Elucidating Protein Structures in the Gas Phase: Traversing Configuration Space with Biasing Methods.

Achieving accurate characterization of protein structures in the gas phase continues to be a formidable challenge. To tackle this issue, the present study employs Molecular Dynamics (MD) simulations in tandem with enhanced sampling techniques (methods designed to efficiently explore protein conformations). The objective is to identify suitable structures of proteins by contrasting their calculated Collision Cross-Section (CCS) with those observed experimentally.

Ultralong transients enhance sensitivity and resolution in Orbitrap-based single-ion mass spectrometry.

Orbitrap-based charge detection mass spectrometry utilizes single-molecule sensitivity to enable mass analysis of even highly heterogeneous, high-mass macromolecular assemblies. For contemporary Orbitrap instruments, the accessible ion detection (recording) times are maximally ~1-2 s. Here by modifying a data acquisition method on an Orbitrap ultrahigh mass range mass spectrometer, we trapped and monitored individual (single) ions for up to 25 s, resulting in a corresponding and huge improvement in signal-to-noise ratio (×5 compared with 1 s), mass resolution (×25) and accuracy in charge and mass determination of Orbitrap-based charge detection mass spectrometry.

Eye lens β-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers.

Eye lens α- and β-/γ-crystallin proteins are not replaced after fiber cell denucleation and maintain lens transparency and refractive properties. The exceptionally high (∼400-500 mg/mL) concentration of crystallins in mature lens tissue and multiple other factors impede precise characterization of β-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe β-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins.

Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ.

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs.

Protein shape sampled by ion mobility mass spectrometry consistently improves protein structure prediction.

Ion mobility (IM) mass spectrometry provides structural information about protein shape and size in the form of an orientationally-averaged collision cross-section (CCS). While IM data have been used with various computational methods, they have not yet been utilized to predict monomeric protein structure from sequence. Here, we show that IM data can significantly improve protein structure determination using the modelling suite Rosetta.

Investigation of Charge-State-Dependent Compaction of Protein Ions with Native Ion Mobility-Mass Spectrometry and Theory.

The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends.

Influenza AM2 Channel Oligomerization Is Sensitive to Its Chemical Environment.

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions.

Approaches to Heterogeneity in Native Mass Spectrometry.

Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from solution after they are transferred into the gas phase. Major improvements in native MS instrumentation and experimental methods over the past few decades have led to a concomitant increase in the complexity and heterogeneity of samples that can be analyzed, including protein-ligand complexes, protein complexes with multiple coexisting stoichiometries, and membrane protein-lipid assemblies. Heterogeneous features of these biomolecular samples can be important for understanding structure and function.

Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA.

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies.

Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions.

Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor.

The dynein light chain 8 (LC8) binds predominantly "in-register" to a multivalent intrinsically disordered partner.

Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized.

Computational Insights into Compaction of Gas-Phase Protein and Protein Complex Ions in Native Ion Mobility-Mass Spectrometry.

Native ion mobility-mass spectrometry (IM-MS) is a rapidly growing field for studying the composition and structure of biomolecules and biomolecular complexes using gas-phase methods. Typically, ions are formed in native IM-MS using gentle nanoelectrospray ionization conditions, which in many cases can preserve condensed-phase stoichiometry. Although much evidence shows that large-scale condensed-phase structure, such as quaternary structure and topology, can also be preserved, it is less clear to what extent smaller-scale structure is preserved in native IM-MS.

Ion Mobility-Mass Spectrometry Reveals That α-Hemolysin from Simultaneously Forms Hexameric and Heptameric Complexes in Detergent Micelle Solutions.

Many soluble and membrane proteins form symmetrical homooligomeric complexes. However, determining the oligomeric state of protein complexes can be difficult. Alpha-hemolysin (αHL) from is a symmetrical homooligomeric protein toxin that forms transmembrane β-barrel pores in host cell membranes.