Circular dichroism (CD) is a technique used for determining the secondary struct
ID: 174424 • Letter: C
Question
Circular dichroism (CD) is a technique used for determining the secondary structure (-helix and -pleatedsheet) of a protein. The authors took CD spectra of the protein at varying temperatures at various wavelengths. Then they plotted the results obtained at 220 nm. The results are an indication of the integrity of the secondary structure of the BN protein. A decrease from 100% indicates a loss of secondary structure integrity. The results are shown below.
a. What do these data tell you about the stability of the protein at various temperatures?
b. The authors next carried out fluorescence studies in order to obtain additional information concerning the three-dimensional structure of the BN protein. Specifically, they were trying to determine if there was an interaction between Tyr 63 and Trp 26, and for this reason, they decided to study the microenvironment of the Trp residue. Only tryptophan and tyrosine are fluorophores, which means they are capable of undergoing fluorescence. They found that a single tryptophan residue emits light at 330 nm upon excitation at 295 nm. There are several ions and small molecules that have the ability to quenchfluorescence. This means that upon excitation at 295 nm, the tryptophan will transfer its energy to the quenching agent rather than releasing the energy in emitted light. Cesium (Cs+) ions, iodide (I- ions) and acrylamide are capable of quenching the tryptophan residue’s fluorescence–that is, if these quenching agents can make suitable contact with the tryptophan. Neither of the quenching agents worked very well. The investigators found that there was no quenching at all with cesium ions and that quenching efficiencies with iodide and acrylamide were very low. What do these observations tell you about the microenvironment of the tryptophan residue? Is an interaction between Tyr 63 and Trp 26 likely?
Circular dichroism (CD) is a technique used for determining the secondary structure (-helix and -pleatedsheet) of a protein. The authors took CD spectra of the protein at varying temperatures at various wavelengths. Then they plotted the results obtained at 220 nm. The results are an indication of the integrity of the secondary structure of the BN protein. A decrease from 100% indicates a loss of secondary structure integrity. The results are shown below. a. What do these data tell you about the stability of the protein at various temperatures? b. The authors next carried out fluorescence studies in order to obtain additional information concerning the three-dimensional structure of the BN protein. Specifically, they were trying to determine if there was an interaction between Tyr 63 and Trp 26, and for this reason, they decided to study the microenvironment of the Trp residue. Only tryptophan and tyrosine are fluorophores, which means they are capable of undergoing fluorescence. They found that a single tryptophan residue emits light at 330 nm upon excitation at 295 nm. There are several ions and small molecules that have the ability to quenchfluorescence. This means that upon excitation at 295 nm, the tryptophan will transfer its energy to the quenching agent rather than releasing the energy in emitted light. Cesium (Cs+) ions, iodide (I- ions) and acrylamide are capable of quenching the tryptophan residue’s fluorescence–that is, if these quenching agents can make suitable contact with the tryptophan. Neither of the quenching agents worked very well. The investigators found that there was no quenching at all with cesium ions and that quenching efficiencies with iodide and acrylamide were very low. What do these observations tell you about the microenvironment of the tryptophan residue? Is an interaction between Tyr 63 and Trp 26 likely?
Explanation / Answer
If temperature is increasing the protein would result to denaturing. The dentaruing graph of Cricular dichroism shows negative curve; the more A (change in absorbance) is negative. At a certain point of temperature the curve will get only in negative A (y axis). X axis is showing wavelength.
The indole group of trypthophan involves in the absorption of UV at 280nm and then emits at 350nm. If the other factors like Cesium (Cs+) ions, iodide (I- ions) and acrylamide do not have sufficient quenching, the tyrosine quenching is playing role. Trypthphan binds to phenolic hydroxyl group of tyrosine. This binding is generally exhibit by allosteric enzymes for their allosteric regulation.