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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 J+Biol+Chem
2015 ; 290
(46
): 27712-22
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Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by
Protein Trans-splicing
#MMPMID26405032
Mehler M
; Eckert CE
; Busche A
; Kulhei J
; Michaelis J
; Becker-Baldus J
; Wachtveitl J
; Dötsch V
; Glaubitz C
J Biol Chem
2015[Nov]; 290
(46
): 27712-22
PMID26405032
show ga
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.
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