Ftm is a novel basal body protein of cilia involved in Shh signalling

In this study we show in mice that Ftm (Rpgrip1l) is located at the ciliary basal body. Our data reveal that Ftm is necessary for developmental processes such as the establishment of left-right asymmetry and patterning of the neural tube and the limbs. The loss of Ftm affects the ratio of Gli3 activator to Gli3 repressor, suggesting an involvement of Ftm in Shh signalling. As Ftm is not essential for cilia assembly but for full Shh response, Ftm can be considered as a novel component for cilium-related Hh signalling. Furthermore, the absence of Ftm in arthropods underlines the divergence between vertebrate and Drosophila Hh pathways.     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.     

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable 'UU172 element' from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses.     

Mathematical treatment of adiabatic fast passage pulses for the computation of nuclear spin relaxation rates in proteins with conformational exchange

Although originally designed for broadband inversion and decoupling in NMR spectroscopy, recent methodological developments have introduced adiabatic fast passage (AFP) pulses into the field of protein dynamics. AFP pulses employ a frequency sweep, and have not only superior inversion properties with respect to offset effects, but they are also easily implemented into a pulse sequence. As magnetization is dragged from the +z to the -z direction, Larmor precession is impeded since magnetization becomes spin-locked, which is a potentially useful feature for the investigation of microsecond to millisecond dynamics. A major drawback of these pulses as theoretical prediction is concerned, however, results from their time-dependent offset: simulations of spin density matrices under the influence of a time-dependent Hamiltonian with non-commuting elements are costly in terms of computational time, rendering data analysis impracticable. In this paper we suggest several ways to reduce the computational time without compromising accuracy with respect to effects such as cross-correlated relaxation and modulation of the chemical shift.     

Crystal structure of RNase A tandem enzymes and their interaction with the cytosolic ribonuclease inhibitor

Because of their ability to degrade RNA, RNases are potent cytotoxins. The cytotoxic activity of most members of the RNase A superfamily, however, is abolished by the cytosolic ribonuclease inhibitor (RI). RNase A tandem enzymes, in which two RNase A molecules are artificially connected by a peptide linker, and thus have a pseudodimeric structure, exhibit remarkable cytotoxic activity. In vitro, however, these enzymes are still inhibited by RI. Here, we present the crystal structures of three tandem enzymes with the linker sequences GPPG, SGSGSG, and SGRSGRSG, which allowed us to analyze the mode of binding of RI to the RNase A tandem enzymes. Modeling studies with the crystal structures of the RI-RNase A complex and the SGRSGRSG-RNase A tandem enzyme as templates suggested a 1 : 1 binding stoichiometry for the RI-RNase A tandem enzyme complex, with binding of the RI molecule to the N-terminal RNase A entity. These results were experimentally verified by analytical ultracentrifugation, quantitative electrophoresis, and proteolysis studies with trypsin. As other dimeric RNases, which are comparably cytotoxic, either evade RI binding or potentially even bind two RI molecules, inactivation by RI cannot be the crucial limitation to the cytotoxicity of dimeric RNases.