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A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1-13) potently inhibits the Nln activity while dynorphin A (1-8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln., (© 2024. The Author(s).)

Electrospray ionization (ESI) mass spectrometry is widely used for interrogating peptides, proteins, and other biomolecular analytes. A growing number of laboratories use molecular dynamics (MD) simulations for uncovering ESI mechanisms by modeling the behavior of highly charged nanodroplets. The outcome of any MD simulation depends on certain assumptions and parameter settings, and it is desirable to optimize these factors by benchmarking computational data against experiments. Unfortunately, benchmarking of ESI simulations is difficult because experimentally generated gaseous ions do not generally retain any features that would reveal their formation pathway [e.g., the charged residue mechanism (CRM) or the ion evaporation mechanism (IEM)]. Here, we tackle this problem by examining the effects of various MD settings on the ESI behavior of the 9-residue peptide bradykinin in acidic aqueous droplets. Several parameters were found to significantly affect the kinetic competition between peptide IEM and CRM. By systematically probing the droplet behavior, we uncovered problems associated with certain settings, including peptide/solvent temperature imbalances, unexpected peptide deceleration during IEM, and a dependence of the ESI mechanism on the water model. We also noted different simulation outcomes for different force fields. On the basis of comprehensive tests, we propose a set of "best practice" parameter settings for MD simulations of ESI droplets. The strategies used here should be transferable to other types of droplet simulations, paving the way toward a more solid understanding of ESI mechanisms.

This study explores the innovative field of pulsed direct current arc-induced nanoelectrospray ionization mass spectrometry (DCAI-nano-ESI-MS), which utilizes a low-temperature direct current (DC) arc to induce ESI during MS analyses. By employing a 15 kV output voltage, the DCAI-nano-ESI source effectively identifies various biological molecules, including angiotensin II, bradykinin, cytochrome C, and soybean lecithin, showcasing impressive analyte signals and facilitating multicharge MS in positive- and negative-ion modes. Notably, results show that the oxidation of fatty acids using a DC arc produces [M + O - H] - ions, which aid in identifying the location of C═C bonds in unsaturated fatty acids and distinguishing between isomers based on diagnostic ions observed during collision-induced dissociation tandem MS. This study presents an approach for identifying the sn -1 and sn -2 positions in phosphatidylcholine using phosphatidylcholine and nitrate adduct ions, accurately determining phosphatidylcholine molecular configurations via the Paternò-Büchi reaction. With all the advantages above, DCAI-nano-ESI holds significant promise for future analytical and bioanalytical applications.