Ion mobility–mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy

Abstract

Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine–Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility–mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft–mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

“… The fact that proteins lacking basic residues favor the CRM over CEM reveals the limitations of using charge state signatures to assess the distribution of folded states . As a result, proteins with few ionizable groups may display diverging charging patterns upon unfolding. Previous studies have shown that point mutations can affect the CIU pattern of native-like protein ions by modulating their structure in solution. ,,, Our findings reveal that even identically folded proteins can exhibit different CIU fingerprints due to altered surface electrostatics during ESI. Because these differences arise from the locations of ESI charges, not from solution folding, care must be taken when using CIU data to probe solution structures. Gas-phase unfolding is routinely used to identify compounds that can stabilize protein complexes by occupying specific binding pockets. ,− We find that moving charged sites can have a dramatic effect on a protein’s conformation in the gas phase.…”

“…(3) Previous studies have shown that point mutations can affect the CIU pattern of native-like protein ions by modulating their structure in solution. 9,15,54,55 Our findings reveal that even identically folded proteins can exhibit different CIU fingerprints due to altered surface electrostatics during ESI. Because these differences arise from the locations of ESI charges, not from solution folding, care must be taken when using CIU data to probe solution structures.…”

“…IM-MS has been successfully applied to characterize the lipid preferences of membrane proteins, 11 , 12 define structural heterogeneity associated with glycosylation, 13 follow structural transitions in disordered proteins, 14 and determine the impact of disease-associated mutations on protein stability. 15 By combining alanine scanning with CIU, it is possible to quantify the contributions of individual residues on the conformational stabilities of protein ions. 16 IM-MS is furthermore employed in the pharmaceutical industry, 17 for example to characterize drug conjugates.…”

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain

“…IM separates the ions based on their size, shape, and charge. In native IM-MS experiments, we can separate different conformations and oligomeric states of membrane protein ions ( 55 , 56 , 57 , 58 , 59 ). All IM-MS data were collected using a Synapt G2 HDMS IM-Q-ToF mass spectrometer (Waters), with a direct infusion nESI source set to positive ion mode.…”

“…In native IM-MS experiments, we can separate different conformations and oligomeric states of membrane protein ions. (55)(56)(57)(58)(59) All IM-MS data were collected using a Synapt G2 HDMS IM-Q-ToF mass spectrometer (Waters, Milford, MA), with a direct infusion nESI source set to positive ion mode. Instrument settings were tuned to dissociate detergent micelles, with minimal perturbation of protein structure prior to the IM separator.…”