Tuesday, April 25, 2017

Week of 4/23/17

Today was prep for my poster session, which is this Thursday. I am very excited to present at RPI as part of the CBIS High School Scholars Program. Rather than describe my preparations, I thought I would post my notes here for you to read and give an idea of what I am going to say this Thursday when I present.

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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

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I hope my notes are somewhat intelligible. I am very excited to present my poster!


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