Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence.
The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca2+-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, United States.
Lauren M
http://orcid.org/0000-0003-1605-8912
LM
Barnett
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, United States.
Thomas E
TE
Hughes
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, United States.
Mikhail
M
Drobizhev
D002118:Calcium / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium