D-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host-guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the D-amino acids, including D-allo-isoleucine and D-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate L-amino acids verifies that the intrinsic backbone conformational propensities of the D-amino acids are the inverse of their cognate L-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties. / NIH

Peptide, plasma free, erythrocyte free and serum protein amino acid arteriovenous differences were quantified across the gastrointestinal tract (GIT) and liver of growing calves during a "steady state" and fasting metabolism. Six Holstein steer calves (136 kg) surgically equipped with cannulae in the aorta, portal vein and hepatic vein were maintained under constant light and fed a natural diet as 24 hourly meals per day. Blood was obtained simultaneously from all cannulae at 9 to 12 d postsurgery during the midpoint of a 1-hr feeding interval and following a 72-hr fast. Most amino acids were added to plasma by the GIT but fasting reduced the magnitude of this addition. GLN and GLU were the only plasma amino acids removed by the GIT. The liver removed a large portion of amino acids in portal plasma but a net splanchnic plasma free amino acid output occurred. Hepatic removal of amino acids, particularly VAL, ILE and LEU, increased after fasting. The GIT generally removed amino acids from the erythrocyte while the liver contributed amino acids to this pool. Peptide amino acids increased as blood crossed the GIT. Responses paralleled those of plasma free amino acids in direction but were threefold greater for peptide amino acids. A net output of peptide amino acids from the splanchnic bed was observed. Generally amino acids were added to the primarily globulin, albumin and mixed protein serum fractions while the pure albumin fraction contributed amino acids to the GIT. Fasting reversed the direction of response in the four fractions monitored. Arteriovenous differences for amino acids in two largely albumin fractions responded oppositely indicating a potential transport role of other proteins in these fractions. A more variable response was observed across the liver with direction and magnitude dependent upon individual amino acids. Fasting resulted in a release of amino acids by the liver into all four fractions. Several blood pools appear to be involved in the interorgan transport of amino acids. The extent and direction of transport varies with tissue, blood pool, nutritional status and amino acid. Quantitatively, peptides appear to be highly involved in interorgan movement of amino acids. / Ph. D.

Urbanization introduces a set of novel stressors for animals in these modified habitats. Developing young, in particular, can be at risk due to increased predation, exposure, and high food intake requirements. Song sparrows (Melospiza melodia) are a model wild songbird system that persist in both urban and rural habitats, and have young that develop very quickly. I investigated how urbanization changes the arthropod community, how that in turn affects nestling diet, and ultimately if there were any differences in nestling amino acid profiles or body condition between habitat types. Prior work found that urban habitats also have far higher rates of brood parasitism, which can additionally restrict food intake for urban nestlings. However, this prior work has also found that urban song sparrow adults avoid consequences for living in urban habitats, so I wanted to see if nestlings were also able to avoid the consequences of urban living. In chapter one, I found our urban arthropod communities had lower average arthropod abundance, biomass, and diversity when compared to rural arthropod communities. Song sparrow nestling diets differed somewhat in composition of arthropod prey items by habitat, but urban and rural nestlings had the same average biomass of stomach contents. In chapter two, I investigated whether different habitat types or brood parasitism altered body size and amino acid concentration of song sparrow nestlings. Only gamma-aminobutyric acid differed, being significantly lower in urban, parasitized nestlings. No groups differed in metrics of body condition. In chapter three, I performed a meta-analysis to investigate how brood-parasitic brown-headed cowbirds (Molothrus ater) affect host clutch size and nest success. Host clutch sizes were lower when parasitized, but surprisingly non-parasitized nests were more likely to fail. Despite lower arthropod food availability and high levels of brood parasitism, urban song sparrow nestlings are not compromised, supporting the conclusion that urbanization does not necessarily have negative consequences for individuals of this species. / Doctor of Philosophy / Urbanization introduces a set of new challenges for animals. Young animals, in particular, can be at risk due to increased predation, exposure, and lack of food. Song sparrows (Melospiza melodia) are a model wild songbird system that persist in both urban and rural habitats, and have young that develop very quickly and rely on parents providing a diet of arthropods, including spiders and insects. I investigated how urbanization changes the arthropod community, how that in turn affects nestling diet, and ultimately if there were any differences in nutrition or body condition. Urban habitats also have higher rates of cowbirds laying their eggs in the nests of other species, which can make it even harder for urban song sparrow nestlings to get enough food. However, this prior work has also found that urban song sparrow adults avoid consequences for living in urban habitats, so I wanted to see if nestlings were also able to avoid the consequences of urban living. In chapter one I found our urban arthropod communities had lower average number and mass of arthropods, when compared to rural arthropod communities. Song sparrow nestling diets differed somewhat in composition of arthropod prey items by habitat, but urban and rural nestlings had the same average biomass of stomach contents. In chapter two, I investigated whether different habitat types or having cowbird 'siblings' resulted in different nutrition and body size of song sparrow nestlings. Out of nearly 30 measures of nutrition only one differed, being significantly lower in urban nestlings with siblings. No groups differed in body size. For chapter three I analyzed the literature to investigate how brown-headed cowbirds (Molothrus ater) affect the number of eggs host birds lay and host nest success. Host clutch sizes were lower when cowbirds were present, but surprisingly, it was nests without cowbirds that were more likely to fail. Despite lower arthropod food availability and high levels of cowbird presence, urban song sparrow nestlings avoided negative consequences, supporting the conclusion that urbanization does not necessarily harm individuals of this species.

NASEM (2021) recently made strides in characterizing effects of 5 individual EAA on milk protein production. However, there are 15 other AA that are incorporated into milk protein, and as such, these AA likely also play significant roles in driving milk protein synthesis, but lack of data prevents their incorporation into current models. A greater supply of AA to the mammary glands does not always mirror AA absorption—the process by which absorbed AA convert into milk protein is variable, and this may be linked to the way the udder regulates AA uptake to preserve intracellular balance. AA transporters housed within the cellular membranes of mammary epithelial cells (MEC), the mammary glands' constituents, are responsible for mediating this intracellular balance. Thus, the objectives of this dissertation were to investigate how AA transport is affected by various AA concentrations using both in vitro and in vivo approaches. In study 1, we evaluated effects of valine and a group of NEAA (AQG; Ala, Gln, and Gly) on exchange transport rates of AA in bovine MEC. High AQG concentrations stimulated Leu, Phe, and Val influx rate parameters, demonstrating that AQG likely increased transport activity for these substrates through exchange transporters. Additionally, high Val concentrations decreased Ile and Leu net uptakes, which occurred via efflux stimulation and transamination downregulation. In study 2, we aimed to identify the effects of 10 EAA and 2 Tyr (CDENSPY) on transport rates and transporter regulation (mRNA expression and protein abundance). Within the physiological AA concentrations used, we were able to measure differential effects of AA on each AA transporter. For example, His stimulated SLC38A2 and SLC38A2 mRNA expression at a decreasing rate; the apex for this curve was reached at a concentration very close to mean plasma concentrations in lactating dairy cows. Therefore, we determined that these transporters may be transcriptionally regulated to regulate intracellular His concentrations. Additionally, all EAA and NEAA groups were involved in significant 2-way interactions on transporter expression and activity. Furthermore, we measured transport rates and rate constants (free of mass influence) of 12 AA to determine important AA on influx, efflux, transamination, irreversible loss, and protein synthesis. We demonstrated competitive inhibition among several AA that share transport systems such as between BCAA. Furthermore, we again demonstrated that NEAA can stimulate transport activity for AA involved in exchange transport. In study 3, we investigated the effects of jugular Lys, Ile, Val, or AQG infusion on mammary AA metabolism and production in lactating dairy cows. Interestingly, Val decreased DMI and milk protein production along with net uptakes of several AA, while the remaining treatments had little metabolic effects. In study 4, we demonstrated that both high protein and starch concentrations independently stimulated milk protein production, but glucose precursor partitioning (lactate, propionate and other) was only affected by starch. In conclusion, we anticipate that nutrition models estimating milk protein production will eventually incorporate up to 20 AA and multiple 2-way interactions; additionally, extremely high concentrations of AA should be prevented to combat negative impacts caused by AA imbalances. However, much more work is required to take steps in this direction. / Doctor of Philosophy / Overfed protein can pose a significant health and environmental risk. Unabsorbed amino acids (AA), the building blocks of protein, are released by dairy cattle into the environment as various nitrogen products. One specific risk is excess runoff of nitrates from dairy farms into nearby water bodies. This contamination can result in serious water quality issues, including eutrophication, which depletes O2 levels in aquatic ecosystems with algae overgrowth, causing dead zones where aquatic life cannot survive. Furthermore, high nitrate levels in drinking water can decreased oxygen availability in humans, in which pregnant women and babies are the most at risk. Finally, the volatilization of N compounds also contributes to air pollution and the formation of greenhouse gases like nitrous oxide, a potent climate-altering compound with global warming potential. Theoretically, feeding an AA profile to precisely match dairy cow requirements would minimize these losses. However, the udder does not take up all available AA. Thus, this research aimed to better understand different AA profiles on AA transport, the route in which AA are taken up by the milk-producing cells in the cow's udder, to ultimately increase efficiency of milk production. In our first study, we demonstrated that non-essential AA (Ala, Gln, and Gly), which can be synthesized in the mammary glands, stimulated Leu, Phe, and Val transport activity within mammary epithelial cells, which could mean that supplemental non-essential AA could increase essential AA transport efficiency. Interestingly, high Val had a negative effect on net uptake (entry minus exit) of Leu and Ile. The second study sought to understand how varying concentrations, within ranges observed in the blood of lactating dairy cows, of 10 different essential AA and 2 non-essential AA groups affected AA transporter expression and activity. We observed greater protein presence and mRNA expression levels of several transporters in response to low availability of their AA transport substrates. Additionally, some AA were involved in stimulating transporter expression and activity when present at high concentrations, such as Leu. There was a plethora of 2-way interactions among AA on transporter protein quantity, mRNA expression, and activity that indicated that the relationship between certain AA will need to be incorporated into future nutrition models. In our third study, we observed that high Val supply decreased the amount that cows ate as well as their milk protein production. This demonstrated that excessive concentrations of certain AA may negatively affect cow metabolism. Lastly, we wanted to investigate the relationship between protein and glucose production in dairy cows, as energy availability is another driver of milk protein production. Our fourth study revealed that dietary protein and starch independently increased milk protein production, yet only starch affected glucose formation. Our findings urge caution against excessive AA concentrations in diets, as imbalances can have negative effects. Overall, we have demonstrated that AA transporters are differentially affected by changes in individual AA supply and various 2-way interactions. This work unveiled the almost unlimited AA interactions that must be further explored to better integrate this knowledge into practical dietary formulations for dairy cows.

The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.

Bio-nanohybrid materials based on the combination of biomolecules and inorganic supports are interesting by their versatile applications in regenerative medicine, drug delivery, bio-engineering and catalysis. In that context, understanding of organic/inorganic interactions offers an important key to design new and more complex bio-systems with modified interactions. In this thesis, amino acids and synthesised poly-amino acids were immobilised in hydrotalcite-like materials under green, controllable and efficient protocols. Additionally, these nanohybrid materials were used as heterogenized catalysts exhibiting high catalytic activity and selectivity in comparison with their counterpart components. All result data showed that the location and nature of the immobilization had an important role in the final chemical properties; moreover, this novel bio-nanohybrid material exhibited excellent synergistic behaviour which was unique for each material and could be modified according with the reaction requirements. All novel bio-nanohybrid materials were easily synthesised, recovered and, in some cases, reused without appreciable deactivation. / Materiales bio-nanohíbridos compuestos por la combinación de biomoléculas y soportes inorgánicos son interesantes por sus versátiles aplicaciones en medicina regenerativa, transporte de medicamentos, bio-ingeniería y catálisis. En este contexto, el entendimiento de las interacciones orgánico/inorgánicas ofrece importante información para el diseño de nuevos y más complejos bio-sistemas con interacciones modificadas. En esta tesis, amino ácidos y poli-amino ácidos sintéticos fueron inmovilizados en materiales tipo hidrotalcita a través de eficientes y controlables protocolos amigables con el medioambiente. Adicionalmente, estos materiales fueron usados como catalizadores heterogeneizados mostrando alta actividad y selectividad en comparación con sus componentes de partida. En este sentido, la localización y naturaleza de la interacción tuvo un importante rol en las propiedades catalíticas finales; además, estos novedosos materiales mostraron un excelente comportamiento sinérgico el cual fue único y pudo ser modificado de acuerdo a las necesidades en reacción. Todos los materiales bio-nanohíbirdos fueron sintetizados y recuperados con facilidad del medio de reacción y en algunos casos fueron reutilizados sin desactivación apreciable.

Thesis (doctoral)--Universitetet i Lund. / Reprints of journal articles inserted in pocket inside back cover.

No description available.

Fourteen Ball State University softball players participated in this study to examine the effects of an amino acid supplement, Rebuild II TM, on strength and performance. Rebuild II TM contains high levels of BCAAs and glutamine. Two groups strength trained for ten weeks with the treatment group consuming Rebuild II TM, and the control group consuming a placebo. Subjects were pre and post-tested on the bench press, squat and machine shoulder press for strength, and performed a 90-foot sprint and a vertical jump for performance measures. LBW was calculated from the subjects body weight and percent fat. Pre and post measures were analyzed using a two way Anova variance with repeated measures test with a significant level set at p< 0.05. Both groups showed significant increases in strength and vertical jump performance, but only the Rebuild II TM group had a significant increase in LBW and decrease in percent fat. It was concluded that with a controlled weight training program, softball players will increase strength and vertical jump performance, and by adding an amino acid supplement to an athletes diet, there may be larger increases in LBW and decreases in percent fat while strength training. / School of Physical Education