Hires SA, Zhu Y, Tsien R (2008)
Optical measurement of synaptic glutamate spillover and reuptake
by linker optimized glutamate-sensitive fluorescent reporters
Proc Natl Acad Sci USA 105:4411-4416

(Translated by Jeff Elhai)

Experiment: Optimization of sensor characteristics

[Need to decrease affinity of glutamate binding protein for glutamate. Site-specific mutagenesis: S73T had KD of 2.5 μM (17x decrease in affinity).	[image] [Picture of GltI bound to glutamate from PDB. Highlight Ser-73.]
[Need to increase response magnitude. Truncate GltI(S73T) on N terminus and C terminus.]	[image] [Need figure for truncation of sequence. Use graphical convention like that below.]
[Measure maximum response to glutamate as in Fig. 1d of all 176 possible truncation combinations. One big winner: 8 amino acids truncated on N terminus, 5 amino acids on C terminus. 44% maximum response to glutamate. Omit Fig. 2e	Show truncations graphically. Label color bar with absolute percentages: 7.1% = blue, 44% = red. Draw line from red box to X- and Y-axes
Performance of final optimized sensor with the following modifications (as described above): S73T, -8N, -5C. Optimized range of sensitivity to glutamate. Optimized range of fluorescent response +/- glutamate.	Change X-axis to 10-9 etc. Draw line from midpoint down to X axis, label KD = 2.5μM. [Color bar. Erase black. Lines to Y axis. Maximum response = 44%]