To further examine to what extent a dodecyl-phosphocholine (DPC) micelle mimics a phosphatidylcholine bilayer environment, we performed 13C, 2H, and 31P NMR relaxation measurements. Our data show that the dynamic behavior of DPC phosphocholine groups at low temperature (12 degrees C) corresponds to that of a phosphatidylcholine interface at high temperature (51 degrees C). In the presence of helical peptides, a PMP1 fragment, or an annexin fragment, the DPC local dynamics are not affected whereas the DPC aggregation number is increased to match an appropriate area/volume ratio for accommodating the bound peptides. We also show that quantitative measurements of paramagnetic relaxation enhancements induced by small amounts of spin-labeled phospholipids on peptide proton signals provide a meaningful insight on the location of both PMP1 and annexin fragments in DPC micelles. The paramagnetic contributions to the relaxation were extracted from intra-residue cross-peaks of NOESY spectra for both peptides. The location of each peptide in the micelles was found consistent with the corresponding relaxation data. As illustrated by the study of the PMP1 fragment, paramagnetic relaxation data also allow us to supply the missing medium-range NOEs and therefore to complete a standard conformational analysis of peptides in micelles.
The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBMGABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBMGABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBMGABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.
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