Download Equilibrium and Free Energy of Protein Denaturation

Estimation of the Free Energy of Stabilization of a Folded Small Globular Protein, Myoglobin:
Determination of the Denaturation Equilibrium
Introduction:
The denaturation of some proteins can be described by a two-state transition model in which the protein exists in either
the native (N) or completely unfolded, denatured (D) conformation. In large and more complex proteins, there may be
multiple unfolding intermediates where only part of the protein is unfolded or the protein adopts a different conformation.
With these more complex protein structures the two-state transition may not apply and it may be necessary to account for
multiple intermediates. The two-state transition model however, can often be applied to small globular proteins. Acid
(pH), heat, and denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate are can be used to unfold and
denature the protein. The denaturation process can be monitored by changes in physical properties of the protein that
can be measured. Spectroscopic changes such as UV absorbance of amino acid side chains or other chromophores
attached to the protein, changes in the fluorescence spectrum, or changes in circular dichroism (the way the protein
interacts with circularly polarized light) have been used to monitor the native and denatured states of the protein. The
aromatic amino acids that are responsible for the absorption spectrum in the UV range are usually buried when the
protein is in the native conformation. Upon unfolding, these amino acids are exposed to solvent and may show a change
in both the wavelength of maximum absorbance and the molar absorbtivity due to the change in chromophore
environment. If the protein has tryptophan residues there is often a change in the fluorescence of the protein. The
denaturation of ribonuclease A, lysozyme, α-lactalbumin and myoglobin by urea and guanidinium chloride fit the two-state
model and show maximum differences in molar absorbtivity at 287, 292, 292 and 409 nm respectively. Chromophores for
ribonuclease A, lysozyme, and α-lactalbumin are aromatic amino acids while the chromophore for myoglobin is heme.
native configuration
denatured
A protein exists in its most stable form. Under biological conditions, this is the folded native state that retains biological
activity. In this experiment, you will determine the free energy that is required to convert the protein from its native
structure to the denatured state. This requires determination of the equilibrium distribution of the protein between the two
states. This will be done in the presence of increasing amounts of denaturant.
Solutions:
1)
2)
3)
0.1M NaH2PO4•H2O with 0.1 M KCl, pH 6.6 (NaOH)
9 M urea in buffer (This solution is nearly saturated. Warm to dissolve and then cool to
room temperature before use.)
protein stock solution - 0.5 mg myoglobin/mL buffer (pH 6.6). Do not shake the protein solution or cause foaming
of the solution. Filter through a small plug of cotton to remove insoluble material.
Abs409nm of 1:10 dilution should be 0.4 to 0.8.
Procedure:
Part 1:
A) Prepare solutions 1-25 as shown in the table below. Calculate the urea concentration for each solution. Mix buffer
and denaturant first, then add the myoglobin. ACCURATE PIPETING IS ESSENTIAL FOR GOOD RESULTS. Allow
solutions to stand for at least 60 min. before reading absorbances. n. Read absorbance at 409 nm.
Solution
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
PBS, pH 6.6
mL
2.70
2.50
2.30
2.10
1.90
1.70
1.60
1.50
1.40
1.30
1.20
1.10
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
9 M urea
mL
0.00
0.20
0.40
0.60
0.80
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.60
2.70
Mb,
0.5 mg/mL
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.30
0.80
4.00
1.20
calculated urea
concentration, M
Abs at 409 nm
2.00 mL solution #24 + 2.00 mL buffer
B) Analyses like the one performed in this lab should include a demonstration that the transition between native and
denatured state is reversible. Solution 24 represents a protein that was denatured at a high urea concentration and then
presented with a diluted urea solution which should allow rapid reversal of denaturation upon decrease in denaturant
concentration. Prepare solution 24 and allow to sit for 45 minutes, then dilute to prepare solution 25. Allow this to sit for
45 minutes before reading absorbance. Solution 5 corresponds to a partially denatured protein. The absorbance of tube
24 should be divided by two for comparision with tubes 5 and 25. Does your protein renature?
Part 2:
Obtain a uv-visible spectra of the native and denatured protein to show the spectral differences in the
two conformations. Use samples 1 and 18 and the visible to near uv region.
Calculations:
A.
Determine Kd: Plot absorbance as a function of denaturant (urea) concentration. The measured absorbance at
each urea concentration is Y. Plotted data should generate a graph like the one below. To calculate Kd you also need the
values for YD (absorbance due to denatured) and YN (absorbance due to native) at each urea concentration. This is done
through extrapolation. At very low urea concentration, the majority of the protein is still in the native configuration and the
measured absorbance (Y) is effectively YN. A line through these points and extrapolated to higher urea concentrations
can be used to determine YN at increasing urea concentrations. In a similar fashion, at high urea concentration, most of
the protein is denatured the measured absorbance (Y) is effectively YD. A line through these points and extrapolated to
lower urea concentration can be used to determine YD at lower urea concentrations. Because at low urea, the amount of
denatured protein is low and hard to determine and because at high urea, the amount of native protein is low and difficult
to determine, those points are only useful for the extrapolation and should not be used to find Kd. The range of interest for
determination of Kd is the linear range where there is a rapid change in absorbance.
B.
Determine ΔGd:
Calculate the fraction of the protein that is denatured at each urea concentration as measured by absorbance using the
formula
Yobs - Yn
fd = Y - Y
d
n
where fd is fraction denatured, Yobs is the observed quantity (absorbance), and Yn and Yd are the values for the native and
fully denatured proteins at each of the urea concentrations, respectively.
Plot fd vs urea concentration.
Calculate the equilibrium ratio for each concentration of urea using the formula
[D]
fd
Yobs - Yn
Keq = [N] = f = Y - Y
n
d
n
Calculate the free energy change for denaturation at each denaturant concentration.
∆Gdenaturation = -RT(ln Keq)
If we assume that ln Keq (and ΔGdenaturation) is a linear function of the denaturant concentration, a plot of ΔGdenaturation versus
urea concentration will be a straight line and the y-intercept will be an estimate of the free energy of denaturation of
myoglobin in the absence of urea, i.e., in buffer alone.