B/B 08 · Protein structure and electrophoresis
Characterizing a Protein
Use gel electrophoresis, pI and refolding data to work out a protein’s structure and stability.
Passage
Proteins are chains of amino acids joined by peptide bonds. The order of the amino acids is the primary structure, local folding into α helices and β sheets is the secondary structure, the three-dimensional shape of a single chain is the tertiary structure, and the association of several chains is the quaternary structure. Covalent disulfide bonds between cysteine side chains, hydrogen bonds, ionic interactions and the hydrophobic effect all help to hold the folded protein together.
Biochemists characterize proteins with several techniques. In SDS-PAGE, a protein is heated with the detergent sodium dodecyl sulfate (SDS), which unfolds the protein and coats it with negative charge in proportion to its length, so that the proteins move through a polyacrylamide gel toward the positive electrode at a rate that depends on size, with smaller proteins moving farther. A reducing agent such as β-mercaptoethanol (BME) can be added to break disulfide bonds. In native electrophoresis, no detergent is used, and a protein’s movement depends on both its charge and its shape. In isoelectric focusing, proteins move through a pH gradient until they reach the pH at which their net charge is zero, which is the isoelectric point (pI).
Part 1. A researcher purifies an enzyme, Protein X, and analyzes it by SDS-PAGE. Molecular mass standards are run on the same gel. Table 1 shows the masses of the bands that are seen.
Table 1. SDS-PAGE of purified Protein X.
| Sample treatment before loading | Mass of band(s) seen (kDa) |
|---|---|
| SDS, no reducing agent | 60 |
| SDS + BME | 30 |
Part 2. In isoelectric focusing, Protein X is found to have a pI of 5.5. The researcher then tests the enzyme activity after different treatments. After each treatment, the denaturing agent is removed by dialysis, and the activity is measured and expressed as a percentage of the untreated enzyme. Table 2 shows the results.
Table 2. Enzyme activity after treatment and removal of the agent.
| Treatment | Activity (% of untreated) |
|---|---|
| 8 M urea for 1 hour, then dialysis | 90 |
| BME alone for 1 hour, then dialysis | 40 |
| Heating to 95 °C for 10 minutes, then cooling | 5 |
A protein’s net charge depends on the pH. An amino acid or peptide gains a proton on each group when the pH falls below the pKa of that group, and loses it when the pH rises above the pKa. The isoelectric point of a protein is therefore the pH at which the positive charges and negative charges balance. A protein that is analyzed in a gel whose pH differs from its pI will carry a net charge and move in an electric field.
Part 3. The researcher also studies a short peptide, Lys-Gly-Gly. The approximate pKa values are 3 for the C-terminal carboxyl group, 9 for the N-terminal amino group, and 10.5 for the side chain of lysine. Urea unfolds proteins by disrupting hydrogen bonds and the hydrophobic effect, but it does not break covalent bonds. Heating to 95 °C also unfolds proteins, and the unfolded chains often clump together as they cool, which keeps them from refolding. The researcher keeps all of the samples at the same protein concentration and uses the same amount of protein in every lane and every activity assay.
Questions
Question 1 of 6
What do the results in Table 1 indicate about the structure of Protein X?
Noncovalent interactions are broken by SDS, so the band without BME would have been 30 kDa.
If the protein were a single 60 kDa chain, BME would not change the mass of the band.
A 60 kDa subunit would show a band at 60 kDa after BME treatment.
Why the answer is what it is
SDS breaks noncovalent interactions, so a 60 kDa band without BME means that the two chains are still linked covalently. BME breaks disulfide bonds and releases a single band at 30 kDa, so the protein is a dimer of two chains of the same size held by disulfide bonds.
Review: Amino Acids · Protein Structure & Folding · Enzymes as Structured Catalysts · Acids & Bases
Question 2 of 6
Why does SDS allow proteins to be separated by mass in SDS-PAGE?
SDS carries a negative charge, so the coated proteins move toward the positive electrode.
SDS does not cleave peptide bonds.
SDS gives proteins a strong negative charge. The charge-to-mass ratio is similar for all of them.
Why the answer is what it is
Because SDS-coated proteins have similar charge-to-mass ratios and similar unfolded shapes, the gel acts as a sieve, and smaller proteins move farther.
Review: Amino Acids · Protein Structure & Folding · Enzymes as Structured Catalysts · Acids & Bases
Question 3 of 6
In a native gel at pH 8.0, in which direction would Protein X (pI 5.5) be expected to move?
pH 8.0 is above the pI of 5.5, not below it.
Above the pI a protein has lost more protons and carries a net negative charge.
Only at the pI is the net charge zero. At pH 8.0 the protein is charged and moves.
Why the answer is what it is
At a pH above its pI, a protein has a net negative charge, so it moves toward the positive electrode (anode) in an electric field.
Review: Amino Acids · Protein Structure & Folding · Enzymes as Structured Catalysts · Acids & Bases
Question 4 of 6
BME alone reduces activity to 40%, but urea followed by dialysis leaves 90%. Which conclusion is best supported?
The enzyme recovered 90% of its activity after urea was removed, so urea did not destroy it.
BME reduces disulfide bonds. It does not cleave peptide bonds.
Nothing in the data shows a change in pI, and BME acts on disulfide bonds.
Why the answer is what it is
Urea unfolds the protein by disrupting noncovalent interactions, but the protein refolds when the urea is removed, as its primary sequence holds the information for its folding. BME breaks disulfide bonds, and the lower activity shows that these bonds are important for the enzyme.
Review: Amino Acids · Protein Structure & Folding · Enzymes as Structured Catalysts · Acids & Bases
Question 5 of 6
What does the recovery of 90% of the activity after urea treatment suggest about the folding of Protein X?
Urea does not break peptide bonds, so the primary structure was not changed.
The experiment used a purified protein and dialysis. No chaperone was added.
Urea unfolds proteins by disrupting hydrogen bonds and the hydrophobic effect.
Why the answer is what it is
If the protein regains activity after the denaturant is removed, the information for folding must be in the primary structure, which was not altered by urea.
Review: Amino Acids · Protein Structure & Folding · Enzymes as Structured Catalysts · Acids & Bases
Question 6 of 6
What is the approximate net charge of Lys-Gly-Gly at pH 7?
At pH 7 the C-terminus is negative (−1), but the N-terminal amino group and the lysine side chain are both protonated (+2).
This would require the lysine side chain to be deprotonated, which happens only above its pKa of 10.5.
The C-terminal carboxyl group (pKa 3) is deprotonated at pH 7 and contributes −1.
Why the answer is what it is
At pH 7: the N-terminal amino group (pKa 9) is mostly protonated (+1), the lysine side chain (pKa 10.5) is protonated (+1), and the C-terminal carboxyl group (pKa 3) is deprotonated (−1). The net charge is +1.
Review: Amino Acids · Protein Structure & Folding · Enzymes as Structured Catalysts · Acids & Bases
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