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

Abstract Protein trans-splicing using split inteins is well established as 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-B and C-D-E-F-G via a splicing site located in the BC loop. The procedure presented here is based on dual expression and ligation in vivo. Global fold, stability and photodynamic were analyzed in detergent by CD-, stationary as well as time-resolved optical spectroscopy. Its fold within lipid bilayers has been probed by high field and DNP-enhanced solid-state NMR utilizing a 13Clabeled retinal co-factor and extensively 13C-15N labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins, for elucidating intramolecular interactions and they offer the possibility towards developing novel labeling schemes for spectroscopic applications.