Frank Sobott

Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study.

The efficiency of heterologous protein production in Escherichia coli (E. coli) can be diminished by biased codon usage. Approaches normally used to overcome this problem include targeted mutagenesis to remove rare codons or the addition of rare codon tRNAs in specific cell lines. Recently, improvements in technology have enabled cost-effective production of synthetic genes, making this a feasible alternative. To explore this option, the expression patterns in E. coli of 30 human short-chain dehydrogenase/reductase genes (SDRs) were analyzed in three independent experiments, comparing the native and synthetic (codon-optimized) versions of each gene. The constructs were prepared in a pET-derived vector that appends an N-terminal polyhistidine tag to the protein; expression was induced using IPTG and soluble proteins were isolated by Ni-NTA metal-affinity chromatography. Expression of the native and synthetic gene constructs was compared in two isogenic bacterial strains, one of which contained a plasmid (pRARE2) that carries seven tRNAs recognizing rare codons. Although we found some degree of variability between experiments, in normal E. coli synthetic genes could be expressed and purified more readily than the native version. In only one case was native gene expression better. Importantly, in most but not all cases, expression of the native genes in combination with rare codon tRNAs mimicked the behavior of the synthetic genes in the native strain. The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.

Structural Basis for Protein-Protein Interactions in the 14-3-3 Protein Family.

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein–protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.

Protein complexes gain momentum

The greatest challenge to structural biologists in the post-genomic era is to decipher both stable and transient interactions of protein complexes. In response to this challenge, significant advances in mass spectrometry have been made in the past two years. With the inception of novel approaches targeted at defining interaction partners, stoichiometry and topology, as well as monitoring real-time reactions, mass spectrometry is well placed to contribute to the understanding of dynamic macromolecular complexes.

Native ion mobility-mass spectrometry and related methods in structural biology☆

Mass spectrometry-based methods have become increasingly important in structural biology - in particular for large and dynamic, even heterogeneous assemblies of biomolecules. Native electrospray ionization coupled to ion mobility-mass spectrometry provides access to stoichiometry, size and architecture of noncovalent assemblies; while non-native approaches such as covalent labeling and H/D exchange can highlight dynamic details of protein structures and capture intermediate states. In this overview article we will describe these methods and highlight some recent applications for proteins and protein complexes, with particular emphasis on native MS analysis. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Protomers of Benzocaine: Solvent and Permittivity Dependence

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid that finds an application as a local anesthetic, is found to adopt in its protonated form at least two populations of distinct structures in the gas phase, and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization process. Here, we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry to yield gas-phase IR spectra of simultaneously m/z and drift-time-resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species: the N- and O-protonated forms. Density functional theory calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution-phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation have been suggested.

Structural and Functional Characterization of the Human Protein Kinase ASK1

Summary Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (Kd ∼ 0.2 μM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.

The flight of macromolecular complexes in a mass spectrometer

The discovery that conditions can be found such that non–covalent macromolecular complexes can survive the transition from solution to gas phase and remain intact during their flight in a mass spectrometer is an intriguing observation. While the nature of the interaction between the components, either ionic, hydrophobic or van der Waals, undoubtedly has an effect on the stability of these gas phase species, the role of small molecules in conferring additional stability is often overlooked. Here we review historical aspects of the development of mass spectrometry for macromolecular complexes with particular focus on the role of small molecules in stabilizing gas–phase complexes. Moreover, we demonstrate how the dissociation of small molecules from subunits within a macromolecular complex can be used to probe the topological arrangement. Overall, therefore, we show that mass spectrometry used in this way is capable of addressing features of the energy landscape not readily accessed by traditional structural biology approaches.

Contactin-associated protein-2 antibodies in non-paraneoplastic cerebellar ataxia

Background Relatively few studies have searched for potentially pathogenic antibodies in non-paraneoplastic patients with cerebellar ataxia. Methods and Results We first screened sera from 52 idiopathic ataxia patients for binding of serum IgG antibodies to cerebellar neurons. One strong-binding serum was selected for immunoprecipitation and mass spectrometry, which resulted in the identification of contactin-associated protein 2 (CASPR2) as a major antigen. CASPR2 antibodies were then found by a cell-based assay in 9/88 (10%) ataxia patients, compared to 3/144 (2%) multiple sclerosis or dementia controls (p=0.011). CASPR2 is strongly expressed in the cerebellum, only partly in association with voltage-gated potassium channels. Conclusions Prospective studies are now needed to see whether identification of CASPR2 antibodies has relevance for the diagnosis and treatment of idiopathic cerebellar ataxia.

Ion Mobility Mass Spectrometry for Extracting Spectra of N-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact, Folded HIV gp120

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

Comparison of CID Versus ETD Based MS/MS Fragmentation for the Analysis of Protein Ubiquitination

Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N(ε) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular z+1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins.