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Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcusxanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence. [ABSTRACT FROM AUTHOR]

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly. [ABSTRACT FROM AUTHOR]

We describe a new approach to measuring the masses of individual macroions. The method employs a pulsed acceleration tube located between two sensitive image charge detectors. The charge and velocity of the macroion are recorded with the first image charge detector. The ion is pulse accelerated through a known voltage drop, and then the charge and velocity are remeasured using the second image charge detector. The mass of the ion is deduced from its charge and its initial and final velocities. The approach has been used to measure masses in the [10.sup.10]-[10.sup.14] Da range with z = [10.sup.3] = [10.sup.6] and m/z = [10.sup.6]-[10.sup.9]. It should be extendable to masses of [less than] [10.sup.6] Da. We have used the method to determine the size and charge of water droplets transmitted through a capillary interface and an aperture interface. The droplets detected from the aperture interface are ~1 order of magnitude smaller in mass than those detected from the capillary interface. The droplets from both interfaces have relatively low charges, particularly with the capillary interface where they are only charged to a small fraction of the Rayleigh limit. These results suggest that the aerodynamic breakup of the droplets plays a significant role in the mechanism of electrospray ionization.

Image charge detection is used to measure the charge and velocity of individual electrosprayed water droplets. The possibility of the negatively charged droplets resulting from a bipolar fission process is considered, where the incorporation of a small negatively charged droplet between two larger positively charged progeny lowers the energy barrier for symmetric fission.

The development of a new ion mobility/mass spectrometry instrument that incorporates a multifield drift tube/ion funnel design is described. In this instrument, individual components from a mixture of ions can be resolved and selected on the basis of mobility differences prior to collisional activation inside the drift tube. The fragment ions that are produced can be dispersed again in a second ion mobility spectrometry (IMS) region prior to additional collisional activation and MS analysis. The result is an IMS-IMS analogue of MS-MS. Here, we describe the preliminary instrumental design and experimental approach. We illustrate the approach by examining the highly characterized bradykinin and ubiquitin systems. Mobility-resolved fragment ions of bradykinin show that b-type ions are readily discernible fragments, because they exist as two easily resolvable structural types. Current limitations and future directions are briefly discussed.

Ion mobility and calorimetry measurements are used to study the nature of the phase transitions in gallium clusters with 29-55 atoms. It is observed that some clusters show the signature of melting without a significant latent heat, and that the transitions are the finite size analogue of a second-order phase transition, so it is concluded that they occur because their solidlike phase is amorphous.

The conformation of the protonated complex formed between AcA7KA6KK and AcEA7EA7, helical alanine-based peptides that incorporate glutamic acid (E) and lysine (K) is examined using ion-mobility mass spectroscopy. Designed interactions between the acidic E and basic K residues help to stabilize the complex, which is generated by electrospray and studied in the gas phase.

The complexes of methane and its fluro and chloro derivatives with lithium hydride are investigated. The results indicate that charge separation can be favorable in the gas phase and hence are discussed in the light of future experimental efforts.

The application of molecular beam deflection time-of-flight mass spectrometry (MBD-TOFMS) to peptide identification is described. The technique permits a simultaneous measurement of molecular mass and electric dipole susceptibility. The mass and susceptibility are not strongly correlated, and the results can be presented as a two-dimensional map. The susceptibility provides a useful way to disperse isobaric and isomeric peptides, and at least for small peptides, the susceptibility is significantly different for different amino acid sequences. Results for peptides in the mass range 1000-2300 Da show that the mass and susceptibility lead to a higher identification score than mass spectra alone.

Helix formation in unsolvated glycine-based peptides containing three alanine residues is examined by using ion mobility measurements. Peptides with three adjacent alanines have the highest helix abundances, and those with well-separated alanines have the lowest.

A report is presented on the studies of the conformations of unsolvated peptides of acetyl, alanine and glycine, over a wide temperature range of 100-410K. It is found that the helix to globule transition that occurs for the unsolvated peptides at 250-350 K temperature is not a direct process and takes place at several intermediates.

Charge detection mass spectrometry (CDMS) is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of their mass-to-charge ratio (m/z) and charge. Masses are determined for thousands of individual ions, and then the results are binned to give a mass spectrum. Using this approach, accurate mass distributions can be measured for heterogeneous and high-molecular-weight samples that are usually not amenable to analysis by conventional mass spectrometry. Recent applications include heavily glycosylated proteins, protein complexes, protein aggregates such as amyloid fibers, infectious viruses, gene therapies, vaccines, and vesicles such as exosomes. [ABSTRACT FROM AUTHOR]

Equilibrium constants for the adsorption of the first water molecule onto a variety of unsolvated alanine-based peptides are measured and (delta)H and (delta)S are determined. Globular Ac-KA(sub n)+H(super +) peptides were found to adsorb water, while no water adsorption was detected for helical Ac-A(sub n)K+H(super +).