September 27, 1995
1. 60 Pts. Amino Acid Sequence Problem
A. Peptide Z had AA composition: Arg, Cys, Glu, Gly, Lys, Met, Phe, Pro, Tyr, Val
Peptide Z had N-terminal = Phe by Dansyl-Cl
Peptide Z had C-terminal = Gln by carboxypeptidase
B. Trypsin gave 3 peptides:
T-1 had AA composition of Val, Glu, Tyr
T-2 had AA composition of Lys, Pro, Phe
T-3 had AA composition of Cys, Met, Arg, Gly
C. Chymotrypsin gave 2 peptides:
Ch-1 had AA composition of Met, Lys, Phe, Pro, Tyr, Gly, Cys, Arg
Ch-2 had AA composition of Val, Glu
D. CNBr cleavage gave 2 peptides:
CB-1 had AA composition of HSL, Lys, Phe, Pro, Gly
CB-2 had AA composition of Glu, Tyr. Val, Arg, Cys
Edman on CB-2 yielded Cys first, then Arg, then Tyr
What is the Amino Acid Sequence of Peptide Z?
Draw Complete Chemical Structure of Peptide Z at pH 7?
What is net charge on Peptide Z at pH 3 and pH 10? (See bottom of page for pKs)
What is the pI of Peptide Z?
2. 30 Pts. Protein Purification (6 Pts for each answer -- be brief but complete!!!)
A. What is affinity chromatography?
B. What is gel filtration chromatography?
C. What is Native PAGE used for?
D. What is SDS-PAGE used for?
E. How do you determine the subunit composition of an enzyme/protein?
3. 10 Pts. Thought Question/Cell Components
Eukaryotic cells have two types of DNA: nuclear and cytosolic/organellar (for example, mammalian cells have DNA in the mitochondria). To use the DNA in the organelles, there must be transcription of organellar DNA and translation of the mRNA made from this DNA using organellar ribosomes and tRNAs.
Why do eukaryotic cells go to all this trouble?
In other words, what is the purpose of organelles having DNA which encodes unique proteins found only in these cellular components?
(Remember that most of the proteins found in mitochondria are encoded in the nucleus, synthesized in the cytosol and imported into the mitochondria.)
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pK values for Amino Acids:
Arg 1.8, 9.0, 12.5 Cys 1.8, 10.8 Glu 2.2, 4.3, 9.5
Gly 2.3, 9.8 Lys 2.2, 9.2, 10.8 Met 2.2, 9.3
Phe 2.6, 9.2 Pro 1.9, 10.6 Tyr 2.2, 9.1
Leu 2.3, 9.7 Gln 2.2, 9.1 Trp 2.4, 9.4
ANSWERS for 1995 EXAM I
1. 60 Pts. Amino Acid Sequence Problem
What is the Amino Acid Sequence of Peptide Z (25 Pts)?
PheProLysGlyMetCysArgTyrValGln
Draw Complete Chemical Structure of Peptide Z at pH 7 (25 Pts)?
No structure is shown here since it can easily be figured out from the sequence.
The charges at pH 7 is: +1 on alpha amino group of Phe, +1 on side chain of Lys, +1 on side chain of Arg, and -1 on the alpha carboxyl group of Gln.
What is net charge on Peptide Z at two pHs (5 Pts)?
pH 3 -- net charge = +2; pH 10 -- net charge = +1
What is the pI of Peptide Z (5 Pts)?
pI = 11.7 (rounded up from average value of 11.65)
pI = 11.7 = (10.8 + 12.5)/2 = ((pK of Lys side chain) + (pK of Arg side chain))/2
2. 30 Pts. Protein Purification (6 Pts for each answer)
Note: the best answers to these questions include small graphics to explain the answers, but none are shown here to make it easier to get the answers out on to the Web quickly. If you want to see the graphics go to Lecture 6 and 7 via the 401 Home Page.
A. What is affinity chromatography?
Affinity chromatography is the protein purification method which takes advantage of the biological activity of an enzyme and is the most unique method we discussed for protein purification. Affinity chromatography is done by preparing an affinity material with the substrate of an enzyme or an inhibitor of it. When the mixture of proteins is poured over this affinity material, only the enzyme with an affinity for the substrate/inhibitor will bind and all non-binding proteins can be washed away. Next the bound enzyme can be eluted by applying the free substrate which the enzyme has a higher affinity for or binds to more strongly than the covalently linked substrate/inhibitor of the affinity material. Finally, the substrate can be removed from the enzyme by dialysis or gel filtration. This is the most effective method for protein purification (at least of those methods we discussed in class) and often allows one to purify an enzyme to homogeneity in a single step.
B. What is gel filtration chromatography?
Gel filtration is the protein purification method which takes advantage of the differences in molecular size of proteins. A gel filtration column is composed of beads with molecular size pores which small proteins can enter and large ones can not. As the protein mixture is passed over the gel filtration column, large proteins pass by the gel beads and elute from the column ahead of the medium sized proteins, which elute before the small ones which spend more time inside the beads. This is basically a method which separates proteins by molecular weight and can be used to obtain an estimate of the native molecular weight of a protein by comparing its elution volume from the gel filtration column to that of standard proteins with known native molecular weight. This can be accomplished by graphing the log of the native molecular weight of the standard proteins versus their elution volumes from the gel filtration column and using the elution volume of the unknown protein to estimate its native molecular weight.
C. What is Native PAGE used for?
Native PAGE (non-denaturing polyacrylamide gel electrophoresis) is a method for determining the purity of an enzyme or protein. In Native PAGE, proteins are separated due to differences in their charge density. Since an enzyme will retain its native shape or conformation during Native PAGE, an enzyme activity stain can be used detect the presence of the enzyme of interest after each purification step. All the proteins separated by Native PAGE can be detected by using a general protein stain. So by comparing the pattern obtained with the enzyme stain to the total protein stain, one can determine if you have succeeded in the protein purification and obtained a pure protein. If you have a pure protein, then you will have only one protein staining band on the Native PAGE gel and it will also stain for the enzyme activity.
D. What is SDS-PAGE used for?
SDS-PAGE (denaturing polyacrylamide gel electrophoresis) is a method for determining the molecular weight of the polypeptide subunit of a protein. SDS-PAGE is done by first denaturing the protein by heating it in the presence of the detergent sodium dodecyl sulfate (SDS) and also with a thiol reductant to break any disulfide bonds in the protein, which will interfere with the molecular weight determination. To determine the subunit molecular weight, the SDS-coated protein must be compared to standard proteins (also denatured with SDS) of known subunit molecular weight run in the same SDS-PAGE gel as the protein of unknown subunit molecular weight. Since all the denatured proteins are assumed to bind SDS to the same degree and in proportion to the length of their polypeptide backbone, they have the same charge density and are separated according to their molecular size during SDS-PAGE due to the sieving effect of the gel (ie large proteins move less in the gel during electrophoresis than the small ones). The results for the standard proteins are graphed using a semi-log plot of their molecular weights versus their mobility (distance moved down the gel during electrophoresis) and the mobility of the unknown protein is used to find its subunit molecular weight.
E. How do you determine the subunit composition of an enzyme/protein?
The subunit composition of an enzyme/protein is obtained by comparing its native molecular weight to its subunit molecular weight. The native molecular weight can be estimated by gel filtration using standard proteins of known molecular weight. The subunit molecular weight can be determined by SDS-PAGE using standard proteins of known subunit molecular weight. The native molecular weight is divided by subunit molecular weight and rounded to the nearest whole number. If the protein has 2 subunits, then it is a dimer; if it has 4 subunits, then it is a tetramer. If the native molecular weight is the same as the subunit molecular weight , then the protein is a monomer. Pure proteins must be used in these analyses since a mixture of proteins will give confusing results. Occasionally, a protein is made of 2 different polypeptide chains (like hemoglobin) and then you may find it has two different subunits - a bit more complicated situation, which is not easily handled by this approach and more sophisticated methods must be used.
3. 10 Pts. Thought Question/Cell Components
We do not really know why organelles have their own DNA. Thoughtful answers get the credit here.
One answer and a potential way to explain exist of mitochondrial DNA and proteins encoded by it, is that these unique organellar proteins encoded by the mitochondrial DNA can not get into the mitochondria due to their unusual properties. In other words, the cellular mechanism for sending proteins into organelles can not cope with some of the proteins needed by mitochondria to function, so these unique proteins must be synthesized in the mitochondrion. This may have to do with the evolutionary origin of mitochondria (and the other complex organelle, the chloroplast), which we think may have been a free living organism like a prokaryote that was captured by the precursor of eukaryotic cells.
Another answer, is that mitochondrial DNA gives the organelle more control over its metabolism and enhances cellular regulation of overall metabolism via coordination of mitochondrial development with total cellular development. Existence of mitochondrial DNA and RNA may allow for communication between the nucleus and the organelles.
A third answer (perhaps a bit beyond this course and more related to genetics and evolution of organisms) may be that cytosolic DNA gives some advantage to eukaryotic cells in reproduction and eukaryotic evolution, since in almost all cases this DNA is maternally inherited. In other words, since the mother donates the egg which has cytoplasm and therefore, cytosolic DNA, and the father does not since the sperm transfers its DNA to the egg without transferring cytoplasm, this mechanism of transferring DNA to the offspring gives some advantage in the evolution of eukaryotic organisms by having two pools of genetic information being passed on unequally from the two parental types.
©Wilbur H. Campbell, 1996; wcampbel@mtu.edu