Macromolecular Mass Spectrometry Research Articles

Structural and Evolutionary Dynamics Facilitate Ordered Protein Complex Assembly

Most proteins can interact with other proteins to form complexes. Is the dynamic assembly of a protein complex connected to the intrinsic flexibility of its component subunits? Can the order of this assembly process be related to evolutionary changes in quaternary structure? Two methodological advances have allowed us to address these questions. First, we introduced a simple method for predicting the free-state flexibility of protein subunits from the structures of protein complexes based on accessible surface area calculations. Second, we showed that we can predict assembly pathways through analysis of intersubunit interface areas, and validated this method by experimentally characterizing the assembly of several heteromers with macromolecular mass spectrometry. using these new methods, we demonstrate that the intrinsic flexibility of protein subunits is crucial for assembly, as it facilitates the conformational changes that enable both the packing together of multiple distinct subunits and the formation of asymmetric interfaces required for cyclic symmetries. We also find that protein complex subunits have a strong tendency to assemble in an order corresponding to their flexibility and magnitude of conformational change, thus suggesting a possible mechanism for control of ordered assembly. Moreover, we reveal that changes in quaternary structure driven by evolutionary gene fusion events tend to occur in such a way so that they mimic the existing order of subunit assembly. This shows that there is selective pressure in the evolution of protein complexes to conserve assembly pathways. Finally, we observe that evolutionarily more recent subunits tend to be more flexible than more ancient subunits within a complex. Together, these results reveal the influence of both evolution and intrinsic structural dynamics on protein assembly and demonstrate, for the first time on a genome-wide scale, the biological importance of ordered assembly pathways.

Comparative Multiplexed Mass Spectrometric Analyses of Endogenously Expressed Yeast Nuclear and Cytoplasmic Exosomes

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear–cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the α/β importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.

Monte Carlo Simulation of Macromolecular Ionization by Nanoelectrospray

Electrospray ionization (ESI) is commonly used in macromolecular mass spectrometry, yet the dynamics of macromolecules in ESI droplets are not well understood. In this study, a Monte Carlo based model was developed, which can predict the efficiency of electrospray ionization for macromolecules, i.e., the number of macromolecular ions produced per macromolecules electrosprayed. The model takes into account ESI droplet evaporation, macromolecular diffusion within the droplet, droplet fissions, and the statistical nature of the ESI process. Two idealized representations of macromolecular analytes were developed, describing cluster prone, droplet surface inactive macromolecules and droplet surface active macromolecules, respectively. It was found that surface active macromolecules are preferentially ionized over surface inactive cluster prone macromolecules when the initial droplet size is large and the analyte concentration in solution is high. Simulations showed that ESI efficiency decreases with increasing initial droplet size and analyte molecular weight, and is influenced by analyte surface activity, the properties of the solvent, and the variance of the droplet size distribution. Model predictions are qualitatively supported by experimental measurements of macromolecular electrospray ionization made previously. Overall, this study demonstrates the potential capabilities of Monte Carlo based ESI models. Future developments in such models will allow for more accurate predictions of macromolecular ESI intensity.

Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry

The stoichiometry of yeast V(1)-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V(1)-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V(1)-ATPase purified from yeast grown on (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V(1)-ATPase is A(3)B(3)DE(3)FG(3)H.

Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.

Improving the Performance of a Quadrupole Time-of-Flight Instrument for Macromolecular Mass Spectrometry

We modified and optimized a first generation quadrupole time-of-flight (Q-TOF) 1 to perform tandem mass spectrometry on macromolecular protein complexes. The modified instrument allows isolation and subsequent dissociation of high-mass protein complexes through collisions with argon molecules. The modifications of the Q-TOF 1 include the introduction of (1) a flow-restricting sleeve around the first hexapole ion bridge, (2) a low-frequency ion-selecting quadrupole, (3) a high-pressure hexapole collision cell, (4) high-transmission grids in the multicomponent ion lenses, and (5) a low repetition rate pusher. Using these modifications, we demonstrate the experimental isolation of ions up to 12 800 mass-to-charge units and detection of product ions up to 38 150 Da, enabling the investigation of the gas-phase stability, protein complex topology, and quaternary structure of protein complexes. Some of the data reveal a so-far unprecedented new mechanism in gas-phase dissociation of protein oligomers whereby a tetramer complex dissociates into two dimers. These data add to the current debate whether gas-phase structures of protein complexes do retain some of the structural features of the corresponding species in solution. The presented low-cost modifications on a Q-TOF 1 instrument are of interest to everyone working in the fields of macromolecular mass spectrometry and more generic structural biology.

Macromolecular mass spectrometry and electron microscopy as complementary tools for investigation of the heterogeneity of bacteriophage portal assemblies

The success of electron-cryo microscopy (cryo-EM) and image reconstruction of cyclic oligomers, such as the viral and bacteriophage portals, depends on the accurate knowledge of their order of symmetry. A number of statistical methods of image analysis address this problem, but often do not provide unambiguous results. Direct measurement of the oligomeric state of multisubunit protein assemblies is difficult when the number of subunits is large and one subunit renders only a small increment to the full size of the oligomer. Moreover, when mixtures of different stochiometries are present techniques such as analytical centrifugation or size-exclusion chromatography are also less helpful. Here, we use electrospray ionization mass spectrometry to directly determine the oligomeric states of the in vitro assembled portal oligomers of the phages P22, Phi-29 and SPP1, which range in mass from 430 kDa to about 1 million Da. Our data unambiguously reveal that the oligomeric states of Phi-29 and SPP1 portals were 12 and 13, respectively, in good agreement with crystallographic and electron microscopy data. However, in vitro assembled P22 portals were a mixture of 11- and 12-mer species in an approximate ratio of 2:1, respectively. A subsequent reference-free alignment of electron microscopy images of the P22 portal confirmed this mixture of oligomeric states. We conclude that macromolecular mass spectrometry is a valuable tool in structural biology that can aide in the determination of oligomeric states and symmetry of assemblies, providing a good starting point for improved image analysis of cryo-EM data.

Probing Genuine Strong Interactions and Post-translational Modifications in the Heterogeneous Yeast Exosome Protein Complex

The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels.