Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry.

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson's disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo-electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins.

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins.

Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433-434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.