To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).protein complex | native mass spectrometry | protein interactions | structural biology | surface-induced dissociation N ative mass spectrometry (MS) has emerged as a powerful structural biology tool. By using "soft" ionization techniques such as nanoelectrospray ionization, noncovalent interactions can be retained, enabling the study of intact protein:protein, protein: ligand, and protein:RNA complexes in the gas phase (1-4). Native MS overcomes many of the barriers associated with traditional protein characterization methods; it requires low sample volumes (3-10 μL) and micromolar or lower concentrations, while also having a broad mass range for analysis, allowing study of small monomeric proteins up to large megadalton assemblies (1,5).Typical MS experiments to study subunit interactions of protein complexes involve first preparing the sample in an aqueous solution at near neutral pH, typically 100-200 mM ammonium acetate. The complex is then introduced intact into the mass spectrometer to measure the mass of the native complex. To obtain subunit connectivity information on the sample, the complex can be disrupted in solution, typically either with small volumes of organic solvent or through alteration of the ionic strength; this destabilizes the protein:protein interfaces and allows measurement of stable subcomplexes (6, 7). This approach, however, targets all species present in solution and can therefore be problematic for heterogeneous samples where it may not be possible to decipher which subcomplex came from which precursor. Alternatively, the complex can be isolated and then dissociated in the gas phase. The most commonly used dissociation method for such studies is collision-induced dissociation (CID). In CID protein ions are accelerated i...
Surface-induced dissociation (SID) is a powerful means of deciphering protein complex quaternary structures due to its capability of yielding dissociation products that reflect the native structures of protein complexes in solution. Here we explore the suitability of SID to locate the ligand binding sites in protein complexes. We studied C-reactive protein (CRP) pentamer, which contains a ligand binding site within each subunit, and cholera toxin B (CTB) pentamer, which contains a ligand binding site between each adjacent subunit. SID dissects ligand-bound CRP into subcomplexes with each subunit carrying predominantly one ligand. In contrast, SID of ligandbound CTB results in the generation of subcomplexes with a ligand distribution reflective of two subunits contributing to each ligand binding site. SID thus has potential application in localizing sites of small ligand binding for multisubunit protein-ligand complexes.
Paper spray (PS) ionization, an ambient ionization method, has previously been explored as a direct and fast method for mass spectrometric analysis of complex mixtures. It has been applied to the analysis of a wide variety of compounds, mostly small molecules. The work reported here extends the application of PS ionization to noncovalent protein complexes on an ion mobility tandem mass spectrometer. Similar mass spectra for protein complexes were obtained by PS ionization and nanoflow electrospray ionization (nESI), indicating that intact protein complexes can be preserved in PS ionization. In addition, collisional cross sections measured by ion mobility provide evidence that the protein assemblies may remain compact by PS ionization. With PS, it is possible to detect hemoglobin tetramer from a blood sample with minimal sample preparation. This is the first report to show that PS ionization is a promising ionization method for nonconvalent protein complexes.
Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. Graphical Abstract ᅟ.
Summary Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher-order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets.
As hypoxia plays a vital role in the angiogenic-osteogenic coupling, using proline hydroxylase inhibitors to manipulate hypoxia-inducible factors has become a strategy to improve the osteogenic properties of biomaterials. Dimethyloxallyl glycine (DMOG) is a 2-ketoglutarate analog, a small molecular compound that competes for 2-ketoglutaric acid to inhibit proline hydroxylase. In order to improve the osteogenic ability of calcined bone calcium (CBC), a new hypoxia-mimicking scaffold (DMOG/Collagen/CBC) was prepared by immersing it in the DMOG-Collagen solution, followed by freeze-drying. All coated CBC scaffolds retained the inherent natural porous architecture and showed excellent biocompatibility. A slow release of DMOG by the DMOG-loaded CBC scaffolds for up to one week was observed in in vitro experiments. Moreover, the DMOG/Collagen/CBC composite scaffold was found to significantly stimulate bone marrow stromal cells to express osteogenic and angiogenic genes in vitro. In addition, the osteogenic properties of three kinds of scaffolds, raw CBC, Collagen/CBC, and DMOG/Collagen/CBC, were evaluated by histology using the rabbit femoral condyle defect model. Histomorphometric analyses showed that the newly formed bone (BV/TV) in the DMOG/Collagen/CBC group was significantly higher than that of the Collagen/CBC group. However, immunostaining of CD31 and Runx2 expression between these two groups showed no significant difference at this time point. Our results indicate that DMOG-coated CBC can promote osteogenic differentiation and bone healing, and show potential for clinical application in bone tissue engineering.
Currently, no unequivocal evidence is given for elucidation of "black box" during the structural transformations of dynamic crystalline materials. Here, three types of mechanisms are revealed for such transformations through X-ray diffraction and optical microscopy; namely, single-crystal to single-crystal (SC-SC), as well as "core-to-core" and "core-on-shell" processes. As confirmed by time-lapse optical microscopy, the latter two cases can be properly ascribed as partial recrystallization processes, while the former one is a continuous process with two different crystal lattices simultaneously maintained in one single crystal. Interestingly, these three distinct pathways can be exquisitely realized by changing only the halogen substituent (from -F, -Cl, to -Br) of the organic ligands in the coordination supramolecular systems.
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors, however, the matrices and high vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-SorbPC) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B-subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS based on differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin B as a model receptor-ligand pair, the minimal detectable concentration of toxin was estimated to be 4 nM. On-plate trypsin digestion of bound cholera toxin B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.
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