12/23/2023 0 Comments Multipass transmembrane protein![]() Integral proteins comprise nearly 20-30% of the protein content of a cell, and these are tightly fixed in the membrane such that researchers often have to resort to more stringent conditions involving chaotropic agents like 6-8M urea, thiocyanate, lithium perchlorate or guanidinium chloride to disrupt the hydrophobic interactions of these proteins with the membrane to facilitate their extraction. Still, in general, the transmembrane domain is composed of hydrophobic residues that generally adopt the configuration of either an alpha helix or a beta-sheet made up of multiple beta-strands. The chemical nature and structure of these domains vary greatly. These transmembrane proteins generally have three different domains: a cytosolic domain (for transmitting signals to the internal of the cell), a transmembrane domain, and an exoplasmic domain that hangs outside the cell, acting as a receptor. (H) Three views of the backbone superposition of TMHC4_R crystal structure and design model.Transmembrane proteins or integral membrane proteins are embedded into the cell membrane and span the lipid bilayer membrane. The transmembrane region is indicated in lines. (G) Cross section through the TMHC4_R crystal structure and electrostatic surface the HRD forms a bowl at the base of the overall structure with a depth of ~20 Å. The outer helices of the transmembrane hairpins tilt off the axis by ~10°. The overall tetramer structures are very similar to the design model, with a helical bundle body and helical repeat fins. (E) Analytical ultracentrifugation sedimentation-equilibrium curves at three different rotor speeds for TMHC4_R fit well to a single species with a measured molecular weight of ~94 kDa. The four protomers are colored green, yellow, magenta and cyan, respectively. (D) Model of designed transmembrane tetramer TMHC4_R with eight transmembrane helices. The data fit to a single ideal species in solution with molecular weight close to that of the designed trimer. (C) Representative analytical ultracentrifugation sedimentation-equilibrium curves at three different rotor speeds for TMHC3. (B) Circular dichroism characterization of TMHC3 the design is stable up to 95☌. Stick representation from periplasmic side (left) and lateral surface view (right) are shown. (A) Model of designed transmembrane trimer TMHC3 with six transmembrane helices. ![]() ![]() Stability and structural characterization of designs with six and eight membrane spanning helices. No apparent unfolding transitions are observed up to 95☌. (D) CD spectra and temperature melt (inset). ‘MW (D)’ and ‘MW (E)’ indicate the molecular weight of the oligomer design and that determined from experiment, respectively. Each data set is globally well fitted as a single ideal species in solution corresponding to the dimer molecular weight. (C) Representative analytical ultracentrifugation sedimentation-equilibrium curves at three different rotor speeds. Line scans (yellow lines in the images) across the membranes show significant increase in fluorescence across the plasma membranes for TMHC2, TMHC2_E and TMHC2_L, but less significant increase for TMHC2_S. (B) Confocal microscopy images for HEK293T cells transfected with TMHC2 fused to mTagBFP, TMHC2_E fused to mTagBFP, TMHC2_L fused to mCherry and TMHC2_S fused to eGFP. Ribbon diagrams are on left, electrostatic surfaces on right, and the neutral transmembrane regions are in gray. Horizontal lines demarcate the hydrophobic membrane regions. (A) Design models with intra- and extra-membrane regions with different lengths. From left to right, designs and data are shown for TMHC2 (transmembrane hairpin C2), TMHC2_E (elongated), TMHC2_L (long span) and TMHC2_S (short span). Government Works.ĭesign and characterization of proteins with four transmembrane helices. Our results pave the way for the design of multispan membrane proteins with new functions.Ĭopyright © 2018 The Authors, some rights reserved exclusive licensee American Association for the Advancement of Science. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |