This is a peptide sequence: SASVELTANAQLTVGPSKASVEIKASLNIT analyze: All the AVLP

Question

This is a peptide sequence:

SASVELTANAQLTVGPSKASVEIKASLNIT

analyze: All the AVLPVIT are hydrophobic amino acids, they tend to alternate with a polar amino acid one by one, so this might be a beta sheet. However, when we look at the PSKA part, that is a HPPH sequence, so this might form a liittle bit alpha helix coiled coil as well, I want to ask:

1) analyze the primary sequence and suggest what secondary structure(s) it may contain

2) What technique(s) will you use to test your hypothesis in 1)?

3) How do you expect the N- and C- termination of this peptide to effect the stability of its folded state? eg. how might the free amine, free acid version compared to the N-acelylated, C-amidated version? (Personally I tend to this form a Beta-sheet, will the there be any difference between a free amine and A N-acelyted on N terminus in a beta sheet conformation? will the there be any difference between a free acid and a C-amidated on C terminus in a beta sheet conformation?)

4)What experiment could do to test your hypothesis in (3) (I guess we can use the CD, do the Thermal Denaturing, detect when the fashion would fold like this picture, but how to explain this experiment accurately?)

Explanation / Answer

1)

Represent polar amino acid by P

non-polar amino acid by H

charged amino acid by C

So, we have following sequence: PHPHCHPHPHPHPHHHPCHPHCHCHPHPHP

On analysis, we find only one possible secondary structures possible : antiparallel beta sheet (due to presence of proline which forms kink and chain fold back on itself)

Though there is PHHP sequence which is typical representation of alpha helic forming domain; however presence of proline does not favour its formation. Remember, proline is destabilizer of alpha helix due to absence of hydrogen bonding.

2.

Following techniques can be used to study secondary structure of proteins

a) Circular dichroism

b) Raman spectroscopies

c) Chemical cross-linking

3.

The N and C termini of the peptide harbors polar residues which are required to stabilize the beta sheet. Daestabilizing the bond can destabilize or denature the beta sheet conformation. e.g. if we acetylate N-terminal amino acid, then free hydrogen on this amino acid is replaced by acetyl group which will hinder the formation of hydrogen bond with -O group of C-terminus. Similarly, C-amidated amino acid will not be able to bond with free H of N-terminus amino. Just draw the structures and you will get answer.

4.

Thermodynamics of protein folding by CD in presence of denaturent

Briefly, circular dichroism is defined as the difference between the absorption of left-handed, R, and right-handed, L, circularly polarized light.the major application of CD today is the determination of the thermodynamics of protein folding and the effects of mutations on protein stability.Essentially the use of CD to follow protein denaturation depends on the fact that the change in ellipticity is directly proportional to the change in concentration of native and denatured forms.The observed ellipticity of a protein, changes when it unfolds.

When the protein is fully folded obs = a1 and when it is fully unfolded obs = a2. In the case of a monomeric protein, the equilibrium constant of folding, K = folded / unfolded.  If we de¢ne K as the fraction folded at a given temperature then

K = / (1 - )

delta G = nRTlnK

where delta G is the free energy of folding and R is the gas constant = 1.987 cal / mol.

In some cases thermal folding and unfolding studies are impractical. The protein may have a very high TM, or may aggregate and precipitate upon heating. In these cases denaturants, such as urea or guanidineHCl, may be added to a protein or peptide to induce unfolding. One obtains K at each concentration of denaturant by assuming that the protein is native in the absence of denaturant, and fully unfolded when there is no further change in ellipticity upon addition of higher concentrations of perturbant. In each case, K, the equilibrium constant of folding is determined from

the ellipticity observed at each concentration of denaturant. K = / (n[C]n-1) (1 - )n

where = (obs - a2) / (a1 - a2)

K, a1 and a2 have the same de¢nitions as in the case of thermal unfolding above. [C] is the total concentration of protein monomers. The free energy of folding is evaluated at every concentration of denaturant using the equation delta G = -nRTlnK

The free energies are plotted as a function of denaturant and extrapolated to zero denaturant to obtained the free energy of folding of the native material. It should be noted that one may obtain different apparent vGs of folding when different denaturants are employed because some denaturants, such as solutions of guanidine-HCl, which have a high ionic strength, disrupt salt bridges in proteins, while others, such as urea, which is uncharged, have a lesser effect on protein charge^charge interactions.

In our experiment, we can use this method to test effect of N-acetylation / C-amidation on folding structure of beta sheet in the peptide.

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