Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2)

Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the alpha 7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with alpha 7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an alpha 7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule

The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H₂: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity.</p> <p>Results</p> <p>Here we report the identification of a new H₂: benzyl viologen oxidoreductase enzyme activity in <it>E. coli </it>that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of <it>E. coli </it>mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes.</p> <p>Conclusions</p> <p>The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of <it>Escherichia coli </it>have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.</p

Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded

Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry

Abstract Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-ProHyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of ±10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results

Free radical-initiated peptide sequencing (FRIPS)-based cross-linkers for improved peptide and protein structure analysis

Free radical-initiated peptide sequencing (FRIPS) has recently been introduced as an analytical strategy to create peptide radical ions in a predictable and effective way by collisional activation of specifically modified peptides ions. FRIPS is based on the unimolecular dissociation of open-shell ions and yields fragments that resemble those obtained by electron capture dissociation (ECD) or electron transfer dissociation (ETD). In this review article, we describe the fundamentals of FRIPS and highlight its fruitful combination with chemical cross-linking/mass spectrometry (MS) as a highly promising option to derive complementary structural information of peptides and proteins. FRIPS does not only yield exhaustive sequence information of cross-linked peptides, but also defines the exact cross-linking sites of the connected peptides. The development of more advanced FRIPS cross-linkers that extend the FRIPS-based cross-linking/MS approach to the study of large protein assemblies and protein interaction networks can be eagerly anticipated

Novel Concepts of MS-Cleavable Cross-linkers for Improved Peptide Structure Analysis

The chemical cross-linking/mass spectrometry (MS) approach is gaining increasing importance as an alternative method for studying protein conformation and for deciphering protein interaction networks. This study is part of our ongoing efforts to develop innovative cross-linking principles for a facile and efficient assignment of cross-linked products. We evaluate two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for automated analysis of cross-linked products. We introduce the bromine phenylurea (BrPU) linker that possesses a unique structure yielding a distinctive fragmentation pattern on collisional activation. Moreover, BrPU delivers the characteristic bromine isotope pattern and mass defect for all cross-linker-decorated fragments. We compare the fragmentation behavior of the BrPU linker with that of our previously described MS-cleavable TEMPO-Bz linker (which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy moiety connected to a benzyl group) that was developed to perform free-radical-initiated peptide sequencing. Comparative collisional activation experiments (collision-induced dissociation and higher-energy collision-induced dissociation) with both cross-linkers were conducted in negative electrospray ionization mode with an Orbitrap Fusion mass spectrometer using five model peptides. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the loss of an intact peptide from the cross-linked products. As the BrPU linker combines a characteristic mass shift with an isotope signature, it presents a more favorable combination for automated assignment of cross-linked products compared with the TEMPO-Bz linker