Concurrent Scientific Session (Mass Spectrometry): Mass Spectrometry Guided Structural Biology
Measuring the stoichiometry of antimicrobial peptides in nanodiscs with native mass spectrometry
Antimicrobial peptides (AMPs) and other membrane active compounds attack pathogens by targeting the lipid bilayer rather than specific proteins, which may have advantages in combating drug resistance. However, the mechanisms of AMP toxicity and selectivity are poorly understood due to challenges in measuring polydisperse interactions of AMPs within lipid environments. Native or noncovalent mass spectrometry (MS) has recently made significant strides in characterizing the stoichiometry of polydisperse protein complexes and membrane protein interactions. However, conventional detergent-based native MS approaches are unsuitable for AMPs because lipid bilayers are essential for complex assembly. Here, we employ lipoprotein nanodiscs as a membrane mimetic to assemble AMP complexes for native MS. The nanodiscs are ionized under non-denaturing conditions to introduce the entire nanodisc complex with embedded AMP into the mass spectrometer. By precisely measuring the mass of the intact nanodisc complexes, we can determine the oligomeric state of the AMP without disrupting the lipid bilayer. As a positive control, we observed that gramicidin A forms dimers exclusively, although multiple dimers can be inserted into the nanodisc with sufficient concentration. Melittin, in contrast, shows no specific complex formation. LL-37 has a more complex equilibrium that seems to show partial specificity for dimer and hexamer complexes. Each of these three AMPs shows different levels of association depending on the types of lipids in the nanodisc. Ultimately, our goal is that these novel measurements of AMP complex stoichiometries will shed light on the structural biology of AMP complex formation and the mechanisms of their selectivity and toxicity.
Native mass spectrometry for the study of membrane proteins
Membrane proteins are essential to mediate the traffic of solutes in and out of the cell and in translating extracellular stimuli into function. Membrane proteins can be challenging to structurally characterize with traditional techniques, due to their low expression yields and difficulty in producing diffraction quality crystals. Here we apply native mass spectrometry to structurally characterize the membrane protein Aquaporin-0 (AQP0), which exists as a tetrameric homooligomer and is found in the eye lens.
Bovine and rabbit AQP0 was isolated and purified from lens tissue, specifically from both the cortex and the nucleus. Samples were prepared in 200 mM ammonium acetate containing twice the critical micelle concentration of the detergent tetraethylene glycol monooctyl ether (C8E4), and analyzed at 1-7 μM. Experiments were performed on either an in-house modified Thermo Exactive Plus EMR (modified to include a quadrupole and SID device) or a Thermo Q Exactive UHMR.
AQP0 is the most abundant membrane protein in the eye lens, comprising >50% of the membrane protein content. Interestingly, there is no protein turnover in lens fiber cells and, therefore, age-related modifications accumulate with time and are likely related to age-related cataract formation. We have isolated AQP0 directly from two distinct regions of eye lens: the cortex which contains “young” fiber cells and the nucleus which contains older fiber cells.
We can introduce the tetramer intact into the gas-phase and can observe different proteoforms from the tetramer including singly and doubly phosphorylated tetramer and truncated species. In addition, we can observe non-covalent lipid binding. CID provides further insight into the proteoforms present at the monomer level and SID provides information on subunit connectivity.
Native MS can provide insight into the structure of membrane protein complexes isolated from eye lens tissue, and provides information on the proteoforms present.
Navigating the strait between efficiency and quality in a mass spectrometry facility
Mass spectrometry continues to advance at a rapid pace. Groundbreaking new techniques seem to appear nearly every month that deepen our impact on biology and bring customers flocking to core labs with new papers and equally new expectations in hand. As a consequence, the goalpost for maintaining "quality" can seem like one that is constantly moving away from the efficiency of established workflows. How can mass spectrometry facilities maintain the throughput required for cost recovery while still meeting today's new definition of "quality"?
Quantifying the Dynamics of L-Kynureninase Orthologs during Catalysis using HDX-MS
The breakdown of tryptophan in eukaryotes and some bacteria can proceed via a L-Kynurenine (Kyn) intermediate. This intermediate has three potential fates; it can be converted to kynurenic acid, hydroxylated to 3-hydroxyl-kynurenine (3OH-Kyn), or hydrolyzed to L-anthranillic acid and alanine. L-Kynureninase (Kynase) is the aminotransferase enzyme responsible for hydrolysis of either Kyn or 3OH-Kyn. Kynase functions as a homodimer, with one chain contributing an essential pyridoxal-5’-phosphate cofactor, and the other chain contributing three catalytic residues. High-resolution structural studies show that while human and bacterial Kynase orthologs are structurally similar, they have distinct catalytic residues. Enzyme studies also identify different substrate specificities, with the human ortholog preferring 3OH-Kyn and the bacterial ortholog preferring Kyn. The basis for this preference is beyond the catalytic residues, as simply switching active sites between orthologs fails to switch substrate preference. The goal of this study was to characterize the dynamics of Kynase enzymes during catalysis. To do this, we have used hydrogen/deuterium exchange coupled to mass spectrometry. Through a substantial series of experiments, we have compared the dynamics of several Kynase orthologs and engineered Kynase mutants. Data sets were collected for enzymes without substrate, as well as with Kyn and 3OH-Kyn. The results identify the determinants of substrate specificity and provide insight into mechanisms of catalysis. This information is invaluable in the design of Kynase-based enzyme therapeutics, as well as our general understanding of enzyme dynamics.