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 researchers observed that the last three amino acids added to the Tat monomer during solid-phase peptide synthesis required more time for linkage than the first several amino acids added. Which of the following best explains this observation?

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 researchers observed that the last three amino acids added to the Tat monomer during solid-phase peptide synthesis required more time for linkage than the first several amino acids added. Which of the following best explains this observation?

Quizplus · Accepted Answer

11ecba2d_0f27_218b_a3bf_e519de93e107_MD0008_00 The side chains of the amino acids in a peptide interact with each other through electrostatic interactions, including ionic attractions and repulsions, hydrogen bonding, London dispersion forces, and steric interference. These interactions can also influence the ability of amino acids and peptides to react with each other. Amino acids with similar charges repel each other, reducing the probability that they will collide in a way that results in a reaction (ie, a productive collision). The passage states that solid-phase peptide synthesis occurs from C-terminus to N-terminus. However, by convention the sequence of a peptide or protein is always written from N-terminus to C-terminus. Therefore, the last three amino acids added to the Tat monomer (ie, those at the N-terminus) were a lysine (K), another lysine, and an arginine (R) residue. These residues are all positively charged and will repel each other, resulting in fewer productive collisions. In contrast, the first several amino acids added were glutamine (Q), proline (P), and another proline, which are neutral and will not experience the same electrostatic repulsion. These amino acids are more likely to collide productively and react more quickly. (Choice A) Figure 1 shows that it is the amino group of the growing peptide (not the added amino acids) that reacts, and that the carboxyl group (not the amino group) of added amino acids reacts. (Choice C) Proline residues are known to add kinks to polypeptide chains, making them unfavorable for α-helices. However, the proline residues in Tat are near the C-terminus of the peptide, and therefore they are among the first amino acids added, not the last. (Choice D) Although charge repulsions make collisions less likely at the end of the Tat peptide, this decreased probability is not related to the length of the growing chain. In general, the length of a peptide chain has no impact on the probability of collisions between molecules. Educational objective: Charged amino acids within a peptide interact with each other. Similarly charged residues repel each other whereas oppositely charged residues attract each other.

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