1.

A protein having single tryptophan residue shows fluorescence emission

spectrum with λmax ~325 nm in water.
a. What could be the reason for the large blue shift observed as compared to
isolated tryptophan molecule?
b. Which fluorescence method would you employ to justify your answer?
Explain the expected observation from the experiment.

a. Tryptophan is buried inside the hydrophobic core of the environment.

b. Fluorescence quenching:

A buried Trp will not be accessible to an aqueous quencher and therefore

little or no decrease in fluorescence is expected.
2. You are working with a protein. You believe that the protein undergoes large
structural changes when it binds to a drug molecule. You synthesise the
protein mutant wherein you incorporate a Trp residue and a Cys residue
through site-directed mutagenesis. You label the cysteine residue with dansyl
moiety (Dansyl makes a FRET pair with Trp as the donor). The distance
between the two sites in the absence of drug molecule is 20 Å. The spectra for
unlabeled (only Trp, no dansyl) and the labeled protein recorded in the
presence of large excess of drug molecule are shown below. Answer the
following questions if the Förster distance is 21 Å, and the donor-alone
lifetime is 3 ns.

a. What is the D–A distance?

b. What do you conclude from the distance?
c. Why was large excess of drug used in this experiment?
d. What is the donor lifetime in the Donor-Acceptor pair?
e. Do you think, measuring fluorescence lifetime rather than intensity is a
better way to determine the FRET efficiency?
 a. What is the D–A distance?

The efficiency of energy transfer, E is given by:

𝑅06
𝐸= 𝑅06 + 𝑟 6

Given: E = 90% = 0.9, R0 = 21 Å

Rearranging the expression for the efficiency of energy transfer
1
𝐸= 𝑟 6
1+ 𝑅
0

1
0.9 = 𝑟 6
1+ 21

𝑟 6 1
1+ = = 1.111
21 0.9

𝑟 6
21
= 1.111 − 1 = 0.111
1
𝑟
= 0.111 6 = 0.693
21

𝑟 = 0.693 × 21 = 14.55 Å
b. What do you conclude from the distance?
The two sites come close to each other upon drug binding.

c. Why was large excess of drug used in this experiment?

To ensure that there is no free protein.
 d. What is the donor lifetime in the Donor-Acceptor pair?

𝐹𝐷𝐴 𝜏𝐷𝐴
𝐸 =1− 0.9 = 1 −
𝐹𝐷 6.8
Γ 𝜏𝐷𝐴
Γ + 𝑘𝑛𝑟 + 𝑘 𝑇 = 0.1
𝐸 =1− 6.8
Γ
Γ + 𝑘𝑛𝑟

𝜏𝐷𝐴 𝜏𝐷𝐴 = 0.68 ns

𝐸 =1−
𝜏𝐷
𝐹𝐷𝐴 𝜏𝐷𝐴
Or directly =
𝐹𝐷 𝜏𝐷
Everything except 𝜏𝐷𝐴 is given

e. Do you think, measuring fluorescence lifetime rather than

intensity is a better way to determine the FRET efficiency?

Yes. Because fluorescence lifetime is independent of fluorophore

concentration.
Pyrenylmaleimide is a convenient fluorescent probe for labeling sulfhydryl residues on
proteins. The fluorescence lifetime of pyrene on a protein sulfhydryl was measured by
determining the relative fluorescence intensity after excitation of pyrene with a flash
lamp. Typical data are given below.
Relative fluorescence Time (ns)
0.716 20
0.513 40
0.264 80
0.189 100

a. Determine the fluorescence lifetime.

b. If the quantum yield is 0.7, what is the natural lifetime?
c. When a ligand is bound to the protein, the fluorescence lifetime is 10% shorter.
What might be the cause of this change?

𝑡
−𝜏
𝐼 𝑡 = 𝐼0 𝑒
You are provided with the
𝐼(𝑡) 𝑡
−𝜏
I(t)/I0 values, just plot ln(I/I0)
=𝑒 against t. For a single
𝐼0
exponential decay, the plot is
𝐼(𝑡) 𝑡 a straight line and slope of the
𝑙𝑛 =−
𝐼0 𝜏 line is (-1/τ).
 𝐼(𝑡) 𝐼(𝑡)
Relative fluorescence Time (ns) 𝑙𝑛
𝐼0 𝐼0
0.716 20 -0.33
0.513 40 -0.67
0.264 80 -1.33
0.189 100 -1.67

1
− = −0.0167
𝐼(𝑡) 𝜏
𝑙𝑛
𝐼0
1
𝜏= = 59.88 𝑛𝑠
0.0167
b. If the quantum yield is 0.7, what is the natural lifetime?

Natural lifetime τn, is the lifetime when all energy is lost through fluorescence i.e.
when all competing processes can be ignored.
𝜏
Natural lifetime, 𝜏𝑛 =
𝑄

59.88
Therefore 𝜏𝑛 = ≈ 85.5 𝑛𝑠
0.7

c. When a ligand is bound to the protein, the fluorescence lifetime is 10% shorter. What
might be the cause of this change?

The ligand could be a FRET acceptor.

4. Completely polarized emission (i.e. anisotropy = 1) is never observed
for fluorescence from homogeneous unoriented samples. Why?

This is because of photoselection during excitation. The molecules that

are not parallel to the plane of polarized light also get excited with cos2
dependence, where  is the angle between the electric field vector of the
plane polarized light and the transition dipole moment of the molecule.

Emission from these molecules will result in perpendicular component

of intensity, thereby causing anisotropy <1.

In fact, the maximum anisotropy for an isotropic solution is 0.4; that too
when the molecules are assumed to be static and the absorption and
emission transition dipoles are parallel.
5. The figure on the right shows the binding of a 16-
residue peptide with a protein. This peptide contains
a single tryptophan residue, which is placed at
positions 1 through 16 in 16 different peptides. This
peptide binds to the hydrophobic patch of
calmodulin. Explain the changes in emission maxima,
Stern-Volmer quenching constant for acrylamide (K),
and anisotropy (r).
The changes in λmax, K, and r are the result of the tryptophan
residue being exposed to or shielded from the water. Increases in
λmax and K indicate increased exposure to water, and decreases in
λmax and K indicate decreases in exposure to water. Increases and
decreases in r indicate a less mobile and more mobile tryptophan
residue, respectively.

The three parameter values show a cyclical behaviour with a period

of about 3.5 amino acid residues per cycle. This suggests that the
peptide is in an α-helical state when bound to protein. The spectral
changes seen are the result of the tryptophan residue being
alternately exposed to water or shielded as its position is shifted
along the peptide chain.
6. A short peptide having a single tryptophan residue is quenched using KI. KNO2 was used to
maintain the constant ionic strength at all quencher concentrations. The quenching data is
given below:
a. Construct a Stern-Volmer plot.
b. Is the quenching dynamic, static or both?
c. Determine the quenching constant(s) (KD if quenching is dynamic, KS if quenching is static,
both KD and KS if both types of quenching takes place).
d. Calculate the observed bimolecular quenching constant. The unquenched lifetime τ0 = 12
ns.
e. Calculate the diffusion limited bimolecular quenching constant and the quenching
efficiency (Given: collision radius (Rf + Rq) is 4 Å, diffusion coefficient of KI = 2 × 10-5
cm2/s, diffusion coefficient of peptide = 10–4 cm2/s).
Concentration of Concentration of Fluorescence
KI (M) KNO2 (M) intensity (AU)
0 1.0 1000
0.05 0.95 204
0.1 0.9 107
0.2 0.8 50
0.3 0.7 30
0.4 0.6 20.5
0.5 0.5 15
0.6 0.4 11.4
0.7 0.3 9.1
0.8 0.2 7.4
0.9 0.1 6.2
1.0 0 5.2
a. Construct a Stern-Volmer plot.

250

200 y = 1.2917x2 + 0.7707x - 2.4448

150

100

50

0
0 5 10 15

b. Is the quenching dynamic, static or both?

The curvature towards F0/F axis suggests that the quenching is both
static and dynamic.
 c. Determine the quenching constant(s)
Quenching constants can be determined by using modified Stern-Volmer equation and
plotting Kapp against [Q].
Given, F0 = 1000 AU
KI conc. Kapp= [(F0/F)-
F F0/F 200
(M) 1]/Q
y = 119.41x + 71.77
0.00 1000.00 1.00 180 R² = 0.9998
0.05 204.00 4.90 78.04
0.10 107.00 9.35 83.46 160
0.20 50.00 20.00 95.00
0.30 30.00 33.33 107.78 140

Kapp
0.40 20.50 48.78 119.45
120
0.50 15.00 66.67 131.33
0.60 11.40 87.72 144.53 100
0.70 9.10 109.89 155.56
0.80 7.40 135.14 167.67 80
0.90 6.20 161.29 178.10
1.00 5.20 192.31 191.31 60
0.00 0.50 1.00 1.50
[Q]
The intercept gives, K1+K2 = 71.77 M-1 (1)
The slope gives, K1K2=119.41 (2)
Substituting the value of K1 = 71.77‒K2 in equation (2)
(71.77-K2)K2 = 119.41 K22 -71.77K2+119.41 = 0 K2 = 1.70 M-1, 70.06 M-1

The two quenching constants are 70.06 M-1, 1.70 M-1

d. The bimolecular quenching constant kq = KD/τ0

Assuming, KD = 1.70 M-1

kq = 1.70 M-1/12 ns = 0.14 × 109 M-1s-1 = 0.014 × 1010 M-1s-1

Assuming, KD = 70.06 M-1

kq = 70.06 M-1/12 ns = 5.84 × 109 M-1s-1 = 0.58 × 1010 M-1s-1

As kq obtained using KD = 70.06 M-1 is comparable to the diffusion controlled

collisional quenching, this is the bimolecular quenching constant.

Further, this establishes that the dynamic quenching constant, KD = 70.06 M-1 while
static quenching constant, KS = 1.70 M-1.
e. The diffusion limited bimolecular quenching constant, k0 is given by:

k0 = 4πRND/1000 =(4×3.14×4×10-8 cm× 6.022×1023×1.2×10-4 cm2s-1)/1000

=363 × 108 M-1s-1

=3.63 × 1010 M-1s-1

Quenching efficiency = (0.58 × 1010)/( 3.63 × 1010 M-1s-1)= 0.16

7. The binding of a small fluorescent dye with a protein is studied using
fluorescence anisotropy. The data is given below.

[Fluorophore] M [Protein] M r
1×10-7 0 0.01
1×10-7 2×10-5 0.20
1×10-7 >>Kd 0.30

a. Determine the dissociation constant (Kd) for the binding assuming that the
bound and free dye has same quantum yield.
𝑓𝐹 𝑞𝐹 𝑟𝐹 + 𝑓𝐵 𝑞𝐵 𝑟𝐵
The anisotropy is given by 𝑟=
𝑓𝐹 𝑞𝐹 + 𝑓𝐵 𝑞𝐵

𝑓𝐹 𝑟𝐹 + 𝑓𝐵 𝑟𝐵
If 𝑞𝐹 = 𝑞𝐵 , the equation reduces to 𝑟=
𝑓𝐹 +𝑓𝐵

If 𝑓𝐹 +𝑓𝐵 = 1

𝑟 = 𝑓𝐹 𝑟𝐹 + 𝑓𝐵 𝑟𝐵
 [Fluorophore] M [Protein] M r
1×10-7 0 0.01
1×10-7 2×10-5 0.20
1×10-7 >>Kd 0.30

When [BSA] = 0 one observes 𝑟𝐹 , and when [BSA] >> Kd one observes 𝑟𝐵

Therefore,
𝑟 = 𝑓𝐹 𝑟𝐹 + 𝑓𝐵 𝑟𝐵

𝑟 = 𝑓𝐹 (0.01) + (1 − 𝑓𝐹 )(0.3)

𝑓𝐹 = 0.345 𝑓𝐵 = 0.655
𝑃 𝐹
𝐾𝑑 =
𝑃𝐹
Since the concentration of protein is much higher than the dye concentration of
the free protein can be assumed to be not depleted by the binding of the dye.

Therefore,
2 × 10−5 𝑀 0.345
𝐾𝑑 = = 1.05 × 10−5 𝑀
(0.655)
b. Determine the dissociation constant (Kd) for the binding assuming that
the bound and free dye has same quantum yield.

If the relative quantum yield of the bound probe is twofold larger

than the free probe, then clearly the concentration of the bound form
is twofold lower.

Therefore,

2 × 10−5 𝑀 0.345
𝐾𝑑 = = 2.1 × 10−5 𝑀
(0.655/2)