title: 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-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the 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 of developing novel labeling schemes for spectroscopic applications.
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and.
authors.forename: Michaela
authors.initials: M
authors.lastname: Mehler
authors.affiliation: Institute for Physical and Theoretical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany.
authors.forename: Carl Elias
authors.initials: CE
authors.lastname: Eckert
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and.
authors.forename: Alena
authors.initials: A
authors.lastname: Busche
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kulhei
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and.
authors.forename: Jonas
authors.initials: J
authors.lastname: Michaelis
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and.
authors.forename: Johanna
authors.initials: J
authors.lastname: Becker-Baldus
authors.affiliation: Institute for Physical and Theoretical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Wachtveitl
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: From the Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance and glaubitz@em.uni-frankfurt.de.
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D047668:Inteins; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D017510:Protein Folding; D019154:Protein Splicing; D017433:Protein Structure, Secondary; D025604:Rhodopsins, Microbial / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin
keywords: intein; membrane protein; photoreceptor; protein folding; protein splicing; proton pump; seven-helix receptor; solid-state NMR