Investigating the denaturation of Myoglobin using Fluorescence – Insights

into Protein Unfolding

The goal of this experiment to probe the denaturation of the heme protein myoglobin through
chemical denaturation and monitor the same using fluorescence. Protein fluorescence derives
from the aromatic amino acids tryptophan, tyrosine, and phenylalanine, but it is usually
dominated by tryptophan fluorescence. The sensitivity for tryptophan fluorescence exceeds
that of the other aromatic amino acids owing to its greater fluorescence quantum yield and
increased molar absorptivity.
In today’s experiment, you will be using equine skeletal myoglobin (Mb) that has two
tryptophan (Trp) residues. The protein will be subjected to different concentrations of the
chemical denaturant urea, with the concentration of urea being varied from 0 – 8 M. In the
native conformation of myoglobin, however, the tryptophan fluorescence is essentially
completely quenched; overlap between the fluorescence emission band (max ~350 nm) of
tryptophan and the   * absorption band (max ~350 nm) of heme allows efficient energy
transfer and leads to a low fluorescence quantum yield.
Energy transfer (Förster Resonance Energy Transfer- FRET) is critically dependent upon the
distance between the fluorescing tryptophan residues and the heme prosthetic group. As the
protein denatures, the aromatic amino acid–heme distance increases and the solvent sensitive
fluorescence increases in intensity and shifts in wavelength. Fluorescence emission maxima
typically occur at ~330 nm for tryptophans in nonpolar environments (such as the protein
interior), whereas maxima at ~350 nm are typical for solvent-exposed residues. At zero and
low concentrations of denaturant (conditions that favor the native state) only very weak, if
any, fluorescence is observed for myoglobin solutions. At higher urea concentrations, a
wavelength shift in the fluorescence maximum occurs with a concomitant increase in the
fluorescence intensity as the aromatic amino acid residues are exposed to the solvent in the
unfolded protein.

Experimental Protocol:
Solution preparation
 Prepare phosphate buffer having pH around 7.4
 Use the buffer to prepare urea concentrations in the range of 0 – 8 M at 1 M intervals.
To ascertain the urea concentrations so prepared, measure the refractive index of the
buffer (without urea) and all the urea solutions prepared. Subsequently, use the table
provided in the following link (http://sosnick.uchicago.edu/gdmcl.html) to get the
actual concentration prepared.
 Dissolve equine skeletal myoglobin (Mb) in each of the above solutions such that the
final concentration of Mb is in the range of 20 - 25 M.
 Allow the solutions to equilibrate for at least 30 minutes.
 Collect the absorption spectrum of each of your samples using tha appropriate blank.
 Proceed for the fluorescence measurements.
Fluorescence measurements:
 You will need two quartz cuvettes, of pathlength 1 cm, polished on all 4 sides.
 One cuvette will be having your blank (buffer + urea) for which you will take the
emission spectrum.
 Take about 2.5 mL of your sample in the second cuvette and measure the emission
spectrum
 Set the excitation wavelength at 295 nm (to avoid excitation of tyrosine residues)
 Collect the fluorescence spectrum of each sample (and blank) within the range of 310
– 560 nm at 1 nm intervals. Set the excitation and emission slits appropriately. In case
the spectra are noisy, increase the integration/dwell time for spectra acquisition.
 While reporting your final spectrum, make sure to subtract the emission spectrum of
the blank from that of the sample.

Theory
A two-state system will be assumed as follows:
N ❑D (1)
↔

with N being the native state of the protein and D being the denatured state, and both of these
are in equilibrium with each other.
The equilibrium constant K is
[ D]
K= (2)
[N]
The fraction of the unfolded state so populated as a function if increasing concentration of
urea can be expressed as
I N −I
f D= (3)
I N −I D

Where I is the measured fluorescence intensity (at 340 nm) observed at a given urea
concentration, IN is the fluorescence intensity of the native state (at 340 nm); and ID is the
fluorescence intensity of the fully unfolded state (at 340 nm), obtained by extrapolation from
the linear regions of the denaturation curve at low and high urea concentrations.

Data Analyses
 Report the equilibrium concentration in a tabular form for each urea concentration
 Plot the (i) absorption spectra and (ii) emission spectra of all your samples. Try to plot
these in two separate panels, one for absorption and one for fluorescence, as a
function of the urea concentration.
  Plot the denaturation curve using I340 (fluorescence intensity at 340 nm) against urea
concentration.
 An alternate way of plotting the denaturation curve is to, based on equation 3,
calculate fD for each urea concentration and plot it as a function of the chemical
denaturant.
 In the experimental section, make sure to mention all he parameters that you used to
collect the emission spectra of the samples.

Discussion:
1. Explain the changes in the emission spectra as a function of the denaturant
concentration. Do you expect to see such changes happening for all proteins
containing Tryptophan residues when denatured by urea?
2. What information can one get from the denaturation profile? From the denaturation
profile, estimate the chemical denaturation midpoint.
3. Explain the probable mechanism by which urea denatures a protein. What is the other
commonly used chemical denaturant and its probable mechanism.
4. What is FRET? Explain the principle of the same and its application in understanding
the conformations of biological macromolecules.

References:
(i) Miao, Y. and Thomas, C. L. Using Myoglobin Denaturation To Help
Biochemistry Students Understand Protein Structure, Journal of Chemical
Education, 2017, 94, 1498−1501.
(ii) Jones, C. M. An Introduction to Research in Protein Folding for Undergraduates,
Journal of Chemical Education, 1997, 74, 1306−1310.