Nano-positioning system for structural analysis of functional homomeric proteins in multiple conformations.
Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target-site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a positioning method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states, with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one per subunit), relative to a single fluorescent acceptor placed in a static site. As first application, the method is used to determine the position of a functional voltage-gated potassium channel's voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA. fbezanilla@uchicago.edu
H Clark
HC
Hyde
Walter
W
Sandtner
Ernesto
E
Vargas
Alper T
AT
Dagcan
Janice L
JL
Robertson
Benoit
B
Roux
Ana M
AM
Correa
Francisco
F
Bezanilla
D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D056831:Coordination Complexes / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D056004:Molecular Dynamics Simulation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051637:Shaker Superfamily of Potassium Channels / Q000737:chemistry*; D012984:Software; D013725:Terbium / Q000737:chemistry; D014982:Xenopus laevis
D056831:Coordination Complexes; D051637:Shaker Superfamily of Potassium Channels; D013725:Terbium