An aptly positioned azido group in the spacer of a protein cross-linker for facile mapping of lysines in close proximity.

Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division.

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside-out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane-bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP). FtsZ-R174D-GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non-permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild-type FtsZ. FtsZ-R174D could also localize in an FtsZ depletion strain. However, in contrast to wild-type FtsZ, FtsZ-R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ-R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

Chemical cross-linking and mass spectrometry for protein structural modeling.

The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described, complementing existing technologies. Based on chemical cross-linking and offering the analytical speed and sensitivity of mass spectrometry these methodologies are thought to contribute valuable tools towards future high throughput protein structure elucidation.

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry.

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane-associated supercomplex of approximately 1 MD. A chaperone-like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade-like arrangement of the building blocks projecting into the intermembrane space.

Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling.

A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.

Influence of infection of cells with bacteria associated with reactive arthritis on the peptide repertoire presented by HLA-B27.

Reactive arthritis (ReA) after infections with various gram-negative bacteria is strongly associated with the MHC class I molecule HLA-B27. It is supposed that the B27 molecule itself plays a role in the pathogenesis of ReA by presenting antigenic peptides to cytotoxic T lymphocytes. The peptide repertoires presented by Salmonella-, Shigella- and non-infected cells were compared to identify such peptides. From the peptides isolated from the B27 molecules of these cells, profiles were generated by reversed-phase chromatography and peaks present in the profiles from infected cells but not in profiles from non-infected cells were studied for their peptide compositions. Some sequences with identity to those in human histone H3, human ribosomal protein S17 and the heavy chain of HLA-B27 itself were detected only in profiles from infected cells. All peptides identified from infected cells contained the B*2705 peptide-binding motif. The data suggest that HLA-B27-positive cells infected with ReA-inducing bacteria show an increased presentation of certain self-peptides. There was no evidence for altered peptide-binding specificity of B27 after infection. However, the interpretations were hampered by the variation in peptide presentation between different experiments.