Fourier transform mass spectrometry

This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.     

Structural and functional role of threonine 112 in a superantigen Staphylococcus aureus enterotoxin B.

Bacterial superantigens are potent T-cell stimulatory protein molecules produced by Staphylococcus aureus and Streptococcus pyogenes. Their superantigenic activity can be attributed to their ability to cross-link major histocompatibility complex class II molecules with T-cell receptors (TCRs) to form a tri-molecular complex. Each superantigen is known to interact with a specific V(beta) element of TCR. Staphylococcal enterotoxin B (SEB, a superantigen), a primary cause of food poisoning, is also responsible for a significant percentage of non-menstrual associated toxic shock syndrome in patients with a variety of staphylococcal infections. Structural studies have elucidated a binding cavity on the toxin molecule essential for TCR binding. To understand the crucial residues involved in binding, mutagenesis analysis was performed. Our analysis suggest that mutation of a conserved residue Thr(112) to Ser (T112S) in the binding cavity induces a selective reduction in the affinity for binding one TCR V(beta) family and can be attributed to the structural differences in the native and mutant toxins. We present a detailed comparison of the mutant structure determined at 2.0 A with the previously reported native SEB and SEB-TCR V(beta) complex structures.     

The ATPase domain of SecA can form a tetramer in solution.

Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)=[monomer](4)/[tetramer]) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore.     

Crystal structure analysis,covalent docking,and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3‐hydroxybutyrate) depolymerase were identified in two high‐resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281–295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3‐hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281–295 in comparison to the apo (substrate‐free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281–295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351–1361. © 2017 Wiley Periodicals, Inc.     

Zinc binding in peptide models of angiotensin-I converting enzyme active sites studied through 1H-NMR and chemical shift perturbation mapping

We report the design and synthesis through solid phase 9-flourenylmethoxycarbonyl (Fmoc) chemistry of the two angiotensin-I converting enzyme active sites possessing the general sequence HEMGHX(23)EAIGDX(3). Their zinc-binding properties were monitored in solution through high-resolution (1)H-NMR. The obtained data were analyzed in terms of chemical shift differences. The results indicate that zinc binds to the HEMGH and the EAIGD characteristic motifs, and suggest possible coordination modes of zinc in the native enzyme.     

The crystal structure of human angiogenin in complex with an antitumor neutralizing antibody

The murine monoclonal antibody 26-2F neutralizes the angiogenic and ribonucleolytic activities of human angiogenin (ANG) and is highly effective in preventing the establishment and metastatic dissemination of human tumors in athymic mice. Here we report a 2.0 A resolution crystal structure for the complex of ANG with the Fab fragment of 26-2F that reveals the detailed interactions between ANG and the complementarity-determining regions (CDRs) of the antibody. Surprisingly, Fab binding induces a dramatic conformational change in the cell binding region of ANG at the opposite end of the molecule from the combining site; crosslinking experiments indicate that this rearrangement also occurs in solution. The ANG-Fab complex structure should be invaluable for designing maximally humanized versions of 26-2F for potential clinical use.     

Preprotein-controlled catalysis in the helicase motor of SecA

The cornerstone of the functionality of almost all motor proteins is the regulation of their activity by binding interactions with their respective substrates. In most cases, the underlying mechanism of this regulation remains unknown. Here, we reveal a novel mechanism used by secretory preproteins to control the catalytic cycle of the helicase 'DEAD' motor of SecA, the preprotein translocase ATPase. The central feature of this mechanism is a highly conserved salt-bridge, Gate1, that controls the opening/closure of the nucleotide cleft. Gate1 regulates the propagation of binding signal generated at the Preprotein Binding Domain to the nucleotide cleft, thus allowing the physical coupling of preprotein binding and release to the ATPase cycle. This relay mechanism is at play only after SecA has been previously 'primed' by binding to SecYEG, the transmembrane protein-conducting channel. The Gate1-controlled relay mechanism is essential for protein translocase catalysis and may be common in helicase motors.     

Comparative proteomic analysis of hypertrophic chondrocytes in osteoarthritis

Background

Osteoarthritis (OA) is a multi-factorial disease leading progressively to loss of articular cartilage and subsequently to loss of joint function. While hypertrophy of chondrocytes is a physiological process implicated in the longitudinal growth of long bones, hypertrophy-like alterations in chondrocytes play a major role in OA. We performed a quantitative proteomic analysis in osteoarthritic and normal chondrocytes followed by functional analyses to investigate proteome changes and molecular pathways involved in OA pathogenesis.

Methods

Chondrocytes were isolated from articular cartilage of ten patients with primary OA undergoing knee replacement surgery and six normal donors undergoing fracture repair surgery without history of joint disease and no OA clinical manifestations. We analyzed the proteome of chondrocytes using high resolution mass spectrometry and quantified it by label-free quantification and western blot analysis. We also used WebGestalt, a web-based enrichment tool for the functional annotation and pathway analysis of the differentially synthesized proteins, using the Wikipathways database. ClueGO, a Cytoscape plug-in, is also used to compare groups of proteins and to visualize the functionally organized Gene Ontology (GO) terms and pathways in the form of dynamical network structures.

Results

The proteomic analysis led to the identification of a total of ~2400 proteins. 269 of them showed differential synthesis levels between the two groups. Using functional annotation, we found that proteins belonging to pathways associated with regulation of the actin cytoskeleton, EGF/EGFR, TGF-β, MAPK signaling, integrin-mediated cell adhesion, and lipid metabolism were significantly enriched in the OA samples (p ≤10⁻⁵). We also observed that the proteins GSTP1, PLS3, MYOF, HSD17B12, PRDX2, APCS, PLA2G2A SERPINH1/HSP47 and MVP, show distinct synthesis levels, characteristic for OA or control chondrocytes.

Conclusion

In this study we compared the quantitative changes in proteins synthesized in osteoarthritic compared to normal chondrocytes. We identified several pathways and proteins to be associated with OA chondrocytes. This study provides evidence for further testing on the molecular mechanism of the disease and also propose proteins as candidate markers of OA chondrocyte phenotype.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9085-6) contains supplementary material, which is available to authorized users.     

Identification of the preprotein binding domain of SecA

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.     

SecA: a tale of two protomers

Bacteria, Archaea and Eukaryotes have evolved a plethora of mechanisms to translocate proteins across their various membranes. The bacterial Sec pathway is ubiquitous and essential for cell viability and is used by most proteins destined for the inner membrane, the periplasm or beyond. In bacteria, Sec system components include the heterotrimers SecY/SecE/SecG and SecD/SecF/YajC and the peripherally associated ATPase motor SecA. SecA in solution is mainly dimeric. Unexpectedly, structures of SecA dimers from different or even the same bacterium do not have a consistent dimerization interface. Analysis of the functional assembled translocase complexes blurs the picture even further as the functional quaternary state of the SecYEG channel is also disputed. Several experimental approaches tried to define the oligomeric state of SecA during preprotein ‘pushing’ through SecYEG. One high‐resolution SecA–SecYEG complex has been visualized. This snapshot might be a step closer to the actual translocating machinery. Nevertheless, because of the use of detergent, the true quartenary state of the translocase might have been disturbed. Hence, even after this and other studies, several issues remain puzzling. New approaches must be combined with current tools to gain insight into the functionally relevant quartenary states of SecA and SecYEG during preprotein translocation.