BL401/CH401 Exam I September 30, 1996

1. 50 Points. Amino Acid Sequence Problem

A peptide had the following amino acid composition obtained by acid hydrolysis and amino acid analysis: Arg, Gly, His, Tyr, Phe, Pro, Ala, Glu, Asp

The peptide was found to have no N- or C-terminal amino acid residue by Dansyl-chloride analysis and carboxypeptidase treatment.

Next treatment with trypsin was negative. However, chymotrypsin treatment gave 2 peptides, called C-1 and C-2.

C-1 had amino acid composition (acid hydrolysis): Arg, Gly, Glu, Pro, Tyr
Edman degradation of C-1 gave Gly in 1st cycle, Gln in 2nd cycle and Arg in 3rd cycle.

C-2 had amino acid composition (acid hydrolysis): His, Phe, Ala, Asp
Edman degradation of C-2 gave Asp in 1st cycle, His in 2nd cycle and Ala in 3rd cycle.

What is the sequence of the peptide?
Draw the full chemical structure of the peptide at pH 7.
What is the net charge on the peptide at pH 1, 5, 7, 10, 13

pK values for amino acids: Gly - 2.4, 9.8; Ala - 2.4, 9.9; Phe - 2.2, 9.2; Tyr - 2.2, 9.1;
Pro - 2.0, 10.7; His - 1.8, 6.0, 9.3; Glu - 2.1, 4.1, 9.5; Gln 2.2, 9.1; Asp 2.0, 3.9, 9.9;
Asn 2.1, 8.8; Arg 1.8, 9.0, 12.5

2. 25 Points - Cell Structure (5 points each short answer)

A. What regulates gene expression in eukaryotes (be brief)?
B. What is a ribosome composed of (in some detail)?
C. What do mitochondria do in the cell of a eukaryote?
D. What is the function of smooth endoplasmic reticulum (ER)?
E. What is the significance of the chloroplast and photosynthesis to life on earth (be brief)?

3. 15 Points - Protein Purification (5 points each short answer)

(Give me more than just a term as the answer; explain your answer briefly but fully)

A. How do you decide if your protein is pure?
B. What is the method used to find subunit molecular weight?
C. What method takes advantage of the enzyme’s biological activity in purifying it?

4. 10 Points Thought Question

Why does mRNA (messenger RNA) exist in prokaryotes and why is it used as a template for protein synthesis? In other words, why not use DNA directly as the template for protein synthesis?

Answers:

1. 50 Pts. Circular Peptide as shown below:

Structures can be obtained from text, notes or Web (see lecture 4)

pH Profile: There are no N- and C-terminal amino acids and so no alpha amino and carboxyl groups. Only side chains will ionize. Three amino acids in sequence ionize: Arg, Asp and His and the pK values of their side chains are 12.5, 3.9, and 6.0 respectively. So the net charge at various pH values are:

	pH 1	pH 5	pH 7	pH 10	pH 13
Asp Charge	0	-1	-1	-1	-1
His Charge	+1	+1	0	0	0
Arg Charge	+1	+1	+1	+1	+1
Net Charge	+2	+1	0	0	-1

2. 25 Pts Cell Structures

Proteins, known as DNA-binding proteins or trans-acting factors, regulate gene expression in eukaryotes is the simplest answer. More complex answers involving control over display of DNA by methylation, nuclear envelope gating of entry to proteins, and other processes could be added to answer.
Ribosomes are composed of a small and large subunit both consisting of rRNA molecules and proteins. More detail could include the types of rRNA and the approximate numbers of proteins in the the small and large subunits of the ribosome. (see lecture 2)
Mitochrondria "burn" products of metabolism like pyruvate to produce energy in the form of ATP. More complex answers include that mitochrondia have their own DNA and partially build themselves by synthesizing their own proteins. (see lecture 2)
Smooth ER provides the cell with an "organic solvent" medium for synthesis and metabolism of water insoluble compounds like cholesterol, fatty acids and xenobiotics like dioxin. (see lecture 2)
Chloroplast and photosynthesis made it possible for oxygen based life to become rather dominant on earth and is basically responsible for the beginning of the food chain since they catalyze the process of converting light energy from the sun into chemical energy in the form of sugars and proteins virtually all non-green organisms consume to exist. (see lecture 2)

3. 15 Pts Protein Purification

Protein purity is determined by native polyacrylamide gel electrophoresis (PAGE) where the enzyme/protein can be stained both for its biological activity and protein character, which allows you to say that the single band of protein found in preparation of pure protein is the enzyme of interest. You can not get the molecular weight of the protein by running a single gel unless you use special conditions. (See Lecture 6/7)
Subunit molecular weight is found by denaturing/SDS PAGE, where the gel is calibrated with proteins of known subunit molecular weight and the electrophoretic mobility of your protein of interest compared to the mobility in the gel of the standard proteins to deduce its molecular weight. It can in general also be obtained by cloning the gene for protein/enzyme of interest and obtaining its amino acid sequence from the DNA sequence of its protein encoding portion. (See Lecture 6/7)
Affinity chromatography takes advantage of an enzyme's biological activity to affect its purification. In this method, the substrate of the enzyme or an inhibitor is covalently attached to a solid support like Sepharose and after, the affinity chromatography "gel" is placed in a column, the mixture of proteins to be resolved is applied. The non-binding proteins are washed away and the enzyme/protein of interest eluted by applying the free substrate which will have a higher affinity for the bound enzyme than the immobilized substrate of the affinity chromatography column matrix or material. Finally, the substrate and enzyme can be resolved from one another by dialysis, gel filtration or a number of other methods. (See Lecture 6/7)

4. 10 Pts Thought Question

In prokaryotes, one strand of DNA for genes encoding proteins is identical to the mRNA, since there are no introns found in prokaryotic protein encoding genes. In theory, it seems possible to directly use the DNA as the template for protein synthesis, which would not work for most eukaryotic genes. The reason DNA probably does not function in this way is related to the origin of prokaryotes in evolution where we suspect that these organisms arose from less efficient ones that acted in the same manner as eukaryotes do today and had the need to make mRNA in order to get rid of introns, which the prokaryotes subsequently got rid during evolutin. You also might answer that the DNA needs to protected and so it is exposed to less damage if mRNA is used for protein synthesis, since the double stranded structure of DNA must be unwound for the genetic code to be read by protein synthesis machinery like ribosomes (The DNA would need to be exposed for a greater length of time than it is to make an mRNA molecule which can be read many times to make any protein molecules from the gene).

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