BIOCHEMISTRY QUESTION PLEASE HELP You have purified a protein (which you call pr
ID: 61130 • Letter: B
Question
BIOCHEMISTRY QUESTION PLEASE HELP
You have purified a protein (which you call protein X) that you suspect to be covalently modified by the addition of a single ubiquitin molecule. You also know that your purified protein is very unique, in that it only contains one lysine amino acid in its entire primary sequence. The molecular weight of the unmodified form of protein X is 60,000 Daltons. Suppose you also know that the c-terminal sequence of ubiquitin is HOOC-Gly-Gly-Lys-Ile-Gln-etc. where the indicated lysine at position 3 is also the only lysine amino acid in the ubiquitin sequence. You are now trying to confirm that your protein does indeed carry this ubiquitin modification. Suppose you add to your purified protein X the enzyme LysC, a serine protease that specifically recognizes the protonated amine group on lysine side chains, and hydrolyzes the peptide bond on the c-terminal side of lysine. Assume the solution is buffered to pH 7 and the pKa of the lysines in question is > 10. Based on the information above, answer the following two questions: If protein X is modified by ubiquitin, how many polypeptide products would you expect to create if it is treated with LysC? Briefly explain why. What would be the approximate molecular weight of the different peptide products?Explanation / Answer
The general weight of an amino acid is 110 da. So the number of amino acids in this protein would be 545.
1. If the protein is ubiquitinated, the molecule attaches to the second amino group in lysine. So after treatment with LysC which cleaves at the COOH end of lysine, we see 4 fragments
2. ubiquitin is a protein which contains 76 amino acids. So after treatment with LysC we get a two fragment peptide, a 74 amino acid fragment. The protein X might be divided into two roughly equal fragments. This depends on the position of the lysine residue. So 545 residues can be divided into 200 and 245 amino acids per fragment.
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