Figure 4.

T1 variants decrease Kv1.3 membrane association, core glycosylation, and T1 folding. A. Membrane-Associated Kv1.3 and core glycosylation. Membrane association efficiency of full-length Kv1.3 protein (red bars, left axis) for the indicated variants after 120 min translation reactions in the presence of microsomal membranes. Fraction core glycosylated is depicted as blue bars (right axis) for each variant. Dots are data points; colored bars are means; error bars are ±SEM. B. Quaternary folding of T1 domains. Multimer formation for WT (lanes 1, 2) and KCNA3 variants (red labels, lanes 3–6). Modification with o-phenyldimaleimide crosslinker (PDM), yields monomer, dimer, trimer, and tetramer, indicated by the number of asterisks, respectively. Two cysteines in the T1 –T1 interface were engineered for this purpose (Figure 1B). C. Quantitative analysis of crosslinking gels. Each bar is the fraction of total protein crosslinked as dimer, trimer, tetramer divided by the sum of monomer, dimer, trimer, and tetramer (n = 3, mean ± SEM), i.e., equivalent to the probability of crosslinking (Pxlink). D. Trimer + Tetramer fraction of crosslinked protein. This fraction is calculated as the sum of trimers + tetramers divided by the sum of dimers, trimers, and tetramers. E. Tertiary folding of T1 domains. Tertiary folding of the T1 WT (lanes 1–3) and R114G variant (lanes 4–6). Full-length constructs containing an engineered cysteine pair (Figure 1B) were translated, then treated with bismaleimide PDM (0.5 mM, Kosolapov and Deutsch, 2003 [13]) and finally pegylated with PEG-MAL or PEG-SH (5 mM, 1 h at 4 °C). Numbers to the right represent doubly (2) or singly (1) pegylated or unpegylated (0) protein. Pfold is 0.90 for WT, 0.55 for R114G.