Search

Native electrospray ionization mass spectrometry (ESI-MS) has emerged as a potent tool for examining the native-like structures of macromolecular complexes. Despite its utility, the predominant “buffer” used, ammonium acetate (AmAc) with pKavalues of 4.75 for acetic acid and 9.25 for ammonium, provides very little buffering capacity within the physiological pH range of 7.0–7.4. ESI-induced redox reactions alter the pH of the liquid within the ESI capillary. This can result in protein unfolding or weakening of pH-sensitive interactions. Consequently, the discovery of volatile, ESI-compatible buffers, capable of effectively maintaining pH within a physiological range, is of high importance. Here, we demonstrate that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) offer buffering capacity at physiological pH where AmAc falls short, with pKavalues of 7.2 and 5.5 for the conjugate acids of DFEA and TFEA, respectively. Native ESI-MS experiments on model proteins cytochrome cand myoglobin electrosprayed with DFEA and TFEA demonstrated the preservation of noncovalent protein–ligand complexes in the gas phase. Protein stability assays and collision-induced unfolding experiments further showed that neither DFEA nor TFEA destabilized model proteins in solution or in the gas phase. Finally, we demonstrate that multisubunit protein complexes such as alcohol dehydrogenase and concanavalin A can be studied in the presence of DFEA or TFEA using native ESI-MS. Our findings establish DFEA and TFEA as new ESI-compatible neutral pH buffers that promise to bolster the use of native ESI-MS for the analysis of macromolecular complexes, particularly those sensitive to pH fluctuations.

ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the treatment of cancer and bacterial infections due to their critical role in mitochondrial and bacterial proteostasis, respectively. As such, there is significant interest in understanding structure–function relationships in this protein family. ClpPs are known to crystallize in extended, compact, and compressed forms; however, it is unclear what conditions favor the formation of each form and whether they are populated by wild-type enzymes in solution. Here, we use cryo-EM and solution NMR spectroscopy to demonstrate that a pH-dependent conformational switch controls an equilibrium between the active extended and inactive compressed forms of ClpP from the Gram-negative pathogen Neisseria meningitidis. Our findings provide insight into how ClpPs exploit their rugged energy landscapes to enable key conformational changes that regulate their function.

ClpXP, a serine protease located in the mitochondrial matrix, regulates mitochondrial proteostasis by degrading damaged or aggregated proteins, including respiratory chain subunits. ClpXP is a bipartite protein complex comprised of the ClpX regulatory particle that caps each end of the ClpP degradation chamber. ClpX recognizes substrates, unfolds them, and feeds them into ClpP for proteolysis. Genetic or chemical inhibition of ClpXP leads to impaired oxidative phosphorylation function and leukemic cell death in vitroand in vivo. Importantly, however, the degradation marker of human ClpXP remains elusive. Since the bacterial homologue of ClpXP recognizes phosphorylated arginine (pArg)-tagged protein for degradation, we hypothesized that human ClpXP might also recognize phosphorylated amino acids as degrons.

Detectable warning surface (DWS) is a panel product installed at the edge of curb ramps to warn visually impaired pedestrians about the proximity of the roadway. Most DWS products are made of polymers and are subjected to outdoor weathering. Therefore, sunlight degradation is an important factor that affects their service life. In this study, the effects of sunlight on material degradation in four DWS products made from polyester, neopentylglycol (NPG), polyurethane, and polyolefin were evaluated by exposing DWS test coupons in a xenon weatherometer. The exposure conditions were largely based on ASTM D 2565 for a duration of 3,000 h. Color change induced by radiation was measured using a spectro-colorimeter. The fastest discoloration was measured in DWS made from polyurethane. The largest color change occurred between 500 and 1,000 h for polyurethane and between 1,000 and 3,000 h for other polymers. The changes of surface appearance observed under a digital microscope were consistent with the discoloration. The reinforcing fibers in DWS products made from polyester and polyurethane were revealed after 3,000 h exposure. Decrease in surface wear resistance was obtained in tested coupons that were exposed to the highest irradiance level. The product made from NPG exhibited the greatest decrease in wear resistance, while that made from polyolefin showed the least change.

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3β3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coliF1-ATPase by hydrogen–deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get “stuck” (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional “greasy bearing” model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20–265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266–432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a “dimer of dimers” quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

The objective of this study was to examine if asphalt rejuvenators can offset the stiffness attributed by the hardened binder from reclaimed asphalt pavement (RAP) and reclaimed asphalt shingles (RAS) in mixtures that incorporate high RAP and RAS content without adverse impact on the performance of the mixtures. Also, to assess, if rejuvenators can help the hardened binder from the RAP/RAS comingle with the virgin binder. Overall, the results showed that asphalt rejuvenators can mitigate the stiffness of the resultant binder. The cracking characteristics of the mixture improved by the addition of the rejuvenators, however, the rutting and moisture susceptibility were adversely impacted at the dosage and the testing conditions used. Also, the tests results at 4°C generally showed that there was blending of the rejuvenated and virgin binder, however, no conclusion could be made at the higher temperatures.

The mitochondrial serine protease, ClpXP, regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in a subset of AML and inhibiting or hyperactivating it kills leukemic cells and stem cells in vitroand in vivo. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation.

Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; UHN: Patents & Royalties.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.