Die Methodik zur Analyse dynamischer Prozesse aus NMR-Relaxationsdaten wird beschrieben und untersucht. Die Dynamik von zwei Proteinen, HNL und hnps-PLA2, wird aus gemessenen 15N-Relaxationsdaten über den modellfreien Formalismus quantifiziert und die Rotationsanisotropie von hnps-PLA2 durch hydrodynamische Rechnungen berücksichtigt. Die Strukturen von HNL und VAT-N werden NMR-spektroskopisch bestimmt und Zusammenhänge zwischen Struktur, Dynamik und Funktion diskutiert. NMR-methodische Neuentwicklungen für Methylgruppenselektion und diagonalfreie 3D NOESY-Spektren werden vorgestellt.     «

Die Methodik zur Analyse dynamischer Prozesse aus NMR-Relaxationsdaten wird beschrieben und untersucht. Die Dynamik von zwei Proteinen, HNL und hnps-PLA2, wird aus gemessenen 15N-Relaxationsdaten über den modellfreien Formalismus quantifiziert und die Rotationsanisotropie von hnps-PLA2 durch hydrodynamische Rechnungen berücksichtigt. Die Strukturen von HNL und VAT-N werden NMR-spektroskopisch bestimmt und Zusammenhänge zwischen Struktur, Dynamik und Funktion diskutiert. NMR-methodische Neuentwic...     »

The present work describes the analysis of the solution structure and dynamics of three proteins as investigated by NMR spectroscopy. Chapter 1 summarizes the theory of relaxation phenomena as required for the practical application of NMR spectroscopy to derive insight into molecular dynamics. The discussion includes the theory of spin relaxation, formalisms for describing statistical motions and simulations of relaxation rates. This chapter also includes practical advice on conducting the relevant NMR experiments, the model-free analysis and hydrodynamic calculations to account for motional anisotropy. Chapter 2 describes the structure and dynamics of the apo-form of human neutrophilic lipocalin, HNL. This extracellular transport protein binds bacterial peptides acting as chemotactics, and moreover possesses enzymatical functions in activating matrix metallo-proteinases. The structure of HNL features the funnel shaped b-barrel characteristic for all lipocalins, which is required for accomodating the ligand. The backbone dynamics was quantified via 15N relaxation experiments and reveals noteable correlations with structural features. Additionally sampled 15N relaxation data of side-chains also displays a high correlation with structural features, such as hydrogen bonds or side-chain stacking. Chapter 3 describes the backbone dynamics of human non-pancreatic synovial phospholipase A2, hnps-PLA2, as investigated by 15N relaxation measurements. hnps-PLA2 hydrolyses the phospholipids of cellular membranes and plays an important role in severe chronical inflammatory processes. The 15N relaxation data indicates significant motional anisotropy which was taken into account by hydrodynamic calculations. The model-free analysis reveals significant motions on the nano- and microsecond timescales. The commonly held assumption that PLA2 proteins are rather rigid owing to their large number of stabilizing cystine disulfide bonds could thus be refuted experimentally for the first time. Chapter 4 presents the high-resolution structure of the N-terminal domain of the archaebacterial AAA protein VAT (VCP-like ATPase of Thermoplasma). VAT-N is the catalytically active domain of this putative chaperone. The structure of VAT-N is composed of two sub-domains of equal size, VAT-Nn and VAT-Nc. VAT-Nn has a double y-barrel structure formed by two babb motifs and displays high internal sequence homology. VAT-Nc features a novel half-open b-sheet structure. Preliminary dynamic examinations confirm that both sub-domains are rigidly attached to form a kidney-shaped overall structure. By comparison with an electron microscopic image, the obtained VAT-N structure could be fitted to the top of the circular VAT hexamer, with the lipophilic contact face between both sub-domains pointing towards the center of the ring. Concomitant phylogenetic analyses and homology-based comparisons indicate that the structure of VAT-N can be generalized to all AAA proteins and might represent a link in the evolution of aspartate proteinases from a simple ancestral babb motif. Chapter 5 describes two new NMR methods developed to optimize spectral resolution for [U-13C, 15N] labeled proteins. The HSQC-based QQF-HSQC module for the selection of methyl groups via a quadruple quantum filter maximally suppresses the detrimental evolution of proton homonuclear couplings, minimizing line widths and optimizing intensities. The QQF-HSQC module thus permits very long constant times for methyl groups whose achievable resolution is then almost exclusively limited by their relatively long transverse relaxation times. A strategy for obtaining maximal resolution with a minimal set of NOESY spectra is delineated in the second part. This minimal set consists of three complementary, doubly heteronuclear edited 3D NOESY experiments, the CCH-, NNH- and NCH-edited 3D NOESY. While their combination contains all possible NOE contacts, the full pseudo-4D information is provided by the addition of a 2D H,H-NOESY or the two better resolved HNH- and HCH-edited 3D NOESY. Both the NCH- and the complementary CNH-edited 3D NOESY employ orthogonal isotope filters and are hence free of diagonal signals. This complete suppression of diagonal signals has great practical importance as these spectra are virtually free of t1 noise, phase errors, baseline errors, and exchange signals between amide and water protons.     «

The present work describes the analysis of the solution structure and dynamics of three proteins as investigated by NMR spectroscopy. Chapter 1 summarizes the theory of relaxation phenomena as required for the practical application of NMR spectroscopy to derive insight into molecular dynamics. The discussion includes the theory of spin relaxation, formalisms for describing statistical motions and simulations of relaxation rates. This chapter also includes practical advice on conducting the relev...     »