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Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels.

Sci Rep. 2019 Aug 29;9(1):12528
Eliane H Yardeni 1 , Thorsten Bahrenberg 2 , Richard A Stein 3 , Smriti Mishra 3 , Elia Zomot 1 , Bim Graham 4 , Kellie L Tuck 5 , Thomas Huber 6 , Eitan Bibi 7 , Hassane S Mchaourab 8 , Daniella Goldfarb 9
Eliane H Yardeni 1 , Thorsten Bahrenberg 2 , Richard A Stein 3 , Smriti Mishra 3 , Elia Zomot 1 , Bim Graham 4 , Kellie L Tuck 5 , Thomas Huber 6 , Eitan Bibi 7 , Hassane S Mchaourab 8 , Daniella Goldfarb 9
+ et al

[No authors listed]

Author information
  • 1 Department of Biomolecular Sciences, Weizmann Institute of Science Rehovot, Rehovot, 76100, Israel.
  • 2 Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.
  • 3 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.
  • 4 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia.
  • 5 School of Chemistry, Monash University, Wellington Road, Clayton, Victoria, Australia.
  • 6 Research School of Chemistry, The Australian National University, Canberra, ACT 2601, Australia.
  • 7 Department of Biomolecular Sciences, Weizmann Institute of Science Rehovot, Rehovot, 76100, Israel. eitan.bibi@weizmann.ac.il.
  • 8 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA. hassane.mchaourab@vanderbilt.edu.
  • 9 Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, 76100, Israel. Daniella.goldfarb@weizmann.ac.il.

摘要


Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.