Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

Abstract

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-015-9936-5) contains supplementary material, which is available to authorized users.

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

“…2 D, right), possibly due to local motion. Previously, we detected such increased mobility of selectivity filter residues, in particular for V76, in ssNMR studies using synthetic (45) as well as native bacterial membranes (46,47), in line with results of previous molecular-dynamics studies (48).…”

1H‐detektierte Festkörper‐NMR‐Studien wasserunzugänglicher Proteine in vitro und in situ

“…Allerdings sind zelluläre ssNMR‐Messungen sehr kompliziert aufgrund der geringen Konzentration der Zielproteine. Zelluläres KcsA wurde in der inneren Membran von E. coli mithilfe der Rifampicin‐Methode exprimiert, um den endogenen spektralen Hintergrund zu reduzieren . Die äußere Membrane wurde entfernt, um die KcsA‐Konzentration zu erhöhen.…”

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

“…However, adequate spectral quality is a challenge in cellular ssNMR studies owing to the low concentration of the target protein. Cellular iFD KcsA was expressed in the E. coli inner membrane using rifampicin to ensure selective 13 C, 15 N‐labeling of KcsA and reduce the endogenous spectral background 11. The outer membrane was removed to increase the amount of KcsA in the sample, which was then incubated in deuterated solvent for three days prior to the ssNMR experiments.…”