Most MmpLs transport lipids and glycolipids of the complex mycomembrane of mycobacteria. This study shows that MmpL10 from Mycobacterium smegmatis, the transporter of the trehalose polyphosphate prec...

The correlation of individual conformational changes in dynamic protein complexes remains challenging as most structural methods rely on averaged information over a large number of molecules. Single molecule FRET is a powerful tool for monitoring such conformational changes. When performed using three distinct probes, it enables the correlation of domain movements by providing up to three simultaneous distance measurements with high temporal resolution. Nevertheless, a major challenge lies in the site-specific attachment of three probes to unique positions within the target protein. Here, we propose an orthogonal triple-labeling strategy that is not compromised by native, reactive amino acid functionalities. It combines genetic code expansion and bioorthogonal labeling of two different noncanonical amino acids with an enzymatic self-labeling SNAP tag. We demonstrate its application by establishment of a 3-color sensor on the human metabotropic glutamate receptor 2, a dimeric, multidomain G protein-coupled neuroreceptor, and describe a previously unknown conformational intermediate state using 3-color single molecule FRET.

Abstract. Initiation of transcription requires the formation of the “open” promoter complex (RPo). For this, the σ subunit of bacterial RNA polymerase (RNA