Correction of spin diffusion during iterative automated NOE assignment

Indirect magnetization transfer increases the observed nuclear Overhauser enhancement (NOE) between two protons in many cases, leading to an underestimation of target distances. Wider distance bounds are necessary to account for this error. However, this leads to a loss of information and may reduce the quality of the structures generated from the inter-proton distances. Although several methods for spin diffusion correction have been published, they are often not employed to derive distance restraints. This prompted us to write a user-friendly and CPU-efficient method to correct for spin diffusion that is fully integrated in our program ambiguous restraints for iterative assignment (ARIA). ARIA thus allows automated iterative NOE assignment and structure calculation with spin diffusion corrected distances. The method relies on numerical integration of the coupled differential equations which govern relaxation by matrix squaring and sparse matrix techniques. We derive a correction factor for the distance restraints from calculated NOE volumes and inter-proton distances. To evaluate the impact of our spin diffusion correction, we tested the new calibration process extensively with data from the Pleckstrin homology (PH) domain of Mus musculus beta-spectrin. By comparing structures refined with and without spin diffusion correction, we show that spin diffusion corrected distance restraints give rise to structures of higher quality (notably fewer NOE violations and a more regular Ramachandran map). Furthermore, spin diffusion correction permits the use of tighter error bounds which improves the distinction between signal and noise in an automated NOE assignment scheme.     

Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays

Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.     

Direct mass measurements of neutron-deficient xenon isotopes using the ISOLTRAP mass spectrometer

The masses of the noble-gas Xe isotopes with have been directly measured for the first time. The experiments were carried out with the ISOLTRAP triple trap spectrometer at the on-line mass separator ISOLDE/CERN. A mass resolving power of the Penning trap spectrometer of of close to a million was chosen resulting in an accuracy of keV for all investigated isotopes. Conflicts with existing, indirectly obtained, mass data by several standard deviations were found and are discussed. An atomic mass evaluation has been performed and the results are compared to information from laser spectroscopy experiments and to recent calculations employing an interacting boson model.PACS: 07.75. + h Mass spectrometers - 21.10.Dr Binding energies and masses - 27.60. + j - 32.10.Bi Atomic masses, mass spectra, abundances, and isotopes     

Elemental composition of fine particulate matter (PM2.5) in Skopje,FYR of Macedonia

Aerosol samples were collected at an urban background site in Skopje, Former Yugoslavic Republic of Macedonia, during four measurement campaigns from December 2006 to October 2007. An impactor was used to collect particulate matter (PM_2.5) aerosol particles and the samples were analyzed for the concentrations of particulate mass, black carbon (BC), and 17 elements. The 12‐h average PM_2.5 concentrations varied in the range 10–140 µg m^?3 with the highest concentrations measured during wintertime pollution episodes and during the summer period. Pair‐wise correlations and crustal enrichment were studied and the data set was analyzed by factor analysis and positive matrix factorization. Major aerosol components were identified as mineral dust (main observed tracers Si, K, Ca, Ti, Fe, Sr, and Rb), combustion (BC, S, K, V, and Ni), traffic‐related aerosol (Pb and Zn), and secondary sulfate combined with mineral dust. Combustion sources dominated during wintertime and were likely due to heavy oil combustion, biomass burning, and other industrial activities within the city area. Mineral dust was observed throughout the year, but the concentrations peaked during the unusually hot and dry summer of 2007. It is concluded that Skopje suffers from serious air pollution due to central and residential heating, the transport sector, and industrial activities within the city, and contributions from mineral dust increase the PM_2.5 concentrations under dry periods. Topography and meteorological conditions aggravate the problems and make the air quality comparable with the conditions in other highly polluted cities in Southern Europe and worldwide. Copyright © 2011 John Wiley & Sons, Ltd.     

DNA based molecular motors

Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid—a process that is driven by one of the most powerful molecular motors.