RT Journal Article SR Electronic T1 In situ distance measurements in a membrane transporter using maleimide functionalized orthogonal spin labels and 5-pulse electron double resonance spectroscopy JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.12.23.473964 DO 10.1101/2021.12.23.473964 A1 Sophie Ketter A1 Marina Dajka A1 Olga Rogozhnikova A1 Sergey A. Dobrynin A1 Victor M. Tormyshev A1 Elena G. Bagryanskaya A1 Benesh Joseph YR 2021 UL http://biorxiv.org/content/early/2021/12/23/2021.12.23.473964.abstract AB Spectroscopic investigation of membrane proteins in their native environment is a challenging task. Earlier we demonstrated the feasibility to measure precise distances within outer membrane proteins in E. coli and native membranes using methanethiosulfonate (MTS) functionalized labels combined with pulsed electron double resonance spectroscopy. Here we show the application of maleimide functionalized Gd(III), nitroxide, and trityl labels for in situ distance measurement using the cobalamin transporter BtuB. These labels enabled distance measurements for BtuB in E. coli and native outer membranes and in the membranes maleimide-Gd-DOTA also is effective. Further, we show that the observable dipolar evolution time can be significantly prolonged in the native environments using the Carr-Purcell 5-pulse electron double resonance sequence. For a nitroxide-nitroxide pair, application of sech/tanh inversion pulses substantially suppressed the 4-pulse artifact at the Q-band frequency. In the case of a nitroxide-trityl pair, Gaussian pump pulses of varying amplitude are sufficient to suppress the artifact to the typical noise level. The feasibility of a range of bioresistant spin labels and the 5-pulse electron double resonance offers promising tools for investigating heterooligomeric membrane protein complexes in their native environment.Competing Interest StatementThe authors have declared no competing interest.