Passage
The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ. When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy. However, Tat delivers cargo to cells with relatively low efficiency. Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads. Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1.
Figure 1 Simplified schematic of one round of solid-phase peptide synthesis. The protecting group is removed during deprotection. The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively. Each variant was allowed to dimerize by disulfide bond formation. Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc) . The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2.
Figure 2 Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours. The cells were then assessed for luciferase activity. Greater luciferase activity indicated more efficient delivery of the pLuc plasmid. The results are shown in Figure 3.
Figure 3 Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity
Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8) :802-807.
-The addition of which reagent would result in the greatest amount of dimerization of the Tat variants?

Question

Passage
The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ. When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy. However, Tat delivers cargo to cells with relatively low efficiency. Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads. Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1.

Figure 1 Simplified schematic of one round of solid-phase peptide synthesis. The protecting group is removed during deprotection. The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively. Each variant was allowed to dimerize by disulfide bond formation. Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc) . The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2.

Figure 2 Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours. The cells were then assessed for luciferase activity. Greater luciferase activity indicated more efficient delivery of the pLuc plasmid. The results are shown in Figure 3.

Figure 3 Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity
Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8) :802-807.
-The addition of which reagent would result in the greatest amount of dimerization of the Tat variants?

Quizplus · Accepted Answer

11ecba2d_0f28_a82c_a3bf_039115e5bac8_MD0008_00 Cysteine (C) residues in proteins and peptides can react with each other to form disulfide bonds. These bonds are commonly (although not always) present in dimers and multimers and help hold protein subunits together. The formation of a disulfide bond occurs through an oxidation-reduction reaction, in which the sulfur atoms of cysteine residues are initially in reduced form and become oxidized. For this reaction to occur, an oxidizing agent must be present. The passage indicates that the Tat variants dimerize by disulfide bond formation. Therefore, the addition of an oxidizing agent such as oxygen gas would help increase the amount of disulfide bond formation and, as a result, the amount of dimerization that occurs. (Choices A and B) The acidity or basicity of a chemical is not directly related to that chemical's ability to act as an oxidizing agent. Disulfide bond formation requires an oxidizing agent, not an acid or a base. (Choice C) Reducing agents such as dithiothreitol are commonly used in gels to break disulfide bonds, not form them. Educational objective: The formation of disulfide bonds between cysteine residues is an oxidation event and requires the presence of an oxidizing agent, such as oxygen gas, to occur.

Passage The HIV-Derived Transactivator of Transcription (Tat) Is a Short Peptide