A thermodynamic study of five homologous HPr proteins derived from organisms inhabiting diverse environments has been undertaken. The aim of this study was to further our understanding of protein stabilization in extremes of environment. Two of the proteins were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus subtilus. Genes for three of these homologous HPr proteins were for the first time cloned from their respective organisms into expression vectors and they were over-expressed and purified in Escherichia coli. Stability measurements were performed on these proteins under a variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent induced denaturation experiments. Stability curves were determined for every homologue and these reveal very similar conformational stability for these homologues at their habitat temperatures. The BstHPr homologue is the most thermostable and also has the highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr. Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of these variants is presented in an attempt to understand the origins of the differences in stability of the HPr homologues.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24326号 / エネ博第454号 / 新制||エネ||85(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Newcastle disease virus (NDV) is one of the paramyxovirus that has been studied at length since this virus infects the birds of all species. NDV is highly virulent in chickens and results in a high mortality rate because the V protein of NDV is found to inhibit the avian immune response system. No drugs are available for treating NDV therefore, determining the structure of V protein would help in developing a drug that can inactivate the V protein, thereby increasing the host immune response against viral infection. The research here is focused on purification and initial structural analysis of the V protein of NDV. The V protein was purified by gel filtration chromatography and the structure was studied using fluorescence and CD spectroscopy, and NMR. The results suggested that the V protein is unstructured. The research also involved purification and structural analysis of the flowering locus T (FT) protein, which is found to play a major role in theninitiation of flowering in plants. Structural analysis of the FT protein may help in finding the possible domains of the FT protein that interacts with other plant proteins, leading ton flowering. The FT protein was purified by ion exchange chromatography and the structure was studied by fluorescence and CD spectroscopy. The fluorescence data suggested that the FT protein may be folded, where as the CD data was inconclusive. More accurate secondary structure information about the protein could be obtained using NMR, but since the concentration of the FT protein was too low (0.007 mM), proper NMR study was not possible.

No description available.

Thesis (M.S.)--Miami University, Dept. of Chemistry and Biochemistry, 2004. / Title from first page of PDF document. Includes bibliographical references (p. 57-64).

It is well-known that the large diversity of protein functions and structures derives from the broad spectrum of physicochemical properties of the 20 canonical amino acids that constitute modern proteins. According to the generally accepted coevolution theory of the genetic code, evolution of protein structures and functions was continuously associated with enrichment of the genetic code, with aromatic amino acids being considered the latest addition to the genetic code to increase structural stability of proteins and diversification of their catalytic functions. The main objective of this master thesis was to test whether enzymatic catalysis could precede the appearance of aromatic amino acids in the standard genetic code. For that purpose, the effect of amino acid alphabet reduction on structure and function of dephosphocoenzyme A kinase (DPCK) was studied. Dephosphocoenzyme A kinase catalyses the final step in the biosynthesis of coenzyme A, a very conserved cofactor. Two aromatic amino acid-lacking mutants of DPCK from a thermophilic bacterium, Aquifex aeolicus, were designed by substituting aromatic amino acid residues by (i) leucines and (ii) various non-aromatic amino acids to best preserve the structural stability of the protein. Wild type protein and the two mutants were cloned and...

No description available.

Coordination of transition metals to peptides, either through the incorporation of unnatural chelating groups or amino acid ligating side-chains, expands the utility of peptides for biological studies. The first part of this project describes induction of α-helical secondary structure in pentapeptides upon side-chain coordination of inert transition metal ions. The second part of this project describes the syntheses of biologically active peptide species that contain a macrobicyclic hexaamine ligand that can complex radioactive metal ions for diagnostic imaging purposes. / Short peptide sequences do not form thermodynamically stable α-helices in water. The capacity of two metal clips, cis-[Ru(NH3)4(solvent)2]2+ and cis [Pd(en)(solvent)2]2+ to induce α-helicity in peptides that are five amino acids long, Ac HARAH NH2 and Ac MARAM-NH2 has been explored. In all cases at pH < 5, the metal ions bind to the side-chains of amino acid residues at positions i, i+4 of the pentapeptides resulting in formation of bidentate macrocyclic species. Circular dichroism and 1H nuclear magnetic resonance data indicate that the metal complexes of Ac-MARAM-NH2 are highly α helical in water, and in the most spectacular case, coordination of Ac-MARAM-NH2 to cis-[Ru(NH3)4(solvent)2]2+ results in up to 80% α-helicity. In contrast, metal complexes of Ac-HARAH-NH2 exhibit significantly less α-helicity in water. / 64Cu-radiolabelled peptides have been investigated for their ability to target specific tissue or cell types. These peptides require a chelating group that binds copper ions strongly. Macrobicyclic hexaamine ligands, based on the compound commonly referred to as “sarcophagine”, have demonstrated extremely high stability under biological conditions. Here we describe the synthesis of diaminosarcophagine chelators with carboxylate groups for conjugation to peptides. These new chelators have been attached to the N-terminus or lysine side-chain of biologically-active peptides, including Tyr3 octreotate, Lys3-bombesin and an integrin targeting peptide. Spectroscopic and voltammetric studies of these species suggest that the conjugated sarcophagine group retains the high metal binding affinity and structural properties of the parent species, diaminosarcophagine. These are among the first sarcophagine-peptide compounds that have been properly characterised. The new sarcophagine-peptide conjugates can be easily radiolabelled with 64Cu2+ over a wide pH range at ambient temperature.

Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

This diploma thesis deals with the study of possible interactions between hyaluronan (HA) and plasmid DNA (pDNA). Plasmid DNA was isolated from E. coli JM109 (pUC19) and resuspended in TE buffer as well as high molecular weight hyaluronan. Individual samples of pDNA, HA and pDNA-HA were characterized by gel electrophoresis, CD spectroscopy and high resolution ultrasonic spectroscopy. Agarose gel electrophoresis examined the effect of the addition of hyaluronan to plasmid DNA on the migration of samples to the positive electrode. Structural changes in pDNA-HA samples were examined using CD spectroscopy. Individual CD spectra describes the dependence of the difference in absorption coefficients for left-hand and right-handed elliptic polarized light at wavelength. High resolution ultrasonic spectroscopy has been used to study interactions. It is an analytical method based on ultrasonic velocity and attenuation. We classify this technique as a non-destructive method because the passing waves do not affect the structure of the analyzed sample.