exam 2019-03-18 Flashcards
Describe the meaning of the following terms or sentences used in protein chemistry.
a) Hydropathy plot
b) Xray structure determined to 1.5 Å resolution
c) ITC
d) TIM barrel
e) Greek key motif
Hydropathy plot shows the hydrophobicity (values above 0) and hydrophilicty(below 0) for each amino acid in a protein
xray structure with 1.5 resolution gives a three dimensional view of the protein and Å is a measure of detail7clarity
ITC measures the heat absorbed/released during chemical reactions which can be used for example to analyze ligand binding
TIM barrel is paralell beta strands with alternating alpha helixes
Greek key motif is antiparalell beta strand connected with loops
What characterize a perfect evolved enzyme?
High catalytic rate constant (kcat)
High substrate specificity
High affnity for substrate
Compatible to it’s enviorment in regards to optimal pH and temperature and stable under physillogivcal conditions
) The figure below shows the four different steps that are included in the reaction
mechanism of the enzyme alkaline phosphatase. Identify four enzyme mechanisms
that are used by the enzyme to catalyze the reaction
There are general acid base catalysis, proximity of reactions components (effective concentration) the enzyme can bind to the two molecules so they are closer to each other and much more likely to bind to each other when they are proximate to each other, electrostatic catalysis, metal ion catalysis and covalent catalysis.
General acid catalysis stabilize a negative charge that needs to be formed during the transition state by the weak acid and stabilise positive charge with a weak base.
electrostatic catalysis is manipulation of charges to accelerate the reaction.
Metal ion catalysis can ehance the nucleophilic attack and stabilize the intermediate
Covalent catalysis creates new covalent bonds that would otherwise not be made that creates a new reaction path with lower activation energy can for example form a better electrophile in an electrophilic attack for example.
User
To purify the protein, you have decided to use Ni-NTA column that binds to a
constructed His-tag (the his-tag was later cleaved off) followed by gelfiltration.
IMPDH was eluted corresponding to a molecular weight of approximately 164 KDa.
An SDS-Page was run to check the purity of protein (Figure 3). Comment on the SDSpage,
what can you say about the structural arrangement of IMPDH?
Is the question and then there is a figure of the SDS page of the purified protein with a ladder and the protien being at 40kD
In SDS page during filtration the subunits of a protein migrate independantly so the whole protein is made out of 4 subunits 164/41=4 (approximatly) o the protein is a tetramer made out of 4 subunits forming the protein
b) To monitor the stability and enzyme activity you want to choose a buffer suitable for
the experiments. Give suggestions of buffer and pH of a suitable buffer for this
experiment.
According to data from Protparam (figure 2) pI =7.70
To avoid problem with aggregation and stability a good rule of thumb is to choose a buffer around one (1) pH unit above or below the isoelectric point, a good choice would be phosphate buffer at pH 6,7 or a Tris buffer at pH 8,7.
5c) IMPDH bind mycophenolate (figure 4) and to address the number of binding sites
and dissociation constant the quenching of the protein fluorescence was studied and
the following results were obtained (Table1). The concentration of IMPDH was 4 μM
throughout the titration.
What are the steps to calculate number of binding sites and dissotiation constant?
- You normalize the data to get R. The formula for that is R=[PL]/[P]total
- Then you check the affinity for the ligand by: R/[L]free
- Then you plot the results, x axis ir R/[L]free and y axis just R
- Calculate slope of the line by y2-y1/x2-x1
- 1/slope=Kd
- Y intercept/slope=number of binding sites
5c) IMPDH bind mycophenolate (figure 4) and to address the number of binding sites
and dissociation constant the quenching of the protein fluorescence was studied and
the following results were obtained (Table1). The concentration of IMPDH was 4 μM
throughout the titration.
What are the steps to calculate number of binding sites and dissotiation constant?
Scatchard plot made and showed straight line and numbr of binding sites to 4.
What type of binding is observed in the above example? Give example how you, by plotting the data can distinguish between different types of binding.
In the schatchard plot there was a straight line and multiple binding sites were asses which shows it’s independant, sigmodial or s shaped curved shows cooperative binding.
As part of the protein characterization a stability profile using chemical denaturation
was performed. Two parameters were monitored, enzyme activity and fluorescence
(plotted as fraction of natively folded protein) (Table 1). Plot the stability of IMPDH
versus denaturant for fluorescence and enzyme activity. What conclusion can you
draw from this plot?
What are the steps in this?
Plot yobs on y axis and denaturant in mol on x axis to see how it denatures. It is one single transitionstate so it shows no intermediate but a two state unfolding process.
d) The fluorescence was monitored at the emission wavelength maximum, why is this better than measuring the fluorescence intensity.
It’s better to look at wavelenght maximum rather then intensity when looking at flourescense like this becasue intensity can be affectd by many things such as the enviorment for example, emission maximum can tell us about the strucutral changes of the protien more accuratly.
As part of the protein characterization a stability profile using chemical denaturation
was performed. Two parameters were monitored, enzyme activity and fluorescence
(plotted as fraction of natively folded protein) (Table 1). Plot the stability of IMPDH
versus denaturant for fluorescence and enzyme activity. What conclusion can you
draw from this plot?
What are the steps in this?
g) Using the same data, calculate relevant parameters for the stability of IMPDH as monitored by fluorescence (plotted as fraction of natively folded protein). The experiments were performed at room temperature (25 C) and R=1.987 Cal K-1 mol-1.
Use the deltaG free energy equation with deltaG=-RTln… etc
deltaG is your y axis and concentration of denaturant is x axis
There are natural variants of IMPDH for example, the deleterious variant R342P (letter R and P indicate the amino acid residue before and after mutation in position 342). Give a reasonable explanation on a structural basis why this variant is deleterious
Proline is uinque in the sense that it’s a beta sheet and alpha breaker, so a deletious mutation like this will cause the whole structure to break.
To get a better understanding of the variant IMPDH R342P you decided to crystallize
the protein and by X-ray crystallography determine the structure. Describe briefly
how you crystallize a protein.
Protein Purification: Obtain a highly pure sample of the protein of interest.
Buffer Optimization: Dissolve the protein in a buffer optimized for stability.
Concentration: Concentrate the protein solution to increase protein concentration.
Screening Crystallization Conditions: Test various conditions to identify optimal crystallization conditions.
Crystal Growth: Set up large-scale crystallization trials to grow crystals over days to weeks.
Crystal Harvesting: Carefully harvest crystals and transfer them to a cryoprotectant solution.
X-ray Data Collection: Flash-freeze crystals and subject them to X-ray diffraction analysis.
Structure Determination: Process and analyze diffraction data to determine the protein’s three-dimensional structure.
Fibrous protein structures in health, disease and aging
A) Amyloid fibrils
In many human diseases proteins misfold and aggregate to form amyloid fibrils in
various tissues including the brain causing neurodegeneration e.g. Alzheimer’s
disease.
Describe the structure and stabilizing interactions of amyloid fibrils (3p)
. The structure of amyloid fibrils typically consists of cross-beta sheets motif which are beta sheets in a paralell or antiparalell matter stacked on to each other. They have hydrogen bonds and wanderwaals interactions as strongest stabilizing factors.
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 …. -XaaYaa-Gly-Xaa-Yaa-Gly-…..
Describe the structure and stabilizing interactions of folded collagen (3p)
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. It’s stabilized by hydrogen bonds and hydrophobic interactions in the center.
c) Explain the molecular mechanism linked to collagen causing wrinkled skin in old people and tough meat from old compared to young animals
It’s due to cross linking. Lysine is modified by an enzyme and form allysine which cross link collagen chains.
