Recently years NMR technique plays an important role in investigation of structures of biochemistry molecules. Especially two-dimension NMR experiments are powerful evidences of the relation of intramolecule of biochemistry. Further more we design a series of experiments about studies of chemical shift and dynamics under different pH values and at different temperature.

Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both immune response and inflammation-related diseases. Viral CC chemokine inhibitor (vCCI) is a poxvirus encoded protein that has been shown to bind tightly and inhibit the action of many CC chemokines. This function suggests that vCCI could be explored as an antiinflammatory therapeutic, a possibility that has been supported in mouse studies. The structure of vCCI in unbound form was determined by others, but to date no structure has been reported of bound vCCI. We report the NMR structure of vCCI in complex with the human CC chemokine MIP-1[beta]. The non-aggregating MIP-1[beta] variant MIP-1[beta] 45AASA48 was used in this complex to allow sufficiently high concentration at pH 7 to carry out the solution structure determination. A combination of NOE distance restraints, torsion angle restraints, and residual dipolar coupling were used to determine the structure of the complex, which also required protein deuteration due to its relatively large size (34kDa). The structure shows that MIP-1[beta] binds to vCCI with 1:1 stoichiometry, forming a complex of 311 amino acids. vCCI uses residues from its [beta]- sheet II to interact with a surface of MIP-1[beta] that includes residues adjacent to its Nterminus, as well as residues in the 20's region, and the 40's loop. The structure of the MIP-1[beta]-vCCI complex reveals for the first time the regions of each protein involved in the interaction, and allows a greater understanding of the strategy used by vCCI to tightly bind numerous chemokines, while retaining selectivity for the CC chemokine subfamily.

Zugl.: Giessen, Universiẗat, Diss., 2009.

Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Zugl.: Charlottesville, Univ. of Virginia, Diss., 2007 / Hergestellt on demand

Habilitation - Universität, Düsseldorf, 1997.

The majority of the work in this dissertation describes the characterization of the nitrophorins (NPs) by NMR spectroscopy.NP7 was studied and compared to NP2 and relevant mutants.NP7 is shown to have three additional amino acids at the N-terminus, which contribute significantly to the protein fold stability and maintenance of NP7 function. The paramagnetic form of the protein has large 1H NMR chemical shifts and broad signals due to protein aggregation. The 1H NMR and CD spectroscopy both revealed that NP7 has mainly the A heme orientation, and the mutation of Glu27 to Val27 leads to an A:B ratio change, from mainly B to mainly or almost exclusively A.NMR investigation of the ferriheme resonances of the low-spin complexes of NP2 and mutant NP2(V24E) with imidazole (ImH), histamine (Hm) and cyanide (CN-) ligands as a function of pH has been completed. Strong chemical exchange cross peaks were observed in the NOESY/EXSY spectra at low pH (pH* 5.5 to 4.0) for the three wt NP2 complexes, which indicate an interchange between two ruffling distortions of the heme. A dramatic change of A:B ratios with pH was observed for the three NP2(V24E) ligand complexes. They both are believed to be a result of a change in protein structure near E53 when it is protonated at low pH.Homo-dimers of NP1 and NP4 were investigated. 1H{15N}-HSQC NMR experimental results revealed that holo-NP4 is a homo-dimer at low pH and a mixture of dimer and monomer at high pH. In contrast, holo-NP4-Hm is monomeric at high pH (6.5 and 7.5). A H-bond between Asp30 and the Hm ligand is responsible for destabilization of the homo-dimer. NP1 was also shown to be a mixture of dimer and monomer at high pH.Heme d1 model complexes were studied. The high-spin form and low-spin forms of the Fe(III) complexes with 2-methylimidazole or imidazole, for monooxo-octaethylchlorin, trans-dioxo-octaethylisobacteriochlorin and 2,7-dioxo-octaethyliso- bacteriochlorin, were characterized by NMR spectroscopy. Spin density maps that show the orbital used for spin delocalization have been generated from Hückel calculations.Preliminary results of additional studies of the nitrophorins and future directions of study are presented.

19F NMR studies of proteins provide unique insight into biologically relevant phenomena such as conformational fluctuations, folding and unfolding, binding and catalysis. While there are many advantages to the use of 19F NMR, experimental challenges limit its widespread application. The focus of this thesis has been to address some of these limitations, including resonance assignment and perturbations arising from fluorine probes, and to develop more robust methods of studying protein topology by 19F NMR. 19F NMR experiments designed to measure local hydrophobicity and exposure were developed and evaluated in two systems, Fyn SH3 and calmodulin, labeled with 3-fluorotyrosine. Paramagnetic effects from dissolved oxygen, solvent isotope shifts from deuterium oxide, and 1H-19F NOEs were each sufficient in establishing relative solvent exposure, while the combination of effects from oxygen and deuterium oxide were able to delineate local hydrophobicity and solvent accessibility of 19F probes. Two NMR based resonance assignment protocols were developed using 13C, 15N-enriched 3-fluorotyrosine and 3-fluorophenylalanine, separately biosynthetically incorporated into calmodulin. In the first approach, isotopic enrichment facilitated two-dimensional heteronuclear experiments based on INEPT and COSY magnetization transfer schemes to correlate the fluorine nucleus to sidechain and backbone 1H, 13C, and 15N atoms, providing complete spectral assignment. The assignment of 3-fluorophenylalanine resonances was achieved using 19F-, and 15N-edited homonuclear NOE experiments to connect the fluorine nucleus to intraresidue and neighboring 1H and 15N resonances. While both strategies were successful, the NOE-based method was vulnerable to alternate relaxation mechanisms, including chemical shift anisotropy and chemical exchange. Structural perturbations arising from uniform incorporation of 3-fluorophenylalanine in calmodulin was thoroughly investigated using 19F and 1H-15N NMR spectroscopy, 15N spin relaxation and thermal denaturation via circular dichroism spectroscopy. While stability was unaffected, NMR experiments revealed increased protein plasticity, minor conformers and line broadening. The merit of fractional fluorine labeling in reducing such disruptions was demonstrated, and labeling levels of 60-75% provided an optimal balance between native-likeness and the usual advantages of 19F NMR in our system. The 19F NMR techniques developed here are broadly applicable and will expand the utility of 19F NMR in studies of protein systems.

1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D2O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration- dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.

1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D2O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration- dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.