Passage
Fluorescent amino acids are useful for labeling proteins because they can be visualized spectroscopically, facilitating the study of protein structure and interactions. The noncanonical amino acid dansylalanine, a fluorescent amino acid that is sensitive to the hydrophobicity of its environment, is a good candidate to be incorporated into proteins because of its small size and fluorescent capabilities. Dansyl chloride and Compound 1 were used to synthesize dansylalanine, as shown in Reaction 1. Dansylalanine absorbs maximally at 340 nm and emits at approximately 540 nm.
Reaction 1 Synthesis of dansylalanineA leucyl-tRNA synthetase from Escherichia coli was engineered to aminoacylate bacterial tRNAs containing a CUA anticodon with dansylalanine. After verifying that the tRNA/synthetase pair does not interact with endogenous amino acids or tRNAs, it was transfected into the yeast Saccharomyces cerevisiae along with a plasmid encoding a mutated human superoxide dismutase (hSOD) . Using this system, dansylalanine was genetically incorporated into hSOD in place of either Gln-16 or Trp-33, and expressed in S. cerevisiae.Position 16 is located on the surface of hSOD at the N-terminus of a β-strand, and position 33 lies in the center of a strand within a β-barrel. To probe changes in the structure of hSOD, researchers exposed the purified protein to guanidinium chloride (GdmCl) to denature the protein at concentrations ranging 0-4.5 M. The experiment was conducted in a 35 mM sodium phosphate buffer at pH 7.2. The fluorescence of hSOD with dansylalanine at either position 16 or 33 was monitored at each GdmCl concentration, and the results of the unfolding experiment are shown in Figure 1.
Figure 1 Plot of dansylalanine fluorescence at different concentrations of GdmCl
Adapted from Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG. A genetically encoded fluorescent amino acid. Proc Natl Acad Sci USA. 2006.
-What is the charge of the N-terminus during the protein unfolding experiment?

Question

Passage
Fluorescent amino acids are useful for labeling proteins because they can be visualized spectroscopically, facilitating the study of protein structure and interactions. The noncanonical amino acid dansylalanine, a fluorescent amino acid that is sensitive to the hydrophobicity of its environment, is a good candidate to be incorporated into proteins because of its small size and fluorescent capabilities. Dansyl chloride and Compound 1 were used to synthesize dansylalanine, as shown in Reaction 1. Dansylalanine absorbs maximally at 340 nm and emits at approximately 540 nm.

Reaction 1 Synthesis of dansylalanineA leucyl-tRNA synthetase from Escherichia coli was engineered to aminoacylate bacterial tRNAs containing a CUA anticodon with dansylalanine. After verifying that the tRNA/synthetase pair does not interact with endogenous amino acids or tRNAs, it was transfected into the yeast Saccharomyces cerevisiae along with a plasmid encoding a mutated human superoxide dismutase (hSOD) . Using this system, dansylalanine was genetically incorporated into hSOD in place of either Gln-16 or Trp-33, and expressed in S. cerevisiae.Position 16 is located on the surface of hSOD at the N-terminus of a β-strand, and position 33 lies in the center of a strand within a β-barrel. To probe changes in the structure of hSOD, researchers exposed the purified protein to guanidinium chloride (GdmCl) to denature the protein at concentrations ranging 0-4.5 M. The experiment was conducted in a 35 mM sodium phosphate buffer at pH 7.2. The fluorescence of hSOD with dansylalanine at either position 16 or 33 was monitored at each GdmCl concentration, and the results of the unfolding experiment are shown in Figure 1.

Figure 1 Plot of dansylalanine fluorescence at different concentrations of GdmCl
Adapted from Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG. A genetically encoded fluorescent amino acid. Proc Natl Acad Sci USA. 2006.
-What is the charge of the N-terminus during the protein unfolding experiment?

Quizplus · Accepted Answer

11ecba2d_1275_fea2_a3bf_59f75a920ac6_MD0008_00 A protein is made up of several amino acids linked through peptide bonds. The free amine group at one end of the peptide and the free carboxyl group at the opposite end are known as the N- and C-termini, respectively. The components of the peptide can take on different charges, depending on the pH of the environment. Under neutral conditions (pH ~7), the N- and C-termini are charged. Neutral pH is above the carboxyl group's pK_a of 2, causing it to become deprotonated and form its conjugate base. The carboxyl group in this form contains a −1 charge. However, the pH is below the amine group's pK_a of 9, so it is fully protonated and exists in the conjugate acid form. A protonated amine contains a +1 charge. This doubly charged species is known as a zwitterion, or a dipolar ion, in which the charges at both ends cancel each other out. The passage states the unfolding experiment is done at pH 7.2. Under these neutral conditions, the N-terminus will be protonated and exist in the conjugate acid form, giving it a +1 charge. (Choice A) Although it is true that the charge of the N-terminus is +1 in the unfolding experiment, the amine group is protonated at pH 7.2. (Choices C and D) The free amine of the N-terminus is protonated, giving it a +1 charge instead a neutral charge. The protonated form of an amine is the conjugate acid rather than the conjugate base. Educational objective: In a neutral environment (pH ~7), amino acids are zwitterions, also known as dipolar ions, because the N- and C-termini are charged (+1 and −1, respectively). The carboxyl group is deprotonated and in its conjugate base form. The amine group is protonated and in its conjugate acid form.

Passage Fluorescent Amino Acids Are Useful for Labeling Proteins Because They