Wxam 2022 Flashcards
1) Give a brief description of following terms and its application in Protein Chemistry
a) Isothermal calorimetry
b) Hill plot
c) Size exclusion chromatography
d) Ramachandran plot
e) Cooperative folding curve
a) IST measures binding and denaturing by measuring energy released and absorbed which occurs durnig reactions like this by measuring changes in temperature
b)hill plot analyzes cooperative binding and on y axis is the logarithm of fractional saturation and x axis logarithm of ligand concentration. A slope greater then one indicates positive cooperation and below one negative cooperetivity. Hill plot analyzes if there is a positve or negative cooperativirty while a schatchard plot shows cooperativity (no cooperativity with straight line) and cooperativity but not what kind with sigmodal line
c)Size excklusion chromotagpry is a type of chromotography (when there is a stationary and mobile phase used to purify samples) that seperates based on size so larger molecules such as proteins get stuck in the stionary phase and smaller molecules rmeoved with the mobile phase
d)ramachandran plot shows for amino acids what type of structure is allowed and not based on their proprties such as size for example small amino acids are more flexible then large ones. It shows phi and psi which is back bone angles.
e) a cooperative folding curve will have a sigmodal shape(sort of an s shaped curve) on a schatchard plot
2) Some proteins have cold denaturation i.e.; the protein unfolds at low temperature.
a) Describe the unfolding process when the temperature is decreased (cold denaturation)
– Which forces are important? (2p)
b) Describe the protein folding process when the temperature is increased again
- Which forces are important? (2p)
Some proteins but not all have cold denaturation. This is caused by the ice causing a more ordered water structure and this causes a loss of entropy which makes the protein less stable. It also causes more effective wander Waals interactions between the water molecules and non polar amino acids (and to some extent the polar ones too.)
At increasing temperatures, the entropy increases and the interactions between the water and amino acids Vanderwaals bonds becomes less effective which strengthens the intramolecular VdW interactions and encourages protein folding.
The driving force is enthalpic for protein folding snce the change in temperature change the bonds between the protein and the water.
a) Thermal induced protein unfolding curves can be analyzed by the
Van’t Hoff equation.
However, a plot of lnK versus 1/T is only linear around Tm (at the midpoint of the transition) and at
other temperatures the plot will be non-linear. Explain why.
The wan hoffs equation looks like: lnK=(-deltaH/RT)/(deltaS/R)
The van hoffs equationis tells us how changes in temperature will affect the equilbrium constant by how temperature will affecting enthalpy and entropy.
The heat capacity (Cp) which is the amount of energy it takes to raise the temperature with one degree is constant close to Tm. But far from Tm it will not be becasue there is a difference in how much heat it takes to raise the temperatue of anunfolded protein
The figure to the right shows the
unfolding curves of protein A measured by
Tryptophan fluorescence and by far UV CD.
From this figure it can be concluded that an
intermediate is formd during the
folding/unfolding process.
Draw a schematic energy diagram (Gibbs
energy (G) versus a reaction coordinate)
for the folding process of protein A during
native conditions.
Discuss the structure of the intermediate formed during the unfolding/folding process of protein A -
explain which parts of the protein structure that are unfolded and which parts of the protein
structure that are folded.
An unfolding curve for a protein denatring that isn’t a two step process would like like unfolded, activation energy intermediate which creates like a stop in the downwards curve, activation energy again and then folded going from high delta G towards low delta G. Delta G in y axis and raction coridnate in x acis.
To determine the binding of a ligand to protein the data below was obtained (Table1).
Use an appropriate graphical method to calculate the number of binding and binding
constant.
There is then a table with column 1 free ligand concentrations increasing and column 2 Rb which shows ligand bound to protein/total protein
What would be the steps to solve this excersise?
b) What type of binding is it?
- So we already have R that is [PL]/[P]total
- R/[L]free
- Plot it with R on y axis and R/L on x axis
- determine slope using y2-y1/x2-x1
- 1/slope=Kd
- (1/Kd)=ka
- Y intercept/slope=number of binding sites
- Look at shape of curve, straight line non cooperative, sigmodal line cooperative
The protein staphylococcal nuclease has been a model protein for protein folding studies. The
function of the protein is to degrade DNA, preferably single stranded DNA and its three-dimensional
structure was described as early as 1971 (Arnone A et al. (1971). J. Biol. Chem. 246 (7): 2303–2316.)
a) Based on the data from three-dimensional structure, amino acid composition and amino acid
sequence of protein staphylococcal nuclease (Figure 1 A-C) give some examples why this is an
excellent model protein for protein folding studies.
Reasons what would make a protein good for protein folding studies is tryptpohan so you can look at the flourescense during degredation and folding, not too big, monomeric and no cysteine which would form displfuldide bonds.
The stability of the protein Staphylococcal nuclease was investigated by chemical denaturation in
urea by monitoring the fluorescence intensity (excitation wavelength 295 nm).
Calculate the relevant parameters for the stability of the protein Staphylococcal nuclease, i.e. CMvalue,
m-value and DGH2O T=298 K and R=1,98 Cal/mol K) from table below (Table 1).
What would be the steps for this analysis?
1. Plot yobs as y and urea as x to find transition point
2. Calculate deltaG on trnasition point usnig the -RTln… formula
3. This will give you a striahg tline and at x intercept will be Cm and y intercept will be deltaGH20 the slope will measure cooperativity, high slope high cooperaitivity
In the above example the fluorescence intensity was monitored at various concentration of the
chemical denaturant, urea. Measuring the protein stability by monitoring the change in fluorescence
intensity can give complicated data to interpret, what is the reason for that?
Suggest other parameters to monitor when using fluorescence instead of fluorescence intensity to
obtain a stability curve.
It’s better to study maximum emission then intensity because as it isn’t affected by experimental errors, denaturants affecting intensity, on linear repsonses etc
Silk is an important textile material. Describe the secondary structure and tertiary packing
structure of a model of silkworm silk fibroin with the repetitive polypeptide sequence ….-
Gly-Ala-Gly-Ala-Gly-Ala-….(2p)
each peptide make a beta strand that is stacked together to make beta sheets ad due to glycine, alanine and serine all found a lot in silk being the smallest maino acids the sheets are packed very tightly. They have strong wanderwaals interactions and a hydrophobic core.
Protein aggregation causes several human diseases, such as Alzheimer’s disease. Around 30
different proteins form amyloid fibrils in humans. What is the common secondary structure
of proteins in amyloid fibril cores?
Beta-sheet conformation. In amyloid diseases like Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes, proteins aggregate and form insoluble fibrils characterized by long, unbranched β-sheet-rich structures. These β-sheets stack together to form the core structure of amyloid fibrils, which are resistant to degradation and accumulate in tissues.
Different fibrous proteins have different means of covalent cross-link chemistry between
fibers to stabilize their structure.
How is alpha-keratin and collagen respectively covalently cross-linked?
Keratins are composed of alpha helices in a coiled-coil motif.
Collagen is high in proline and glycine and proline is a beta and alpha helix breaker so it has a triple helix structure. The prolines are what gives the cracks and turns and glycine is in the center.
Collagen composes skin, bone, artery walls, cartilage etc. and is the most abundant protein
in animals. Collagen is composed of repetitive sequences where every third amino acid is a
glycine, which can be described as variants of the sequence …. -Xaa-Yaa-Gly-Xaa-Yaa-Gly-…..
Describe the secondary structure of folded collagen? (3p)
Alpha-Keratin = disupfide bonds (1p), collagen = allolysine aldehyde condensation (1p)
Collagen is an important component of skeletal bone, the inherited disease Osteogenesis
imperfecta, OI, “Brittle bone disease” results from mutations in the Collagen I genes
(COL1A1 and COL1A2. Collagen I is a large protein with >1000 amino acids. The most severe
mutations in COL1A1 gene occur when one Gly is mutated to Asp, Arg, Ser or Ala.
Why is Collagen I so sensitive to such point mutations that replaces one Glycine for another
amino acid? (1 p)
Why are Collagen I mutations Autosomal dominant (disease occurs in heterozygote carrier
with one mutant allele and one wild type allele? (1 p)
Most severe point mutation is glycine since it’s the smallest amino acid and if replaced with another maino acid it will effect the entire structure of the protien. It’s autosomal dominant because it is so sensitive for mutations, even with one gene coding for the right protein the other gene coding for the wrong protein will affect the entire protein structrue
Describe the secondary structure of folded collagen? (3p)
Collagen has a repetitive primary sequence in which every third residue in glycine. Pro (and Hyp) and Lys are also abundant in the Xaa and Yaa positions. Each chain forms a lefthanded helix with 3.3 residues per turn. Three left-handed polypeptide chains supercoil in a right-handed manner to make a triple-helix. Hydrogen bonds occur between the amide group of one Gly and the backbone carbonyl group on adjacent chains. The close packing of chains also results in van der Waals interactions between side chains. (3p)
