The amino acid L-proline plays a critical role in many biological processes. Therefore, efforts have been made to understand and control its influence. Since glycosylation is a common post-translational modification known to affect the characteristics of peptides and proteins, in a series of experiments, the effects of glycosylation on the properties of L-proline in peptides have been investigated. A conformationally constrained C-glucosyl proline hybrid is introduced, which has the capacity to vary the N-terminal amide equilibrium in model peptides through derivatization of the carbohydrate scaffold. For the first time, a comprehensive study of the effects of O-glycosylation on the kinetics and thermodynamics of prolyl amide isomerization is reported. The O-glycosylation of 4-hydroxy-L-proline has different effects on amide isomerization depending on the stereochemistry of the 4-hydroxyl group, which alters the orientation of the glycan with respect to the prolyl side chain. 4S-Galactosylation of 4-hydroxy-L-proline affects both the amide isomer equilibrium and the rate of amide isomerization, whereas 4R-galactosylation does not measurably influence either parameter. However, close contacts between the carbohydrate and prolyl rings lead to changes in the conformation and stability of longer peptides. As an expansion on these initial model studies, the effects of prolyl O-glycosylation on the properties of model peptides of two extremely important structural proteins are investigated. O-Galactosylation of 4R-hydroxy-L-proline residues in collagen model peptides does not preclude formation of the collagen triple helix, where the anomeric linkage of the Hyp O-glycan has slightly different influences on the conformational stability of the peptides. Also, the O-galactosylation of Hyp residues in polyproline model peptides causes a large increase in conformational stability. In both cases, interactions between the glycan and the peptide backbone and changes in hydration are implicated in contributing to the conformational stabilization of the peptides. These studies demonstrate that both natural and unnatural glycosylation of L-proline can be used as a means to control amide isomerization, and can increase the conformational stability of peptides, properties that will likely contribute to the development of new biomaterials. Also, these experiments provide further insight into the broad role glycosylation plays in affecting peptide and protein structure.

The amino acid L-proline plays a critical role in many biological processes. Therefore, efforts have been made to understand and control its influence. Since glycosylation is a common post-translational modification known to affect the characteristics of peptides and proteins, in a series of experiments, the effects of glycosylation on the properties of L-proline in peptides have been investigated. A conformationally constrained C-glucosyl proline hybrid is introduced, which has the capacity to vary the N-terminal amide equilibrium in model peptides through derivatization of the carbohydrate scaffold. For the first time, a comprehensive study of the effects of O-glycosylation on the kinetics and thermodynamics of prolyl amide isomerization is reported. The O-glycosylation of 4-hydroxy-L-proline has different effects on amide isomerization depending on the stereochemistry of the 4-hydroxyl group, which alters the orientation of the glycan with respect to the prolyl side chain. 4S-Galactosylation of 4-hydroxy-L-proline affects both the amide isomer equilibrium and the rate of amide isomerization, whereas 4R-galactosylation does not measurably influence either parameter. However, close contacts between the carbohydrate and prolyl rings lead to changes in the conformation and stability of longer peptides. As an expansion on these initial model studies, the effects of prolyl O-glycosylation on the properties of model peptides of two extremely important structural proteins are investigated. O-Galactosylation of 4R-hydroxy-L-proline residues in collagen model peptides does not preclude formation of the collagen triple helix, where the anomeric linkage of the Hyp O-glycan has slightly different influences on the conformational stability of the peptides. Also, the O-galactosylation of Hyp residues in polyproline model peptides causes a large increase in conformational stability. In both cases, interactions between the glycan and the peptide backbone and changes in hydration are implicated in contributing to the conformational stabilization of the peptides. These studies demonstrate that both natural and unnatural glycosylation of L-proline can be used as a means to control amide isomerization, and can increase the conformational stability of peptides, properties that will likely contribute to the development of new biomaterials. Also, these experiments provide further insight into the broad role glycosylation plays in affecting peptide and protein structure.

Hydroxyproline as a modified amino acid can serve as a carbon and nitrogen source for certain microorganisms. Its primary isomer trans-4-hydroxy-L-proline is found in the root nodule of legume plants. Hydroxyproline (Hyp) catabolism has been characterized in bacteria and animal cells. In bacteria, trans-4-hydroxy-L-proline (trans-4-L-proline) is converted to the central metabolite α-ketoglutarate (α-KG) by four reactions. The Hyp catabolism pathway has been identified in the nitrogen-fixing legume endosymbiont Sinorhizobium meliloti. hypS is one of the transcripts in the 14 hyp gene cluster on the pSymB megaplasmid, and was annotated to encode a putative malate/L-lactate dehydrogenase. In this study, purified HypS was assayed on different substrates and the reaction products were characterized. It was demonstrated that HypS can oxidize L-proline and reduce Δ1-pyrroline-2-carboxylate, but not on L-malate. Noticeably unlike the wild type strain, a hypS- mutant strain failed to grow on D-proline. The ability of D-proline to support grow of an L-proline auxotroph, together with the substrate specificity of HypS, strongly suggests that hypS is involved in the metabolism of D-proline to L-proline in S. meliloti. The possible role of HypS in the catabolism of Hyp or related compounds remains to be determined. / Thesis / Master of Science (MSc)

While chemical isomers typically have distinct properties, differentiating between them is often an analytical challenge, especially for mass spectrometric methods. Infrared multiple photon dissociation (IRMPD) spectroscopy and ion mobility spectrometry (IMS) can be useful in analysis of such isomeric compounds; however, experimental results alone do not directly provide in-depth structural information. In this thesis, computational chemistry is first used to explain experimental results and understand the conformational preference of the gas phase ions formed from the lithiation of cis-3, cis-4 and trans-4 hydroxyproline isomers and then used in a predictive manner to evaluate IRMPD spectroscopy and IMS as potential paths forward for the characterization of isomeric dye species. Finally, theoretical methods are used to begin to understand the dissociation pathways of lithiated hydroxyproline isomers in the gas phase, which is ongoing.

Hydroxyproline (trans-4-hydroxy-L-proline (4-L-Hyp)) can be used by certain microorganisms as a source of carbon and nitrogen. The nitrogen fixing bacterium, Sinorhizobium meliloti carries a cluster (hyp cluster) of 14 genes responsible for the transport and degradation of this amino acid in the cell. The biological functions of several gene products in the hyp cluster are still unknown. So far, it is known that the conversion of trans-4-hydroxy-proline to α-ketoglutarate, one of the intermediate of the TCA cycle, occurs in four enzymatic reactions. The whole hyp cluster is up regulated in the presence of 4-hydroxy-proline in the media. Previous studies have shown several other 4-hydroxy-proline-inducible genes. One of these genes, smc04388, has been annotated as a putative omega amino transaminase. The role of this omega transaminase in the main catabolic pathway of 4-hydroxy-proline has not been investigated. In order to address this, two mutant strains; a single smc04388 mutant and a double smc04388 hypD mutant were created. Growth curves of these mutants in minimal media showed that the Smc04388 protein is not required for the growth of S. meliloti in the presence of trans-4-hydroxy-L-proline as the sole carbon and nitrogen source. The Smc04388 protein was overexpressed as a Strep-tagged and purified from S. meliloti. The purified enzyme showed amino transaminase activity with pyruvate and α-methylbenzylamine. In addition, an enzymatic reaction using the product of the second enzyme of the 4-hydroxy-proline pathway, Δ1-pyrroline-4-hydroxy-2-carboxylate, was carried out to test the activity of Smc04388 with this compound. Mass spectrometry analysis of this reaction mixture revealed the formation of L-alanine from pyruvate and Δ1-pyrroline-4-hydroxy-2-carboxylate, suggesting the utilization of this compound as an amino donor by the Smc04388 transaminase. It was also shown that transamination activity in cell extracts increase in the absence of Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase, the enzyme that catalyzes the conversion of this compound in the 4-hydroxy-proline pathway. These results confirmed the hypothesis that the Smc04388 omega amino transaminase is involved in a secondary pathway related to the known catabolic pathway of 4-hydroxy-proline in bacteria. Further understanding of this secondary pathway will contribute to the study of the metabolism of 4-hydroxy-proline in bacteria. In addition to this, the complete characterization of the Smc04388 omega amino transaminase could have practical application in the pharmaceutical industry. / Thesis / Master of Science (MSc)

Abstract Collagen prolyl 4-hydroxylase is the key enzyme in the biosynthesis of collagens, a family of extracellular matrix proteins. Vertebrate collagen prolyl 4-hydroxylases are α2β2 tetramers, the β subunit being identical to the multifunctional protein disulphide isomerase (PDI). Several isoforms of the catalytic α subunit have been identified in various organisms. Prolyl 4-hydroxylases have also been isolated from plants, where they hydroxylate proline-rich structural glycoproteins of the cell walls. The structural and functional properties of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase are characterized here. Data obtained from NMR studies indicate that the domain consists of five α helices and one short β strand, this structure being quite different from those of other proline-rich peptide-binding modules. Several residues involved in the binding of a short synthetic peptide were also identified by NMR. Kd values for the binding of several synthetic peptides to the α(I) and α(II) domains were determined by surface plasmon resonance and isothermal calorimetry, and the results indicated that the binding properties of the type I and type II collagen prolyl 4-hydroxylase tetramers can mainly be explained by the binding of peptides to this domain rather than to the catalytic domain. The peptide-substrate-binding domain of human type I collagen prolyl 4-hydroxylase was also crystallized. The crystals were well ordered and diffracted to at least 3 Å, the asymmetric unit most probably containing a domain dimer. The genome of Arabidopsis thaliana was found to encode at least six putative prolyl 4-hydroxylase polypeptides, one of which was cloned and characterized here as a recombinant protein. All the catalytically critical residues identified in animal prolyl 4-hydroxylases were also conserved in this plant prolyl 4-hydroxylase, and their mutagenesis led to inactivation of the enzyme. The recombinant plant enzyme was effective in hydroxylating poly(L-proline) and several synthetic proline-rich peptides. Surprisingly, contrary to previous reports on plant prolyl 4-hydroxylases, the collagen-like peptides were found to be good substrates, the enzyme preferentially hydroxylating prolines in the Y positions of the -X-Y-Gly- triplets, thus resembling the vertebrate collagen prolyl 4-hydroxylases even in this respect. The recombinant plant prolyl 4-hydroxylase also hydroxylated peptides representing the N and C-terminal hydroxylation sites present in the hypoxia-inducible transcription factor α. The fact that these peptides contain only one proline residue indicated that a poly(L-proline) type II conformation was not required for hydroxylation.

No description available.

We present the synthetic studies of coerulescine, horsfiline, and streptorubin B in this report. And we also present the new synthesis of 3-arylpyrrolines and cis-3,4-diarylpyrrolidines via an easy and straightforward pathway in this report. All of these researches are using 4-hydroxypiperidine and 4-hydroxyproline as the starting material.

Typically chronic injury leads to hepatic fibrosis. No effective antifibrotic drugs have been approved, but herbal drugs have potential on the therapy of hepatic fibrosis. The objective of this study used TAA-induced liver fibrosis mouse as a model to elucidate whether aqueous extract of the root of Pluchea indica (PIAE) can reduce liver fibrosis triggered by TAA. Mice were intraperitoneally injected with TAA (200 mg/Kg) three times per week as the TAA group, and those of injected with PIAE once per week as the treatment group. Three PIAE dosages of low- (0.5 mg/ml), medium- (1.0 mg/ml), and high- (1.5 mg/ml) doses were applied. Control mice were intraperitoneally injected with phosphate-buffered saline (2 ml/Kg) three times per week. Mice were sacrificed after 4 or 8 week treatment. Mice serum glutamyl pyruvic transaminases (GPT) were increased in the TAA group while the treatment group effects were declined after 4 or 8 weeks. H&E, Reticular fiber, and Sirius red staining revealed that TAA induced liver fibrosis and fibrotic lesions were reduced by PIAE treatment. Hydroxyproline assay showed that TAA increased collagen contents and PIAE significantly decreased collagen contents after 4 or 8 weeks. Collagen £\1 and £\-SMA mRNA levels were decreased after 4- or 8- week PIAE treatments. The protein levels of ED2, £\-SMA, p53, and phospho-p53 were all significantly declined on 4 or 8 weeks after PIAE treatment. In conclusion, these results demonstrated that the aqueous extract of P. indica shows anti-fibrotic effects on fibrogenesis of mouse liver.

The amino acid (2S, 4R)-4-hydroxyproline (Hyp) plays a critical role in animal kingdom as structural protein collagen. It is ubiquitous in plant cell walls performing various functions such as structural assembly, plant hormones, plant growth, defense against pathogens, etc. Glycosylation of Hyp is often seen in plant cell walls with L-Arabinofuranose and D-Galactopyranose and not in animal kingdom. Glycosylation is a post-translational modification, which affects characteristics of proteins and peptides. The main objective of this thesis is to synthesize various L-arabinofuranosylated hydroxyproline model amides and investigate their thermodynamic and kinetic properties of cis/trans amide isomerization. These results are compared with the previous research of D-galactopyranosylated hydroxyproline model amides, which may provide an insight to structural implications for their stability and conformations of peptides and specificity in plants. Both - and -L-arabinosylation of Hyp resulted in the stabilization of trans rotameric state at room temperature while the α-anomer leads to cis rotamer stabilization at higher temperature. Similarly, both unnatural 4S-hydroxyproline (hyp) building blocks resulted in stabilization of trans rotamer but α-anomer shows exo configuration instead of endo. This result shows a reverse trend when compared to galactosylated hydroxyproline building blocks as previous research results in our group. Our results may provide further insight to the role of glycosylation on protein structure and stability in plants.