The coronavirus SARS-CoV is the primary cause of the life-threatening severe acute respiratory syndrome (SARS). With the aim of developing therapeutic agents, we have tested peptides derived from the membrane-proximal (HR2) and membrane-distal (HR1) heptad repeat region of the spike protein as inhibitors of SARS-CoV infection of Vero cells. It appeared that HR2 peptides, but not HR1 peptides, were inhibitory. Their efficacy was, however, significantly lower than that of corresponding HR2 peptides of the murine coronavirus mouse hepatitis virus (MHV) in inhibiting MHV infection. Biochemical and electron microscopical analyses showed that, when mixed, SARS-CoV HR1 and HR2 peptides assemble into a six-helix bundle consisting of HR1 as a central triple-stranded coiled coil in association with three HR2 ␣-helices oriented in an antiparallel manner. The stability of this complex, as measured by its resistance to heat dissociation, appeared to be much lower than that of the corresponding MHV complex, which may explain the different inhibitory potencies of the HR2 peptides. Analogous to other class I viral fusion proteins, the six-helix complex supposedly represents a postfusion conformation that is formed after insertion of the fusion peptide, proposed here for coronaviruses to be located immediately upstream of HR1, into the target membrane. The resulting close apposition of fusion peptide and spike transmembrane domain facilitates membrane fusion. The inhibitory potency of the SARS-CoV HR2-peptides provides an attractive basis for the development of a therapeutic drug for SARS.

We modified and optimized a first generation quadrupole time-of-flight (Q-TOF) 1 to perform tandem mass spectrometry on macromolecular protein complexes. The modified instrument allows isolation and subsequent dissociation of high-mass protein complexes through collisions with argon molecules. The modifications of the Q-TOF 1 include the introduction of (1) a flow-restricting sleeve around the first hexapole ion bridge, (2) a low-frequency ion-selecting quadrupole, (3) a high-pressure hexapole collision cell, (4) high-transmission grids in the multicomponent ion lenses, and (5) a low repetition rate pusher. Using these modifications, we demonstrate the experimental isolation of ions up to 12 800 mass-to-charge units and detection of product ions up to 38 150 Da, enabling the investigation of the gas-phase stability, protein complex topology, and quaternary structure of protein complexes. Some of the data reveal a so-far unprecedented new mechanism in gas-phase dissociation of protein oligomers whereby a tetramer complex dissociates into two dimers. These data add to the current debate whether gas-phase structures of protein complexes do retain some of the structural features of the corresponding species in solution. The presented low-cost modifications on a Q-TOF 1 instrument are of interest to everyone working in the fields of macromolecular mass spectrometry and more generic structural biology.

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental. HBV capsids are unusual in that they exhibit two distinct icosahedral geometries, nominally composed of 90 and 120 dimers with masses of Ϸ3 and Ϸ4 MDa, respectively. Here, a mass spectrometric approach was used to determine the masses of both capsids to within 0.1%. It follows that both lattices are complete, consisting of exactly 180 and 240 subunits. Nanoindentation experiments by atomic-force microscopy indicate that both capsids have similar stabilities. The data yielded a Young's modulus of Ϸ0.4 GPa. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.atomic force microscopy ͉ collision-induced dissociation ͉ macromolecular mass spectrometry ͉ virus assembly ͉ viral structural biology H epatitis B virus (HBV) is a major cause of liver disease in humans (1), with Ͼ350 million people suffering from chronic infection. For the development of new antiviral drugs, further insight into the replication cycle and assembly pathway of the virus is needed (2). Moreover, there is a growing interest in HBV and other viral particles as vehicles for drug delivery and as platforms for nanoparticle technology (3). In this context, precise biophysical characterization of these particles represents essential basic information.HBV has an enveloped virion. Single-stranded viral RNA is packaged into the assembling capsid and, within this compartment, is reverse-transcribed into DNA (4, 5). The DNAcontaining nucleocapsid then proceeds to envelopment. Both in vivo and in vitro, the capsid protein (cp) forms icosahedral capsids of two sizes, corresponding to triangulation numbers of T ϭ 3 and T ϭ 4 (6), nominally consisting of 180 and 240 subunits, respectively (7-10). Cp has a 140-residue N-terminal core domain connected to a 34-residue ''protamine domain'' by a 10-residue linker (11). The protamine domain binds RNA, whereas the core domain is necessary and sufficient for capsid assembly. The ratio of T ϭ 3 to T ϭ 4 capsids produced depends on the length of the linker and the conditions of assembly: The smaller T ϭ 3 capsid becomes progressively more abundant as the linker is shortened (12). The building-block for capsid formation is a dimer stabilized via an intermolecular four-helix bundle (13-15) and a disulfide bond within the bundle (Cys61). However, dimerization and assembly also occur in the absence of the disulfide, e.g., when Cys61 is replaced with Ala (10, 12). The capsid has protruding spikes at the dimer interfaces that display most of the antigenic epitopes and holes at the symmetry axes that allow infusion of nucleotides for reverse transcription (7, ...

The structural analysis of macromolecular functional protein assemblies by contemporary high resolution structural biology techniques (such as nuclear magnetic resonance, X-ray crystallography, and electron microscopy) is often still challenging. The potential of a rather new method to generate structural information, native mass spectrometry, in combination with ion mobility mass spectrometry (IM-MS), is highlighted here. IM-MS allows the assessment of gas phase ion collision cross sections of protein complex ions, which can be related to overall shapes/volumes of protein assemblies, and thus be used to monitor changes in structure. Here we applied IM-MS to study several (intermediate) chaperonin complexes that can be present during substrate folding. Our results reveal that the protein assemblies retain their solution phase structural properties in the gas phase, addressing a long-standing issue in mass spectrometry. All IM-MS data on the chaperonins point toward the burial of genuine substrates inside the GroEL cavity being retained in the gas phase. Additionally, the overall dimensions of the ternary complexes between GroEL, a substrate, and cochaperonin were found to be similar to the dimensions of the empty GroEL-GroES complex. We also investigated the effect of reducing the charge, obtained in the electrospray process, of the protein complex on the global shape of the chaperonin. At decreased charge, the protein complex was found to be more compact, possibly occupying a lower number of conformational states, enabling an improved ion mobility separation. Charge state reduction was found not to affect the relative differences observed in collision cross sections for the chaperonin assemblies.

Phosphatidylcholine (PC), a major lipid class in the membranes of eukaryotes, is synthesized either via the triple methylation of phosphatidylethanolamine (PE) or via the CDP-choline route. To investigate whether the two biosynthetic routes contribute differently to the steady-state profile of PC species, i.e., PC molecules with specific acyl chain compositions, the pools of newly synthesized PC species were monitored by labeling Saccharomyces cerevisiae with deuterated precursors of the two routes, (methyl-D3)-methionine and (D13)-choline, respectively. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that the two PC biosynthetic pathways yield different sets of PC species, with the CDP-choline route contributing most to the molecular diversity. Moreover, yeast was shown to be capable of remodeling PC by acyl chain exchange at the sn-1 position of the glycerol backbone. Remodeling was found to be required to generate the steady-state species distribution of PC. This is the first study demonstrating a functional difference between the two biosynthetic routes in yeast.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n 5 67), men without PCa (n 5 76). As a control, urine was collected from men after radical prostatectomy (n 5 13), women (n 5 16) and patients with prostate cancer without digital rectal exam (n 5 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.Prostate-specific antigen (PSA, KLK3) is a protein that is commonly used in daily practice to aid urologists in diagnosing prostate cancer (PCa). Although PSA has a high sensitivity, it lacks specificity and therefore causes unnecessary biopsies. Furthermore, PSA is a poor prognostic marker.1 To increase specificity and distinguish between the clinically insignificant cancers and the ones that are clinically relevant, novel markers have to be identified. Recent studies have shown that extracellular vesicles and particularly the vesicles from endosomal origin, referred to as exosomes, could help us in identifying novel tissue-specific markers. Moreover, also their presence and number might be indicative of disease. 2-4Quantifying the number of exosomes and characterizing them on single particle level remains challenging. To determine the number of exosomes in body fluids or measure an exosomal marker of interest, purification and concentration steps are often needed. Isolation of exosomes is most commonly performed by ultracentrifugation, filtration, precipitation or antibody-based capture technologies. Most of these protocols are still under development, labor intensive and limited with respect to efficient isolation or purity of the final exosomal preparation. 5 Measuring the numbe...

The hepatitis B virus (HBV) is a major cause of liver disease in humans [1] and its non-infectious capsid is of interest for nanotechnology, including for drug-delivery applications. A precise biophysical characterization of these particles is of importance not only for these applications, but also because it may provide further insight into the replication cycle and assembly pathway of the virus, and thus contribute to the future development of drugs. [2,3] The HBV capsid protein (cp) forms icosahedral capsids of two sizes in vivo and in vitro (with triangulation numbers of T = 3 and T = 4 [4] that contain 180 and 240 subunits, respectively [5][6][7][8] ). The capsid protein has two domains-a core domain (amino acids 1-140) and a "protamine domain" (amino acids 150-183)-connected by a 10-residue linker; [9] of these, the core domain is necessary and sufficient for assembly of the capsid. The length of the linker and the conditions under which assembly take place determine the ratio of the T = 3 and T = 4 capsids obtained.

Type I cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) is involved in the nitric oxide/cGMP signaling pathway. PKG has been identified in many different species, ranging from unicelölular organisms to mammals. The enzyme serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses, ranging from smooth-muscle relaxation to neuronal synaptic plasticity. In the absence of a crystal structure, the three-dimensional structure of the homodimeric 152-kDa kinase PKG is unknown; however, there is evidence that the kinase adopts a distinct cGMP-dependent active conformation when compared to the inactive conformation. We performed mass-spectrometry-based hydrogen/deuterium exchange experiments to obtain detailed information on the structural changes in PKG I alpha induced by cGMP activation. Site-specific exchange measurements confirmed that the autoinhibitory domain and the hinge region become more solvent exposed, whereas the cGMP-binding domains become more protected in holo-PKG (dimeric PKG saturated with four cGMP molecules bound). More surprisingly, our data revealed a specific disclosure of the substrate-binding region of holo-PKG, shedding new light into the kinase-activation process of PKG.