Exam 2023 Flashcards
d) From Fig. 3 describe which amino acids are most important for forming native contacts in the transition state of the folding process (red color amino acids show abnormal ф – values and can be ignored in this exercise!). You do not need to give the exact number of the amino acids but you should tell their name/color and explain where approximately in the structure they are located.
The figure then shows a protein which amino acids labelled by color ranging from white corresponding to 0.0 in ф value and 1.0 dark blue in ф value.
Which amino acids are most important for forming native contacts, why and what does native contacts even mean?
When an unfolded protein start to fold to form the native protein the proteinis undergoing structural rearrengment to attain native confirmation. During this transition state native contatcs sart to form, which contributrs to stabilization of native structure.
So high ф indicicate these amino acids are crucial to form native contacts during the folding process which stabilizes the structure and enables continious correct folding to form the native protein.
The value of βT (between 0-1) is a measurement of exposed surface in the transition state and is calculated by the formula:
BT=1-mu/(mu-mf)
The βT- value for WT GlpG was found to be 0.23 and the folding stability in water ΔGH2O = -7.39 kcal/mol. Draw a schematic folding energy diagram of the wild type protein. What conclusions can be drawn about the structure of the transition state from this βT value?
What does this formula with mu and mf and BT mean and how would you plot this?
How does BT value change the apperance of the plot?
The plot is G on y axis and reactions cordinate on x axis. x axis is mf then a line down te middle of the transition phase then mu. mf is the unfolded state and mu the folded. deltaGH20 is the energy difference of unfolded and folded. Large negative values means bigger difference of energy between unfolded and folded.
Larger BT means mf is larger and mu smaller smaller BT means the opposite. Larger BT value means transistion state resembles native state and smaller means it resembles the unfolded more.
a) Above picture shows a secondary structure representation of the designed, symmetric, single domain globular protein Symfoil-4T. Mutant positions that were utilized in an φ-value analysis are highlighted and colored to indicate the experimentally derived φ-value (Blaber et al 2015 The protein society). Describe the structure of the transition state in the folding pathway of this protein assuming a two-state folding/unfolding mechanism. Motivate your answer.
It then shows a diagram of the protein with color coded proteins based on their phi values ranging from 0-1
High phi values are most important for foring native contacts which will stabilize the transistion state and help the proten form it’s native structure.
The value of βT (between 0-1) is a measurement of exposed surface in the transition state and is calculated by the formula:
The βT - value was found to be 0.71 and the folding stability in water ΔGH2O = -44.5 kJ/mol. Draw a schematic folding energy diagram of the Symfoil-4T protein in water. What conclusions can be drawn about the structure of the transition state from this βT value?
A βT value of 0.71 means it’s quite high so smaller mf is larger then mu
b) Show how the mutations at positions 39, 62 and 115 affect the energy diagram of WT.
So there is a mutated and wild type protein
39 has 0 phi value, 62 1 and 115 0.5
The plot for wild type and mutant will follow the typical denatured, transisiton state, native curve with deltaG on y axis and reaction cordinate on x axis.
0 phi value means they touch during transistion stage and has no native contacts, 1 means the space between transistion state and folded protein is equally big and ithad formed all it’s native contacts during transistion state 0.5 means the transistion state is half the size f the folded protien space and it has formed half of it’ native contacts.
How do you analyze kex experimentally?
Amide proton exchange experiments involve the determination of the rate constant (kex) for the exchange of amide protons between the solvent and the protein. The experimental procedure includes labeling the protein with 15N dissolving the protein in a deuterated buffer (D2O), and recording HSQC spectra over time. The decrease in intensity of individual cross peaks in the spectra is analyzed, and the curve is fitted to a single exponential decay to calculate Kex
what does native contact mean?
Native contacts means the contacts that the
amino acid has in the fully folded (native)
protein - i.e. both secondary and tertiary bonds.
a amino acid with a high phi value means it’s important for forming native contacts during protein folding which means it creates stability during the protien folding process to ensure the unfolded protein can fold correctly and reach native state
write a plot for kinetic analysis of the folding if we have the infomration that it’s a wild type protein, lnkfh20 is -1.42, lnkuH20 is -9.64, mf -1.68 and mu 0.5 deltaGH20 -20.6kJ/mol and BT=0.77
Writing a plot like this y axis will be G and x axis reactions coordinate. it also means that a lot of the native contacts has been formed during the transisiton state so the structure of the protein is compact and resembles the native protein. deltaGH20 is -20.6kJ/mol which is the difference in free energy before and after the unfolding to folding. So the more energy is released during folding the larger the gap will be between these two stages. The difference between U at the beginning and the transition phase if kfh20 and the Kuh20 is the difference between the energy of the folded and transistion state. This means that there is a lot larger energy difference between the folded state of the protein compared to the transisiton phase(kuH20) then the kfH20 unfolded and the transistion state.mf will always be larger then mu.
We have a wild type protein and a mutated protein. The lnkfh20 is -1.42 for wild type protein and -2.49 for mutated protein. lnkuH20 is -9.64 for wild type and -9.62 for mutated. The formula is phi=…
The phi value is 0.98
How would the plot look, and interpret what the data point means
They always start at the same energy level in the plot (G in y axis, reaction cordinate on x axis, mf then transistion point then mu as arrows underneath the energy diagram) but lnkFH20 the mutated protein will need more energy to reach transistion state since it has a larger - value then wild type and then it’s folded state lnkuH20 is almost the same. This means that the mutated protein will reach a higher energy state during transistion but will go down equally much as the wild type but as it reaches a higher energy state the line on the plot for the mutated one will be above the wild type line. Phi is 0.98 so that shows that the difference between transistion state and folded protein is almost equally large between wild type and mutated protein. A high phi value also shows that it has formed almost all native contacts during the transistio state. mf will always be larger then mu.
There is one native protein and one wild type. lnkfH2O is -1.42 for wild type and -1.45 for mutated. lnkuH2O is -9.64 for wild type and mutated one is -8.41. Phi is 0.02. What does this mean and how will energy plot look?
phi is 0.5 how does the plot look then+
since phi is so low no native contact is formed for mutated protein during TS and therefore transistion state they will touch the liner and after transistion state there will be a difference in energy states.
closeto 0 they touch at transistion state then large differene, 0.5 the space at folded state is twice the size of transistion state space and close to 1 equally large
What is the difference between krc and kex
- krc is the exchange of the amide proton when fully exposed or completely unfolded
- krc has been determined by measure exchange on small peptides and it
depends on the adjacent amino acids, temperature and pH. - kex is the experimentally determined exchange at it depends on the
opening event of the structure and on krc. so it measures the overall proton exchange in both exposed and partially exposed regions.
Three ways for the NH to be exposed to the solvent
How to calculate keq?
Calculste ∆Gop?
The amide proton can be exchanged as a result of global unfolding, and that it Krc when the protein has been completely unfolded and there exchanges protons and then refolds. Then exchange can occur during partial unfolding before it refolds, but also fluctation. kex is for all these three ways of amide exchange.
use the formula Keq=kex/krc
Use the formula -RTlnkex/krc
Question 2.
For some proteins the folding process occurs via an intermediate state that is
called molten globule. Describe the structure of this intermediate.
- Compact structure
- Containing most of its secondary structure
- Lacking tertiary structures
- Has a hydrophobic core
sometimes the protein doesn’t have a two state folding/unfolding but an intermediate state. The intermediate state has gotten it’s secondary state such as alpha helixes and beta sheet sbut not yet the ertiary stucture as that refers to the overall full protien structure.
Question 3.
You have decided to do the experiment amide proton exchange to look at the stability of your protein at
individual amino acid position.
Question 3a. Describe the experimental procedure to determine the value of kex.
kex looks at amide proton exchange in bort the completely unfodled and partially unfolded regions. To achieve this the protein is:
Label your protein with 15N
* Dissolve your protein in D2O buffer
* Start to record HSQC spectra as many as possible over time
* Analyze the decrease in intensity over time for each individual cross peak
nitrogen-15 isotopes
Make the curve into a single exponential decay to calculate kex
nitrogen-15 isotopes is used to incorperate in the proteins backbone since it can be measured in NMR spectroscopy.
Using HSQC spectra you can measure the exchange of amide protons to deterium from D20 over time and the peks will decrease as a result of this.
You can then analyze theresult to see how fast and for which peak it went the fastest and slowest to shrink and this corresponds to how quickly the exchange rate was.
There are three main ways for the amide proton to be exposed to the solvent and these are
i) global unfolding, ii) partial unfolding and iii) local fluctuation. Describe what happens to the structure
of the protein during these three events.
Global unfolding, H to D exchange, refolding with D instead of H. Partial unfolding exchange refolding and fluctation it doesn’t denature it just exchanges H to D.
