Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated.

Significance: Two of the most important issues in Parkinson’s disease (PD) research are the identification of mechanisms underlying α-synuclein cell-to-cell transfer in the nervous system and the discovery of early diagnostic biomarkers. Both of these issues are addressed in our current manuscript. Using multiple approaches, we present that α-synuclein interacts with lipoproteins within human cerebrospinal fluid and can be taken up by cells in such a state. Moreover, using cerebrospinal fluid samples from 3 large and independent cohorts of patients, we demonstrate that apolipoprotein E is elevated in early, not yet medicated, patients with PD. Finally, using postmortem brain tissue, we provide preliminary histological evidence that apolipoprotein E is enriched in a subpopulation of dopaminergic neurons of human substantia nigra.