🧬AP Biology
1. Proteins are essential macromolecules that perform a diverse range of functions within living organisms, and their function is strictly dependent on their three-dimensional structure.
Researchers are investigating the structural stability of Protein X, a secreted protein found in certain bacteria. They hypothesize that a disulfide bond between two cysteine residues is critical for the protein's stability. To test this, they created a mutant version of Protein X in which the two cysteine residues were replaced with alanine residues.
In their first experiment, the researchers purified both the wild-type Protein X and the mutant protein. They incubated samples of each protein at various temperatures ranging from 20°C to 80°C for 30 minutes and then measured the percent of protein that remained folded (functional). The results are shown in Figure 1.
To determine whether the disulfide bond formed in the wild-type protein is intermolecular (between two separate polypeptide chains) or intramolecular (within a single chain), the researchers performed a second experiment. They analyzed the wild-type and mutant proteins using SDS-PAGE gel electrophoresis under two conditions: non-reducing (which leaves disulfide bonds intact) and reducing (which breaks disulfide bonds). The results are represented in Figure 2.
Describe how the R-groups of amino acids contribute to the tertiary structure of a protein.
Figure 1. Effect of temperature on the stability of wild-type and mutant Protein X after 30-minute incubation. Percent of protein remaining folded is plotted versus temperature. Error bars show ±2 standard errors (±5 percentage points) at every temperature for both proteins.
Identify the dependent variable in the experiment shown in Figure 1.
Justify why the researchers compared the thermal stability of the mutant protein to the wild-type protein instead of comparing it to a protein with a random amino acid sequence.
Based on Figure 1, describe the effect of the amino acid substitution on the thermal stability of Protein X.
Figure 2. SDS-PAGE analysis of wild-type and cysteine-to-alanine mutant Protein X under non-reducing and reducing conditions. Lane 1 is a molecular mass marker (kDa).
Identify the independent variable in the researchers' second experiment (results shown in Figure 2).
Based on Figure 2, identify the specific lane(s) containing Protein X in a multimeric (dimer) state.
Based on the data in Figure 2, the monomeric form of Protein X has an approximate molecular mass of 30 kDa (30,000 Daltons). Assuming the average molecular mass of an amino acid is 120 Daltons, calculate the approximate number of amino acids in the monomeric protein.
Researchers claim that the formation of the disulfide bond in Protein X is intermolecular rather than intramolecular. Using evidence from Figure 2, support the researchers' claim.
Justify the claim that replacing cysteine with alanine prevents the formation of disulfide bonds based on the chemical properties of their R-groups.
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