Study of the binding of D-mannose to the lectin domain of the
FimH subunit of Type 1 pili
1. Saturation binding experiment
Stock solutions
1. Radioactive [H3]-D-mannose (Hot mannose)
24.7 Ci/mmol, 1 mCi/ml in H2O
From the data above, the mannose concentration is 40.5 M (Is this correct?)
2. Non-radioactive D-mannose (Cold mannose)
CM(Cold mannose)=5x10-4 M
3. Protein
Calculate the concentration of your sample using a spectrophotometer. For this you have
to know the theoretical value of the extinction coefficient of FimH-LD: the absorbance
value at 280 nm (A280nm) of 1 mg/ml solution of the protein. This is counted based on
the amount of aromatic residues. The sequence of FimH-LD is shown bellow.
FACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDY
VTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLT
PVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTHHHHHH
Create a new protein molecule “FimH-LD” in VNTI and look at the “Analysis” folder of
the text panel. You will find here the extinction coefficient and molecular weight of the
protein. To prepare the working solution dilute your protein sample with TBS buffer (see
bellow) to the concentration of about 5x10-7 M (see example bellow).
Example:
C(protein)=0.8 mg/ml, MW=17.7 kDa → CM(protein)=4.5x10-5 M
To create working solution, dilute this stock 100 times to CM(protein)=4.5x10-7 M (e.g.
add 1.98 ml buffer to 20 l of 4.5x10-5 M solution)
4. TBS buffer
20 mM Tris-HCl (pH 7.4), 150 mM NaCl
Hot+Cold mixtures
The dissociation constant (Kd) is expected to have value between 1 and 5 M. Hence, to
construct a good saturation curve, we need to study binding in 2-30 M range of mannose
concentrations. To avoid using too much of the Hot mannose we will prepare a Hot+Cold
mixture as following:
Take 15 l Hot mannose (do it under the hood in the radioactive room, wearing double
gloves! Use a tip with a filter!) and mix it with 85.5 l Cold mannose from the stock. The
total mannose concentration will be:
CM(total)= (15 l x 40.5 M + 85.5 l x 500 M)/( 15+85.5 M)=433 M
This is the “Hot+Cold” stock. Prepare 7 tubes with 25 l water in each. Add 50 M Hot+Cold
mixture into the first tube and pipette it 2 times. Withdraw 50 M from the first tube and add
to the second. Repeat the same procedure for the third and the other tubes. At the end you will
have 7 dilutions of Hot+Cold mixture (in M):
1
288
2
192
3
128
4
85.3
5
56.9
6
37.9
7
25.3
Save the remaining 50 l of the “Hot+Cold” stock for the inhibition binding assay!
Final mixtures
Prepare two equal 10 l aliquots of each Hot+Cold mixture in new tubes and add 90 l
protein working solution (#3) into all these tubes. You will have 14 mixtures, having protein
concentration of about 0.4 M and following final concentrations of mannose (in M):
28.8
28.8
19.2
19.2
12.8
12.8
8.53
8.53
5.69
5.69
3.79
3.79
2.53
2.53
Incubate reaction mixtures for 15-25 min at room temperature
Filtration
Pre-wet 16 nitro-cellulose filters in a Petri dish with TBS. Assemble filtration device and
adjust vacuum (this will be demonstrated). For each reaction mixture, place 90 l mixture on
the filter and wash filter immediately with 1 ml cold TBS. The protein will bind to the filter,
whereas the unbound mannose will pass thought the filter into the flask.
Transfer 1 l from dilutions 2 and 3 of Hot+Cold mixture onto two dry filters, respectively.
These two filters will be used to count the total mannose radioactivity.
Place all filters on a hand wiping paper and dry them for 1 h.
Measuring radioactivity
Add 5 ml scintillation solution into 16 counting tubes. Place filters into these tubes using
forceps. Incubate for 30-60 min. Place the tubes into -radiation counter racks and start
counting by choosing the “START” mode and pressing “1” and “R” subsequently. Help the
old device to move the racks and print the results (this will be demonstrated).
Calculations (use MS-Excel and Origin)
1. Saturation curve
Estimate average values (you have two repeats for each mannose concentration). Plot the
values against total mannose concentration (28.8, 19.2 …M). Fit the data into the
following equation:
y=P1x/(P2+x)
P1 will be proportional to the total amount of binding sites (or concentration of the folded
protein in our case), whereas P2 will be equal to the dissociation constant Kd.
2. Scatchard plot
(1) Estimate radioactivity of 1 M solution from the data of counting 1 l Hot+Cold
mixtures from dilutions 2 and 3.
E. g.:
#2 counts: 20000 cpm
#3 counts: 13000 cpm
Multiply these values by factor 10, because you added 10 l of Hot+Cold mixtures into
your final mixtures:
#2 counts: 200000 cpm
#3 counts: 130000 cpm
Estimate radioactivity of 1 M solution by dividing the obtained values with the values of
concentration in dilutions 2 and 3:
#2 counts: 200000 cpm/19.2 M=10410 cpm/M
#3 counts: 130000 cpm/12.8 M=10150 cpm/M
Average value is 10280 cpm/M
(2) Using the radioactivity of 1 M solution estimate the total radioactivity for other
dilutions (for #2 and #3 you already calculated 200000 and 130000 cpm, respectively)
(3) Estimate counts for unbound mannose by subtracting counts of the bound mannose
(counts from the filters) from the total radioactivity:
E.g.:
In dilution #2 the total radioactivity is 200000 cpm (as we counted above). The filter gave
counts 1000 cpm. Hence, unbound mannose should give counts 200000-1000=199000
cpm
(3) Estimate the ratios of bound and free mannose by dividing the counts of the bound
mannose with the counts of the unbound mannose.
E.g. for #2 dilution: 1000/199000=0.0052
(4) Estimate concentration of the bound mannose by dividing the filter counts with the
radioactivity of 1 M solution.
E.g. for #2 dilution: 1000 cpm/10280 cpm/M=0.097 M
(5) Plot the values of the ratios of bound and free mannose against the concentration of
the bound mannose. Make a linear fit of the data. Estimate the total amount of sites (at
the ratio of bound and free mannose equal 0) and Kd (from the slop of the line). Find
the value of the total amount of sites from the saturation curve fit by dividing P1
coefficient with the radioactivity of 1 M solution. Are the values of total amount of
sites and Kd obtained with Scatchard plot and Saturation curve fit similar?
2. Inhibition binding experiment
Our well-established radioactive mannose-FimH-LD binding system can be used to determine
dissociation constants for the binding of FimH-LD to different unlabeled ligands. For
example, we can determine the dissociation constants for the binding of FimH-LD to Cold
mannose. Mannose again? Well, it is not the lack of my fantasy. Such an experiment will help
us to check that the method works. Theoretically, the value obtained from the inhibition
experiment should be equal to that obtained from the binding experiment.
Diluted solution of Hot+Cold mixture
Transfer 40 l of “Hot+Cold” stock (from the biding experiment) into a new tube and add 160
l water. This 5-times diluted “Hot+Cold” stock will be directly used in the experiment.
Dilutions of Cold mannose
Prepare dilutions of Cold mannose in 7 tubes as following. Add 25 l undiluted Cold
mannose from the stock into the first tube. Add 25 l water in the other 6 tubes. Next, add 25
l undiluted Cold mannose from the stock into the second tube (total volume is now 50 M).
Withdraw 25 l from this tube and transfer into the third tube. Pipette the mixture, withdraw
25 l and transfer to the fourth tube and so forth. Finally you should have the following
dilutions of Cold mannose in M:
1
500
2
250
3
125
4
62.5
5
31.25
6
15.6
7
7.8
In addition, prepare a tube #8 with 25 l water (infinite dilution).
Protein
Prepare diluted solution of the protein (working solution) as before (see binding exp.)
Experimental mixtures
Prepare two equal 10 l aliquots of each Cold mannose mixture in new tubes and add (1) 90
l protein working solution and (2) 10 l of 5-times diluted “Hot+Cold” stock into all these
tubes. You will have 16 mixtures, having protein concentration of about 0.4 M, “Hot+Cold”
mannose of 7.75 M, and following final concentrations of the inhibiting Cold mannose (in
M):
45.5
45.5
22.7
22.7
11.36
11.36
5.68
5.68
2.84
2.84
1.42
1.42
0.71
0.71
0
0
Next,
1.
2.
3.
4.
Incubate the mixtures at room temperature for 30 min
Filtrate the mixtures through the nitro-cellulose filters (as before)
Dry the filters for 1 h
Place the filters into the counting tubes containing 5 ml scintillation solution. Incubate
for 30-60 min. Place the tubes into -radiation counter racks and start counting by
choosing the “START” mode and pressing “1” and “R” subsequently. Help the old
device to move the racks and print the results (this will be demonstrated)
Calculations (use MS-Excel and Origin)
1. Estimate average values (you have two repeats for each mannose concentration). Plot the
values against Cold mannose concentration (45.5, 22.7 …M). Fit the data into the
following equation:
y=P1/(1+x/P2), where P2 should be equal to IC50.
2. Calculate the dissociation constant using equation Ki=IC50/(1+LT/Ki).
3. Switch from the linear scale to the logarithmic scale for the concentration of the
inhibiting Cold mannose in the the plot. Approximate the data with sigmoid and determine
IC50 and Ki again. Are the new values similar to those obtained from the hyperbolic
approximation? Is the Ki value similar to that of Kd obtained in the binding experiment (2-3
times difference is acceptable)?