---------------------------------------------------------------------------------------------------------------------
Protein folding is one of the most important biological
processes
For many proteins to perform their biological processes,
they must fold into a specific 3D structure
This process is called protein folding – are interested in
how folding processes throughout this process
If we want to study protein folding, we need to perturb this
process – otherwise it is stuck at equilibrium
There are sever types of perturbants
Chemical denaturants (urea, guanidine hydrochloride)
Temperature, changing pH
But in our lab, we use pressure
Talk about how pressure perturbs folding – due to internal
cavities in internal structure
In the folded protein, packing is imperfect
In folded structure, due to imperfect packing of atoms,
there are solvent excluded cavities (where water cannot enter)
Pressure acts to eliminate these cavities (bc pressure
minimizes volume) and unfold the protein
From this pressure study, we can obtain volumetric
properties of the protein
Pressure only acts on the cavities – cavities are different
for each protein – this way, pressure perturbs cavities of different proteins
differentially
Pressure effect has local feature, therefore it reveals more
detailed information regarding the protein folding process
Why not chemical? Temperature?
Chemical has global effect
Protein we study: PP32
PP32 belongs to acidic nuclear phospho protein family
Tumor suppressor – good for cancer research (biological
significance)
We’re more about physical significance
Leucine-rich pp protein
Repeat protein (5 repeats – arrows) conserves leucine
residues within repeats
Repeats are similar in structure and sequence (look similar
– leucine rich at one part of arrow? Same
for next arrow)
Cavity in center of structure (empty – no water) spheres
We study this protein because it is a repeat protein so its
overall architecture is linear and simple – easy to study
Simple linear architecture abundant in local contact,
lacking global interaction (by folding studies)
Protein folding is matter of structural energetic
interaction
Also good for NMR – use fluorescence (tryptophan, terasine,
phenylalanine)
Only rings can accept fluoresce
Quantum yield – how much light is emitted (give 10 photons,
how may do you get back)
Tryptophan has higher quantum yields than other two – so low
we can’t see it
Unfortunately, there is no tryptophan in this protein
Tryptophan residue introduced at c terminus for fluorescence
measurements
Yellow c terminus
PP32 has two capping domains – on two termini (stabilize
protein)
Two black sticks are two residues: aspartic acid 146 and
tyrosine 131
Numbers are residue number (area around first top loop is
approx. 18)
Why are they there? Side chains close to one another- hydrogen bonding between the two
Keeps the structure stable and stabilizes the entire protein
(there naturally)
How the fluorescence works
Tryptophan likes to be excited with wavelength 290
nanometers
Gives emission spectrum which are dependent on micro
environment around that residue
Environment is determined by protein structure
Therefore when proteins are folded and unfolded, we get
different emission spectra (different
environment)
Folded is lower peak –
Usually, folded has higher peak, but in this case – we think
there are histanine residues around the
tryptophan which quenches the fluorescence
(when it is folded) – when protein unfolds, quenching effect dissipates
Folded and unfolded states have different emission spectra –
can be differentiated
With the mutations (change Y131 to F and D146 to L) – we can
break up the hydrogen bond and unfold the protein
Take fluorescence measurement, increase pressure to new
level, protein unfolds to some degree, let it
reach new equilibrium, take next
fluorescence measurement, repeat
That’s how the instrument works -> water pumped in and
increases pressure
For each measurement, we increase the pressure and protein
starts to unfold -> takes time to unfold
Excitation wavelength at 290 nm
In fact, when Yi increased pressure, he didn’t take full
emission spectra – takes too long
Just one – 340 nm
Averaged equilibrium values for intensity graph
Protein dissolved in urea (helps to unfold), water, bis-tris
(pH buffer) – more denaturant added, easier for pressure to unfold protein, DTT
(reducing agent – prevents proteins from aggregating)
Urea facilitates protein unfolding
pH 6.8 20 degrees
What we did: took emission spectra of this protein after the
system reaches equilibrium
How do you know it reached equilibrium?
Monitored intensity at 340 nm as function of time ->
signal doesn’t change anymore
Take intensity at 340 nm and plotted it as a function of
pressure – going past 340, intensity is the same (asymptote)
At each pressure, take the value at 30 nm -> plot 340 as
function of pressure
Sigmoidal (s curve)
Unfolded – higher value
Folded – lower value
Two state model – looks at unfolded versus folded (we follow two state mode to analyze data)
Transition state is population weighted average of two
states – percent unfolded and percent folded average gives transitional value
Delta g value varies with amount of urea
---------------------------------------------------------------------------------------------------------------------
I hope my notes are somewhat intelligible. I am very excited to present my poster!
