Talk:20.109(S13):Assess protein function (Day8)

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(Analysis, W/F)
Current revision (14:42, 24 April 2013) (view source)
 
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|Negative cooperativity, lower affinity in target mutant
|Negative cooperativity, lower affinity in target mutant
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|-
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+
|Yellow
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|T79P
-
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|K77E
-
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|Kd = 0.1531, n = 1.4785
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|Kd = 0.8493, n = 1.5848
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|Kd = 0.0382, n = 1.1695
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|Design mutant: higher affinity, lower positive cooperativity
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|Green
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|T79P
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|M71R
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|Kd = 0.117, n = 1.50
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|Kd = 0.889, n = 4.05
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|Kd = 0.229, n = 0.684
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|T79P showed initial, slight increase in fluorescence, then sharp decrease. M71R showed sharply decreased cooperativity and increased Kd compared to WT.
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|Red
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|D24H
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|G25Q
 +
|Kd = 0.1232, n = 1.3583
 +
|Kd = 0.7541, n = 3.9963
 +
|Kd = 0.0415, n = -0.4091
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|Fluorescence rises to a peak then falls off at end
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| D24H
| D24H
| D22E
| D22E
-
| Kd= 0.0778, n= 1.2080
+
| Kd= 0.0781, n= 1.2420
-
| Kd= 0.6353, n= 3.7325
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| Kd= 0.5804, n= 2.3194
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| Kd= 0.6892, n= 7.8230
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| Kd= 0.6807, n= 7.0509
| Affinity decreased while the hill coefficient increased significantly. No extreme outliers.
| Affinity decreased while the hill coefficient increased significantly. No extreme outliers.
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|-
Line 139: Line 144:
| Kd =  0.3729 n = 0.3830  
| Kd =  0.3729 n = 0.3830  
| Kd =  0.3488 n= 0.9085
| Kd =  0.3488 n= 0.9085
 +
Low fluorescent values
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|Pink
|Pink
Line 148: Line 154:
|Low fluorescence relative to wt,  Affinity increased significantly or IPC was unable to refold properly.
|Low fluorescence relative to wt,  Affinity increased significantly or IPC was unable to refold properly.
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|Purple
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|M12S
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|M51T
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|Kd=0.1276, n=1.4726
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|Kd=0.3430, n=3.009
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|Kd=0.1191, n=1.2544
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|Yellow
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|M124S
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|M51T
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|Kd = 0.0739; n = 1.2038
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|Kd = 0.3859; n = 3.0814
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|Kd = 0.1403; n = 1.6126
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|Higher fluorescence compared to WT. Similar affinity as WT, higher affinity than reference.
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|Red
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|D24H
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|D22E
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|Kd = 0.0919, n = 1.858
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|Kd = 0.7099, n = 6.5600
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|Kd = 0.6134, n = 2.3619
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|Orange
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|D24H
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|D22E
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|Kd = 0.0916, n = 1.2896
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|Kd = 0.7357, n = 4.3991
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|Kd = 0.6766, n = 7.2874
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|Decreased affinity, increased cooperativity
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|Green
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|T79P
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|K77E
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|Kd = 6.75e-8M, n = 1.32
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|Kd = 9.30e-7M, n = 5.34
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|Kd = 1.84e-8M, n = 1.08
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Current revision

Contents

Raw Data

Raw calcium titration data is posted below.


Day 7 data file, T/R Day 7 data file, W/F
Media:S13-M2D7-TR-Red.txt Media:S13-M2D7-WF-Red.txt
Media:S13-M2D7-TR-Org.txt Media:S13-M2D7-WF-Org.txt
Media:S13-M2D7-TR-Ylw.txt Media:S13-M2D7-WF-Ylw.txt
Media:S13-M2D7-TR-Grn.txt Media:S13-M2D7-WF-Grn.txt
Media:S13-M2D7-TR-Blu.txt Media:S13-M2D7-WF-Blu.txt
N/A Media:S13-M2D7-WF-Pnk.txt
Media:S13-M2D7-TR-Prp.txt Media:S13-M2D7-WF-Prp.txt
Media:S13-M2D7-TR-Plt.txt Media:S13-M2D7-WF-Plt.txt


Analysis, T/R

Please be mindful of significant figures when you fill in this table. Do not state far more precise numbers than you can believe.

Group Colour D24H, E67K, T79P, or M124S? X#Z Mutant Most believable KD; n for WT Most believable KD; n for reference Most believable KD; n for X#Z mutant Comments (e.g., very low fluorescence)
Plt M124S D80R WT: 0.0976; 1.4751 M124S: 0.4115; 3.9341 D80R: 1.2991; 2.8465 small difference between 0% and 100% for our mutant, affinity decrease and cooperativity increase
Orange D24H G25Q 0.1462, n = 0.8310 0.6759, n = 2.5734 0.7118; n = -0.2595 Very low fluorescence; oscillating trend...
Purple M124S M144R KD = 0.0672, n = 1.1286 KD = 0.2682, n= 3.3655 KD = 0.3583, n= 4.1618 Higher cooperativity, lower affinity in target mutant
Blue M124S M71R KD = 0.1175, n = 1.4880 KD = 0.3377, n = 3.3863 KD = 0.2246, n = 0.6741 Negative cooperativity, lower affinity in target mutant
Yellow T79P K77E Kd = 0.1531, n = 1.4785 Kd = 0.8493, n = 1.5848 Kd = 0.0382, n = 1.1695 Design mutant: higher affinity, lower positive cooperativity
Green T79P M71R Kd = 0.117, n = 1.50 Kd = 0.889, n = 4.05 Kd = 0.229, n = 0.684 T79P showed initial, slight increase in fluorescence, then sharp decrease. M71R showed sharply decreased cooperativity and increased Kd compared to WT.
Red D24H G25Q Kd = 0.1232, n = 1.3583 Kd = 0.7541, n = 3.9963 Kd = 0.0415, n = -0.4091 Fluorescence rises to a peak then falls off at end

Analysis, W/F

Analysis, W/F

Please be mindful of significant figures when you fill in this table. Do not state far more precise numbers than you can believe.

Group Colour D24H, E67K, T79P, or M124S? X#Z Mutant Most believable KD; n for WT Most believable KD; n for reference Most believable KD; n for X#Z mutant Comments (e.g., very low fluorescence)
Blue D24H D22E Kd= 0.0781, n= 1.2420 Kd= 0.5804, n= 2.3194 Kd= 0.6807, n= 7.0509 Affinity decreased while the hill coefficient increased significantly. No extreme outliers.
Platinum E67K R126P Kd = 0.0970 n =1.0639 Kd = 0.3729 n = 0.3830 Kd = 0.3488 n= 0.9085

Low fluorescent values

Pink D24H G25Q Kd = 0.1232, n = 1.3583 Kd = 0.7541, n = 3.9963 Kd = 0.0415, n = -0.4091 Low fluorescence relative to wt, Affinity increased significantly or IPC was unable to refold properly.
Purple M12S M51T Kd=0.1276, n=1.4726 Kd=0.3430, n=3.009 Kd=0.1191, n=1.2544
Yellow M124S M51T Kd = 0.0739; n = 1.2038 Kd = 0.3859; n = 3.0814 Kd = 0.1403; n = 1.6126 Higher fluorescence compared to WT. Similar affinity as WT, higher affinity than reference.
Red D24H D22E Kd = 0.0919, n = 1.858 Kd = 0.7099, n = 6.5600 Kd = 0.6134, n = 2.3619
Orange D24H D22E Kd = 0.0916, n = 1.2896 Kd = 0.7357, n = 4.3991 Kd = 0.6766, n = 7.2874 Decreased affinity, increased cooperativity
Green T79P K77E Kd = 6.75e-8M, n = 1.32 Kd = 9.30e-7M, n = 5.34 Kd = 1.84e-8M, n = 1.08
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