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100 Cards in this Set

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What is a typical saturation binding curve?
It is a plot of the concentration of the free ligand at equilibrium [A] against the number of possible ligands (Q) per protein molecule
In a typical saturation binding curve, what is the maximum value according to?
The maximum value (Y-axis) tends to have a maximum value according to the number of binding sites (Q) on the protein
What is the simplest example of this?
The protein has one binding site for the ligand, and Q is equal to 1.
What does this curve allow?
It can be used to determine likely interactions between drug molecules and their target cells in the body
What is the Dissociation constant equal to?
The dissociation constant (Kd) is equal to r = 0.5
So what is the equation for r?
r = [A] / ( Kd + [A] )
What does it mean at r = 0.5?
It means half the enzyme molecules are bound to ligand and you get the free concentration of ligand at equilibrium equal to the equilibrium dissociation rate
What is the corresponding [A] equal to?
It is equal to the number you get when you drop a line perpendicularly from r = 0.5
So, all this summed up relating affinity of interaction is?
This graph allows you to directly measure the affinity of the interaction and to compare equivalent plots of varying-affinity interactions
Describe what the value for Kd actually is?
It provides you with a quantitative way to measure the affinity of the interaction
If you wanted to compare different interactions, how would you set the experiment up?
You can compare plots between different interactions by using the same ligand but different enzymes which have different affinities for that ligand
What does a higher value of Kd mean?
It means less tight binding or lower affinity binding
What does a lower Kd value mean?
It means there is a higher binding affinity
What is a more accurate way to measure Kd?
A double-reciprocal plot allows a very accurate way to measure this
What is difficult to supply in order to get a good saturation binding curve?
You must supply enough ligand concentration [A] to get up the curve
What is the equation for a double reciprocal plot?
r / [A] = (1 / Kd ) - (r / Kd)
Describe how to relate the double reciprocal plot to the general form of a straight curve, and provide relationships between one equations variables to the others?
y = mx + c is the equation for a straight line, and c is the constant, m is the slope of the line, and x and y are variables. c = (1/Kd). m = -1/Kd. x and y are r and (r/[A])
Why does plotting a double reciprocal plot give you a negative curve?
The slope of the line (m) is equal to -1/Kd. A negative slope has a negative value, and since Kd cannot be less than 0, it must be negative.
Will a ligand with a higher affinity for the enzyme have a steeper slope or a more shallow slope? Why?
It will have a steeper slope because 1/Kd is larger, which makes Kd smaller
How would one setup an equilibrium dialysis experiment?
Separate the proteins from solution, a reservoir of the ligand, using a dialysis membrane
What is a dialysis membrane?
It is a membrane that is semi-permeable so the ligand can cross but the protein cannot
What do you fill the beaker with that the reservoir floats upon?
Fill the beaker with a reservoir of ligand solution
What is the bottom of the boat sealed by?
The dialysis membrane
What do you put into the boat?
Protein you wish to test
What will diffuse across the membrane, and until when?
The ligand molecules will diffuse back and forth across the membrane until the concentration of ligand in solution equilibrates between the beaker solution and the solution inside the boat
Will there be an equal amount of ligand in the reservoir and in the boat at equilibrium?
No. more ligand will end up inside the boat because you will have unbound ligand in equilibrium plus whatever ligand has bound to the protein
What do you do now that the equilibrium has been reached?
Measure [A] in the cup. Measure the equal concentration of the ligand in the beaker plus what has bound to the enzyme
With the data in hand, what do you plot?
Plot r against r / [A], with r bewing the number of ligands bound per protein, which is the little extra bit in the cup.
What is r equal to?
It is equal to the concentration of the ligand over the concentration of the protein
What now?
Run the experiment over again with different concentrations of ligand, and then plot them all
What is a second method to obtain good data on protein / ligand affinities?
Surface Plasma Resonance Imaging
What is the premise of it?
Tether one of the two interacting partners to a surface, and then run a solution of the other interacting (untethered) across the surface
What happens when they meet?
The interaction between tethered molecules and flowing molecules will bind the two together
What does this do, and how does this allow you to obtain affinity values?
When they bind, it changes the refractive index in the surface. Plot this change against the time curve of an association reaction and then a dissociation reaction, thereby directly measuring the rate constants of the association and dissociation. The Kd is the ratio of the two measures
What does centrifugation provide?
In this case ultra-centrifugation is used to sediment protein molecules
What do the rates of sedimentation depend on?
They depend on the size and shape of proteins or complexes, so if two protein molecules bind together, their size will be bigger than either one protein separately.
By measuring the rates, what can be derive?
We can derive the molecular weight of the complex and the complex's shape
If you rotate the rotor more slowly, what does this provide?
It provides a mechanism to create an equilibrium between sedimentation and homogenizing
What is the difference?
Sedimentation moves toward the bottom while the solution is homogenized
What does the model of the shape of the eventual concentration curve predict?
It gives us information about equilibrium dissociation
Does a higher affinity of a drug mean its better, and why or why not?
Not always because another important factor aside from efficacy is how specific the drug is
Describe a drug like this?
A drug could have a high affinity to bind very quickly, but it could be very unspecific to what it binds to, causing side effects
What is the goal of pharmacology in this regard?
The goal is to make the gap between the binding of drug to target and binding of drug to non-target be as wide as possible
Can we plot the interaction of a ligand with a protein containing multiple binding sites, and if so, how?
We can, via the same double reciprocation plot: r = n [A] / (Kd + [A]) and r / [A] = (n/Kd)-(r/Kd)
With this equation, what do you do with a protein with multiple binding sites to plot this?
Plot r / [A] versus r, and you end up with the same straight line as with a single-binding site proteins, with affinity as the slope
What is the intercept on the x-axis? What does this value mean?
n is the value, and it is the number fo combined binding sites for that particular protein
Why is it assumed that the lines in double reciprocation plots will be straight, and why is it so that this is not always the case?
The assumption of a straight line is based on the assumption that the binding of the ligand to one place on the protein is independent to anywhere else on the protein, but an enzyme may have different affinities at different binding sites, which may be related
Describe an example involving this and antibody immune response.
Inject a mutagen into an animal or human, and allow time for the immune response, and take blood. What you will find is thousands of different antibodies that contribute to the response, each with different affinities for the antigen. The antibodies with the highest affinities will bind first, and the lower affinity antibodies will bind afterward. Each type of antibody that joins in binding has a lower affinity, the slope gets less and less steep producing a curve.
What does this imply about experimentation?
Make sure there is a single level of variability, and not more than a single level of variability, in order to minimize this.
What is cooperativity?
When a protein binds to a ligand the binding affects the affinity for subsequent bindings
What is cooperativity the structural basis for?
It is the structural basis for interactions between one binding site and another
What does cooperativity require first and foremost?
It always requires the binding of the ligand to one part of the protein to cause the initial conformation change, in either the positive or negative direction
What does a positive movement signify for the protein?
It is a move toward a higher affinity
What does a negative movement signify?
It is a move toward a negative direction
If the binding of the ligand increases the affinity of subsequent bindings, what will the plot form?
The plot will form a convex line
What does negative cooperativity form?
Where the affinity gets lower after the first binding produces a concave shaped plot
What is one effect of negative cooperativity in regards to sensitivity for the ligand?
As the ligand concentration gets higher and higher, the protein becomes rapidly insensitive to the concentration of that ligand
During positive cooperativity, does the response to an activator produce a very broad or very narrow range of ligand concentration, and what does this essentially function as?
It will be a very narrow range of ligand concentration, so that the whole system acts like a molecular switch, where tiny concentration changes make a big change in the activity of the protein that is modulated
What are hemoglobin and myoglobin?
They are O2 carriers in the blood
What is the equation for myoglobin carrying Oxygen in the blood?
Mb + O2 <---> MbO2
What is the equation for the equilibrium dissociation constant of myoglobin?
K = [Mb][O2] / [MbO2]
How does hemoglobin relate to myoglobin?
It is homologous to myoglobin
What does hemoglobin carry?
Oxygen
What do we speak of oxygen in the blood as instead of [O2]?
We speak of it as oxygen tension, or partial pressure of O2, which is pO2
What is yO2?
yO2 is used in these discussions rather than the regular r for the proportion of binding sites bound by O2
What is the equation for yO2?
yO2 = [O2] / (K + [O2])
What do you plot in this case to find affinity curves?
Plot the partial pressure of oxygen against the value of Y
For myoglobin, what will the plot appear to be?
It will be a simple saturation plot, with the top of the curve hitting y = 1, where all sites possible are bound
What is the shape for the hemoglobin curve? And why is it this way?
The plot is S-shaped due to positive cooperativity
Describe the portions of the curve?
The curve is made up of two binding curves- one with high affinity and one with low affinity. It starts at low affinity, but due to the cooperativity it moves to a high affinity
What is this curve known as?
A hybrid curve
Near alveolar pressure in the lungs, what is the pressure and will myoglobin be fully or minorly bound to O2? Hemoglobin?
The alveolar pressure here is about 100 millitorr, and it will be essentially fully bound to O2. Hemoglobin as well
In tissues where pO2 is much lower at about 40 millitorr, describe the situation with hemoglobin and myoglobin.
The pO2 is around 40 millitorr, and the oxygen tension is lower, so the O2 is offloaded by hemoglobin in the tissues and moves to bind with myoglobin
How cooperative is the binding of oxygen by hemoglobin? Describe the linearized plot.
It is very cooperative, with log (y/[1-y]) against log10[pO2]. (y/[1-y]) is a fraction of sites filled divided by the fraction of sites not filled. The log is used to linearize the data.
What is max binding for Hb?
4, giving y a maximum value of 4, and a minumum value of 1.
The proteins give what shaped lines in this plot?
They give a positive slope, where the line is further to the right in the case of molecules with weaker and weaker binding affinity
Where will the plot be in the case of low values of oxygen tension, where oxygen just starts binding but still has weak binding?
The plot will be close to the right-hand side
As oxygen tension rises what happens to the plot?
The affinity will increase and converge with the high-affinity line
What does the slope of the line in-between high and low relate?
The affinity slope is the measure of cooperativity of the molecules
What is the maximum value and what is its meaning?
The maximum value is 4, for the number of binding sites, and you would get this value if you had perfect cooperativity
The minimum value will always be? Range for value of slope?
The value of the slope being between 1 and 4.
What is this value called?
The Hill Coefficient
What is the value for a normal adult hemoglobin?
2.8
What does a 2.8 mean?
It means it's a pretty efficient system, but not perfect
Where is the iron embedded in the molecular level on these molecules?
It is embedded in a porphyrin ring, which is comprised of 4 pyrol rings, with the iron coordinated in th emiddle
What shape is the structure?
Distinctly flat
Describe the structure of myoglobin. Name the number and type of the helices, the direction of the helices, and their names.
There are 7 alpha helices, lettered N-terminus to C-terminus, A-G.
Where is the porphyrin wedged?
It is wedged between the E and the F helices
What is noteworthy about this location?
It is inside a hydrophobic pocket
What is hemoglobin's structure?
It is a set of four Mb-like subunits
Hwo many polypeptides are there in Hemoglobin?
4 polypeptides, with each being homologous to myoglobin, each with a porphyrin ring and tetrahedral to one another.
What happens when hemoglobin changes from the deoxy form to the oxyform?
There is a conformational change
Each apple in his discussion was what?
Each apple formed a dimer of two subunits, one alpha and one beta subunit
In hemoglobin, how many dimers come together?
2 come together
What happens when the first O2 binds?
When an O2 binds to one subunit, a transition is transferred to all the subunits so that one dimer shifts in behind the other
What is Fe coordinated by, and what does it form?
It is coordinated by nitrogens and one histidine on the back side, and it forms a 5-coordinated ion
Once O2 binds what is the form?
Once O2 binds the ion is 6-coordinated
The O2 also binds what when it binds Fe?
It also binds the histidine, pushing Fe from displaced to closer to the ring
What does this do, moving the histidine and Fe?
Dragging the histidine pulls an alpha helix with it, and movement of the alpha helix is tranferred to the other side, breaking H-bonds and sulfide bridges