Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing.

ABSTRACT: Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labele

SUBMITTER: Mehler M 

PROVIDER: S-EPMC4646914 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature