[Show abstract][Hide abstract] ABSTRACT: CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1–Csm5) with an uneven stoichiometry and a single crRNA of variable size (35–53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
[Show abstract][Hide abstract] ABSTRACT: The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation.
[Show abstract][Hide abstract] ABSTRACT: Taking chicken Ovalbumin as a prototypical example of a eukaryotic protein we use high-resolution native electrospray ionization mass spectrometry on a modified Exactive Orbitrap mass analyzer to dissect qualitatively and semi-quantitatively 59 proteoforms in the natural protein. This variety is largely induced by the presence of multiple phosphorylation sites, and a glycosylation site that we find to be occupied by at least 45 different glycan structures. Mass analysis of the intact protein in its native state is straightforward and fast, requires very little sample preparation, and provides a direct view on the stoichiometry of all different co-appearing modifications that are distinguishable in mass. As such this proof-of-principal analysis shows that native electrospray ionization mass spectrometry in combination with an Orbitrap mass analyzer offers a means to characterize proteins in a manner highly complementary to standard bottom-up shot-gun proteome analysis.
[Show abstract][Hide abstract] ABSTRACT: Electrospray ionization coupled to native mass spectrometry (MS) has evolved into an important tool in structural biology to decipher the composition of protein complexes. However, the mass analysis of heterogeneous protein assemblies is hampered because of their overlapping charge state distributions, fine structure, and peak broadening. To facilitate the mass analysis, it is of importance to automate pre-processing raw mass spectra, assigning ion series to peaks and deciphering the subunit compositions. So far, the automation of pre-processing raw mass spectra has not been accomplished; Massign was introduced to simplify data analysis and decipher the subunit compositions. In this study, we develop a search engine, AutoMass, to automatically assign ion series to peaks without any additional user input, e.g. limited ranges of charge states or ion mass. AutoMass includes an ion intensity-dependent method to check for Gaussian distributions of ion series and an ion intensity-independent method to address highly overlapping and non-Gaussian distributions. The minimax theorem from Game theory is adopted to define the boundaries. With AutoMass, the boundaries of ion series in the well-resolved tandem mass spectra of Hepatitis B virus (HBV) capsids and those of mass spectrum from CRISPR-related cascade protein complex are accurately assigned. Theoretical and experimental HBV ion masses are shown agreement up to ~ 0.03%. The analysis is finished within a minute on a regular work station. Moreover, less well-resolved mass spectra, e.g. complicated monomer/dimer mass spectra and norovirus capsid mass spectra at different levels of desolvation are analyzed and the boundaries can be defined. In sum, this first-ever fully automatic program reveals the boundaries of overlapping ion peak series and can further aid developing high throughput native MS and top-down proteomics.
[Show abstract][Hide abstract] ABSTRACT: The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.
[Show abstract][Hide abstract] ABSTRACT: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed
in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H
monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The
fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each
subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions.
Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized
chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation
and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted
strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
In this work we present a detailed study on the influence of surface modifications for luminescent silver (Ag) clusters. Ag clusters (25 atoms) capped with dihydrolipoic acid show a distinct absorbance spectrum with several sharp transitions, and relative broad deep red luminescence with a quantum yield of 5% combined with a remarkably long luminescence lifetime of ~3 μs at room temperature. Both pH and the presence of coordinating ligands influence the absorbance spectra and fluorescence intensity. A strong increase in luminescence intensity up to 45% quantum yield could be induced by coordination with PEG ligands.
the surface coordination of the Ag clusters strongly influences the optical properties.
[Show abstract][Hide abstract] ABSTRACT: Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.
PLoS ONE 09/2012; 7(9):e46499. DOI:10.1371/journal.pone.0046499 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/ CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy and small angle X-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA (crRNA). Recently, a cryo-electron microscopy structure of Cascade revealed that the crRNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry (IMMS) to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.
[Show abstract][Hide abstract] ABSTRACT: Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.
[Show abstract][Hide abstract] ABSTRACT: The chromosome of the pathogenic Gram-positive bacterium Streptococcus pneumoniae contains between six to 10 operons encoding toxin-antitoxin systems (TAS). TAS are widespread and redundant in bacteria and archaea and their role, albeit still obscure, may be related to important aspects of bacteria lifestyle like response to stress. One of the most abundant TAS is the relBE family, being present in the chromosome of many bacteria and archaea. Because of the high rates of morbility and mortality caused by S. pneumoniae, it has been interesting to gain knowledge on the pneumococcal TAS, among them the RelBE2Spn proteins. Here, we have analyzed the DNA binding capacity of the RelB2Spn antitoxin and the RelB2Spn-RelE2Spn proteins by band-shift assays. Thus, a DNA region encompassing the operator region of the proteins was identified. In addition, we have used analytical ultracentrifugation and native mass spectrometry to measure the oligomerization state of the antitoxin alone and the RelBE2Spn complex in solution bound or unbound to its DNA substrate. Using native mass spectrometry allowed us to unambiguously determine the stoichiometry of the RelB2Spn and of the RelBE2Spn complex alone or associated to its DNA target.
Proteins Structure Function and Bioinformatics 07/2012; 80(7):1834-46. DOI:10.1002/prot.24081 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study.
[Show abstract][Hide abstract] ABSTRACT: A full-scale remediation facility including a detention basin and a wetland was tested for retention of heavy metals and Poly-Aromatic Hydrocarbons (PAHs) from water drained from a motorway in The Netherlands. The facility consisted of a detention basin, a vertical-flow reed bed and a final groundwater infiltration bed. Water samples were taken of road water, detention basin influent and wetland effluent. By using automated sampling, we were able to obtain reliable concentration averages per 4-week period during 18 months. The system retained the PAHs very well, with retention efficiencies of 90-95%. While environmental standards for these substances were surpassed in the road water, this was never the case after passage through the system. For the metals the situation was more complicated. All metals studied (Cu, Zn, Pb, Cd and Ni) had concentrations frequently surpassing environmental standards in the road water. After passage through the system, most metal concentrations were lower than the standards, except for Cu and Zn. There was a dramatic effect of de-icing salts on the concentrations of Cu, Zn, Cd and Ni, in the effluent leaving the system. For Cu, the concentrations even became higher than they had ever been in the road water. It is advised to let the road water bypass the facility during de-icing periods.
[Show abstract][Hide abstract] ABSTRACT: We report an efficient strategy to conjugate methacrylamide moieties to the lysine units of lysozyme for co-polymerization and subsequent triggered release from hydrogels. Two novel linker molecules, containing an ester bond and/or a disulfide bond for temporary immobilization, were synthesized and conjugated to lysozyme. Lysozyme was successfully modified with on average 2.5 linker molecules per protein molecule, as evidenced by MALDI-TOF and by titration of the free amine groups, while spectral analysis verified the preservation of the protein structure. Next, methacrylated dextran (Dex-MA) was polymerized in presence of native or modified lysozyme to yield hydrogels. The release of native and modified lysozyme from Dex-MA hydrogels was studied in acetate buffer (pH 5, in absence of any trigger) and only a minor fraction (~15%) of the modified lysozyme was released, whereas ~74% of the native lysozyme was released. This indicates successful immobilization of the majority of the modified lysozyme in the hydrogel network. Upon hydrolysis of the ester bonds or incubation with glutathione to reduce disulfide bonds of the linker molecules that conjugate the lysozyme to the gel network, the modified lysozyme was mobilized and released from the hydrogel to the same extent as native lysozyme. These data were confirmed by fluorescence recovery after photobleaching experiments. This approach appeared to be highly interesting for temporary immobilization and subsequent glutathione triggered intracellular delivery of proteins from hydrogels.
[Show abstract][Hide abstract] ABSTRACT: To investigate the in vitro release of octreotide acetate, a somatostatin agonist, from microspheres based on a hydrophilic polyester, poly(D,L-lactide-co-hydroxymethyl glycolide) (PLHMGA).
Spherical and non-porous octreotide-loaded PLHMGA microspheres (12 to 16 μm) and loading efficiency of 60-70% were prepared by a solvent evaporation. Octreotide release profiles were compared with commercial PLGA formulation (Sandostatin LAR(®)); possible peptide modification with lactic, glycolic and hydroxymethyl glycolic acid units was monitored.
PLHMGA microspheres showed burst release (~20%) followed by sustained release for 20-60 days, depending on the hydrophilicity of the polymer. Percentage of released loaded peptide was high (70-90%); > 60% of released peptide was native octreotide. PLGA microspheres did not show peptide release for the first 10 days, after which it was released in a sustained manner over the next 90 days; > 75% of released peptides were acylated adducts.
PLHMGA microspheres are promising controlled systems for peptides with excellent control over release kinetics. Moreover, substantially less peptide modification occurred in PLHMGA than in PLGA microspheres.
Pharmaceutical Research 07/2011; 29(1):110-20. DOI:10.1007/s11095-011-0517-3 · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.
Nucleic Acids Research 07/2011; 39(18):8052-64. DOI:10.1093/nar/gkr498 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.
Proceedings of the National Academy of Sciences 06/2011; 108(25):10092-7. DOI:10.1073/pnas.1102716108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.
[Show abstract][Hide abstract] ABSTRACT: An efficient strategy is reported to introduce methacrylamide groups on the lysine residues of a model protein (lysozyme) for immobilization and triggered release from a hydrogel network. A novel spacer unit was designed, containing a disulfide bond, such that the release of the protein can be triggered by reduction. The modified proteins were characterized by MALDI-TOF MS, titration of free NH(2) residues and spectral analysis. The modification reaction is well controlled, and the number of introduced functions can be tailored by changing the reaction conditions. Gel electrophoresis experiments showed that the methacrylamide modified protein can be immobilized in a polyacrylamide hydrogel and subsequently released by reduction of the spacer by which the protein was grafted to the polymeric network.