Obtaining chemical shift anisotropy (CSA) principal values from large biomolecular systems is often a laborious process preparing many singly isotopically labeled samples and performing multiple... Show moreObtaining chemical shift anisotropy (CSA) principal values from large biomolecular systems is often a laborious process preparing many singly isotopically labeled samples and performing multiple independent CSA measurements. We present CSA tensor principal values measured in the biomolecular building blocks tyrosine(.)HCl, histidine(.)HCl, and all-E-retinal, both isotopically labeled and unlabeled forms at 17.6 T. The measured tensor values are identical for most carbon sites despite significant dipolar couplings between the spins. Quantum mechanical simulations of all arbitrary three spin system were used to evaluate the accuracy of direct CSA measurement as a function of applied magnetic field strength and molecular parameters. It was found that for a CSA asymmetry of 0.2 or more, an accurate measure of the CSA parameters is obtained when the CSA anisotropy is more than six times the largest dipolar coupling in frequency units. If the CSA asymmetry is more than 0.5, this requirement is relaxed, and accurate results are obtained if the anisotropy is more than three times the dipolar coupling. While these limits are insufficient for measurement of CSA's for alpha-carbons and aliphatic sidechain sites in proteins at current field strengths. they open the way for routine systematic CSA measurements of sites with relatively large CSA tensor values in extensively isotopically labeled biomolecules in widely available magnetic fields. (C) 2004 Published by Elsevier Inc. Show less
We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely... Show moreWe have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a “solid state protein film” maintains its nativelike conformation and ET function, even after removal of the aqueous solvent. Show less
This thesis consists of eight chapters. In Chapters 2 and 3, theapplication of AFM to biological systems is explored, both in air and inliquid. Chapter 2 deals with atomic force microscopy in air... Show moreThis thesis consists of eight chapters. In Chapters 2 and 3, theapplication of AFM to biological systems is explored, both in air and inliquid. Chapter 2 deals with atomic force microscopy in air and, as anexample, contains a study of filaments found in dividing yeast cells.Chapter 3 describes atomic force microscopy in liquid, both in contactmode and in tapping mode.In Chapters 4, 5, and 6 we discuss all steps that have to betaken in order to make the high-resolution MRFM experimentdescribed in Chapter 7 possible. As a first step, Chapter 4 provides amethod for immobilizing well-separated single molecules for AFMstudies in liquid. Next, in Chapter 5, the use of carbon nanotubes asAFM tips for molecular recognition force microscopy is discussed, anda description is given of the fabrication process of single-walled andmulti-walled carbon nanotube AFM tips. In Chapter 6, the last step isdiscussed: chemical characterization and functionalization of carbonnanotubes. In Chapter 7, all steps come together, and first results ofmolecular recognition force microscopy experiments on pea lectin andon mannan binding lectin are shown and discussed.To conclude, Chapter 8 provides a summarizing discussion andan outlook. Show less
Partly biosynthetic site-directed isotopically C-13 enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically... Show morePartly biosynthetic site-directed isotopically C-13 enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4-C-13], [1,4-C-13] and [2,3-C-13] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual 13C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4-C-13]-succinic acid or [2,3-C-13]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4-C-13]-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by [2,3-C-13]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures. Show less
Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging... Show moreNowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population. (C) 2005 Wiley-Liss, Inc. Show less
