Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains.

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation.

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy.

Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a ß-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal ß-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation.

GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases.

4,4',4''-(Methane-triyl)triphenyl tris-(2,2,5,5-tetra-methyl-1-oxyl-3-pyrroline-3-carboxyl-ate) benzene tris-olvate.

In the asymmetric unit of the title compound, C(46)H(52)N(3)O(9)·3C(6)H(6), two of the benzene solvent mol-ecules are located in general positions and two are disposed about inversion centers. One of the benzene mol-ecules on an inversion center was grossly disordered and was excluded using the SQUEEZE subroutine in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148-155]. In addition, one of the 2,2,5,5-tetra-methyl-1-oxyl-3-pyrrolin-3-ylcarbonyl groups is disordered over two orientations with refined occupancies of 0.506 (2) and 0.494 (2). The 1-oxyl-3-pyrroline-3-carboxyl-ate groups are essentially planar, with mean deviations from the planes of 0.026 (2), 0.012 (2), 0.034 (4) and 0.011 (4) Å. In the crystal structure, mol-ecules are connected by five weak inter-molecular C-H⋯O and four weak inter-molecular C-H⋯π(benzene) inter-actions.

Biphenyl-4,4'-diyl bis-(2,2,5,5-tetra-methyl-1-oxyl-3-pyrroline-3-carboxyl-ate).

In the title compound, C(30)H(34)N(2)O(6), the complete molecule is generated by a crystallographic 2/m symmetry operation. The 1-oxyl-3-pyrroline-3-carboxyl-ate group lies on a mirror plane. The dihedral angle between the ring planes of the biphenyl fragment is constrained by symmetry to be zero, resulting in rather short intramolecular H⋯H contact distances of 2.02 Å. In the crystal, molecules are connected along the a-axis direction by very weak intermolecular methyl-phenyl C-H⋯π interactions. The C-H bond is not directed to the center of the benzene ring, but mainly to one C atom [C-H⋯C(x - 1, y, z): H⋯C = 2.91 Šand C-H⋯C = 143°].