TY - JOUR
T1 - Nano-positioning system for structural analysis of functional homomeric proteins in multiple conformations
AU - Hyde, H. Clark
AU - Sandtner, Walter
AU - Vargas, Ernesto
AU - Dagcan, Alper T.
AU - Robertson, Janice L.
AU - Roux, Benoit
AU - Correa, Ana M.
AU - Bezanilla, Francisco
N1 - Funding Information:
H.C.H. designed analysis and modeling, performed calculations, wrote analysis software, and wrote the paper; W.S. designed and performed experiments; E.V. performed modeling; A.T.D. and J.L.R. generated acceptor clouds; B.R. designed modeling; A.M.C. designed and performed experiments; and F.B. designed experiments and developed methods and instrumentation. W.S., E.V., B.R., A.M.C., and F.B. participated in manuscript preparation. We thank Jérôme J. Lacroix for toxin preparation, Ludivine Frezza for construct preparation, and Ramón Latorre and Robert A. Hyde for discussing the manuscript. This work is supported by the National Institutes of Health (Grants GM068044, GM030376, GM062342, and U54GM087519).
PY - 2012/10/10
Y1 - 2012/10/10
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/84867345137
U2 - 10.1016/j.str.2012.08.022
DO - 10.1016/j.str.2012.08.022
M3 - Article
C2 - 23063010
AN - SCOPUS:84867345137
SN - 0969-2126
VL - 20
SP - 1629
EP - 1640
JO - Structure
JF - Structure
IS - 10
ER -