Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL.
Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.
Max-Planck-Institut für Neurobiologie, Martinsried, Germany.
Anselm
A
Geiger
Luigi
L
Russo
Thomas
T
Gensch
Thomas
T
Thestrup
Stefan
S
Becker
Karl-Peter
KP
Hopfner
Christian
C
Griesinger
Gregor
G
Witte
Oliver
O
Griesbeck
D019943:Amino Acid Substitution; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057446:Hydrodynamics; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013696:Temperature; D019209:Troponin C / Q000737:chemistry*; D014443:Tyrosine / Q000378:metabolism
D019209:Troponin C; D014443:Tyrosine; D002118:Calcium