Solutes alter the conformation of the ligand binding loops in outer membrane transporters.
The binding and recognition of ligands by bacterial outer membrane transport proteins is mediated in part by interactions made through their extracellular loops. Here, site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy were used to examine the effect of stabilizing solutes on the extracellular loops in BtuB, the vitamin B12 transporter, and FecA, the ferric citrate transporter. EPR spectra from the extracellular loops of FecA and BtuB arise from dynamic backbone segments, and distance measurements made by double electron-electron resonance indicate that the second extracellular loop in BtuB samples a wide range of conformations. These conformations are dramatically restricted upon substrate binding. In addition, the EPR spectra from nitroxide labels attached to the extracellular loops in BtuB and FecA are highly sensitive to solutes, and at every site examined the motion of the label is significantly reduced in the presence of stabilizing osmolytes, such as polyethylene glycols. For the second extracellular loop in BtuB, the solute-induced structural changes are small, but they are sufficient to bring spin-labeled side chains into tertiary contact with other portions of the protein. The spectroscopic changes seen by SDSL suggest that high concentrations of stabilizing solutes, such as those used to generate membrane protein crystals, result in a more compact and ordered state of the protein than is seen under more physiological conditions.
Kim
Miyeon
M
Department of Chemistry and Biophysics Program, P.O. Box 400319, McCormick Road, University of Virginia, Charlottesville, USA.
Xu
Qi
Q
Murray
David
D
Cafiso
David S
DS
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002118:Calcium; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D018583:Pliability; D011092:Polyethylene Glycols; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry*; D013113:Spin Labels; D013816:Thermodynamics; D014805:Vitamin B 12
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; C028589:FecA protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D011956:Receptors, Cell Surface; D013113:Spin Labels; D011092:Polyethylene Glycols; D014805:Vitamin B 12; D002118:Calcium