SB-BB

Passage 7

Follicle stimulating hormone (FSH) is a peptide hormone known to activate FSH G protein-coupled receptors (FSHRs) on ovarian cells. However, FSHRs have recently been found on osteoclasts, and their activation stimulates osteoclastogenesis and bone resorption. Although estrogen activity inhibits these effects, high levels of FSH, despite normal levels of estrogen, are associated with bone density loss.

A peptide consisting of a 13-residue sequence located within the receptor-binding domain of FSH was synthesized (FSH_pep, Figure 1). An antibody (FSH-Ab) was then designed to bind the specific sequence of FSH_pep. The antibody was also able to bind FSH.

Figure 1 Structure of FSH_pep (Note: Underlined and bolded residues are found at the receptor–hormone interface.)

The specificity of FSH-Ab for either FSH_pep or FSH was compared using an enzyme-linked immunosorbent assay (ELISA), in which an enzyme-linked antibody was used to detect FSH-Ab that had bound either FSH_pep or FSH. When enzyme substrate was added, a fluorescent product formed. The intensity of the fluorescence was measured (Figure 2), and increased absorbance was indicative of increased binding.

Figure 2 FSH-Ab binding to (A) FSH_pep and (B) FSH (Note: Absorbance scales are different.)

L. Zhu et al., “Blocking Antibody to the β-subunit of FSH Prevents Bone Loss by Inhibiting Bone Resorption and Stimulating Bone Synthesis,” PNAS. ©2012 National Academy of Sciences.

Questions

46. Based on the passage, FSHRs are found on cells of which type(s) of tissue?

1. Connective
2. Epithelial
3. Nervous

1. I only
2. III only
3. I and II only
4. II and III only

47. Which conclusion is supported by the data presented in Figure 2?

1. The K_d for binding of FSH_pep by FSH-Ab is lower than the K_d for binding of FSH by FSH-Ab.
2. IgG has a higher affinity for FSH than for FSH_pep at all concentrations tested.
3. FSH binding sites for FSH-Ab are saturated when FSH reaches a concentration of 10µg.
4. The affinity of FSH for FSH-Ab is greater than the affinity of FSH_pep for FSH-Ab.

48. Which statement best explains why the absorbance levels for FSH differ from those for FSH_pep?

1. The FSH-Ab binds non-specifically to more than one site on FSH_pep.
2. IgG competitively inhibits the binding of FSH-Ab to FSH.
3. The FSH-Ab cooperatively binds FSH.
4. The tertiary structure of FSH limits FSH-Ab binding interactions.

49. Based on the FSH_pep sequence, which amino acid substitution in the FSHR binding domain is most likely to have the greatest effect on reducing bone density loss in the presence of high levels of FSH?

1. K4R
2. R8D
3. V2L
4. Q12N

50. Which of the following reasons does NOT describe why FSH_pep was included in the ELISA experiment?

1. To act as a positive control to confirm that the assay was functional
2. To provide a baseline against which to evaluate the affinity of FSH-Ab for FSH
3. To generate data to support that the FSH-Ab binds to the receptor-binding domain of FSH
4. To determine the amount of FSH-Ab that is most effective as a therapeutic treatment

51. Which statement describes a characteristic of FSH?

1. FSH does not require transport proteins to remain soluble in the bloodstream.
2. FSH enters the bloodstream by diffusing across the plasma membrane of endocrine cells.
3. FSH is synthesized from cholesterol.
4. FSH is derived from a single amino acid.

52. Two gel electrophoresis analyses are performed on a sample of purified protein with unknown structure: SDS-PAGE (1 band appears) and SDS-PAGE under reducing conditions (2 bands appear). Which prediction about the protein is directly supported by these results? The protein:

1. is posttranslationally glycosylated.
2. contains a high proportion of charged residues.
3. contains no disulfide bonds.
4. is composed of multiple subunits.

53. Which type of interaction does NOT contribute to the stabilization of the tertiary structure of a protein?

1. Disulfide bond
2. Phosphodiester bond
3. Hydrogen bond
4. Salt bridge

54. Which type of inhibitor does NOT alter the K_M/V_max ratio of an enzyme?

1. Competitive
2. Uncompetitive
3. Noncompetitive
4. Mixed

55. An enzyme is more effectively inhibited by uncompetitive inhibitors when:

1. the substrate concentration is decreased.
2. the substrate concentration is increased.
3. the inhibitor concentration is increased.

1. I only
2. III only
3. I and III only
4. II and III only

56. Which peptide sequence is most likely found in a transmembrane helix of a protein?

1. Ala–Ile–Phe–Val–Leu
2. Ala–Thr–Lys–Asn–Leu
3. Lys–Thr–Arg–Asn–His
4. Val–Thr–Pro–Tyr–Ser

Answers

Question 46

Solution: The correct answer is C. This is a Biology question that falls under the content category “Assemblies of molecules, cells, and groups of cells within single cellular and multicellular organisms.” The answer to this question is C because the passage states that FSH regulates the release of estrogen from the ovaries and directly stimulates osteoclast activity. Ovarian cells are epithelial cells (II), and osteoclasts are connective tissue cells (I). The passage provides no information to suggest that nervous tissue cells (III) are stimulated by FSH. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall the different types of tissues formed from eukaryotic cells.

Question 47

Solution: The correct answer is A. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is A because the scale on the graphs show that FSH-Ab binds FSH_pep with a higher affinity than it binds FSH. Higher affinity corresponds to a lower K_d. It is a Data-based and Statistical Reasoning question because you are asked to draw a conclusion from experimental data.

Question 48

Solution: The correct answer is D. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because the absorbance levels show that FSH-Ab binds FSH_pep at higher levels than FSH. The difference between the two is that FSH is a fully folded protein, whereas FSH_pep is a peptide sequence. Thus, the most likely explanation for why FSH-Ab binds FSH at lower levels than FSH_pep is because the tertiary structure of FSH interferes with FSH-Ab binding. It is a Scientific Reasoning and Problem Solving question because you are asked to demonstrate your understanding of protein interactions by proposing an explanation for the results of an experiment.

Question 49

Solution: The correct answer is B. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because FSHR activation increases bone density loss. Therefore, to decrease loss, the substitution should disrupt binding. The R8D substitution replaces a positively charged residue with a negatively charged residue. This would most likely disrupt binding of FSH to the FSHR and the subsequent stimulation of osteoclast activity. It is a Scientific Reasoning and Problem Solving question because you are asked to apply your knowledge of the properties of amino acids in order to solve a problem.

Question 50

Solution: The correct answer is D. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because the use of FSH_pep would provide no useful information about the drug’s therapeutic use, as the peptide does not exist in vivo; further studies using FSH would need to be performed to accomplish this. The use of the FSH_pep acted as a positive control (A) to show that the assay was working as expected (the FSH-Ab was binding its target sequence), because the FSH-Ab was designed to bind the specific sequence of FSH_pep. Its use provided a baseline to determine how the binding affinity of FSH-Ab for FSH compared to that of the peptide alone, which would be expected to be maximal (B). The use of FSH_pep provided data to support that the binding of FSH-Ab to FSH, as observed in the assay, was most likely due to the binding of the antibody to the specific receptor-binding sequence and not another region of FSH (C). It is a Reasoning about the Design and Execution of Research question because you are asked to demonstrate an understanding of a given experimental design.

Question 51

Solution: The correct answer is A. This is a Biology question that falls under the content category “Structure and functions of the nervous and endocrine systems and ways in which these systems coordinate the organ systems.” The answer to this question is A because peptide hormones are hydrophilic and soluble in blood. Hormones that must bind transport proteins are steroid proteins, which are lipophilic. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall the properties of the different classes of hormones.

Question 52

Solution: The correct answer is D. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because a reducing agent is used during SDS-PAGE to cleave disulfide bonds. The appearance of 1 band on SDS-PAGE without the reducing agent and 2 bands on SDS-PAGE with a reducing agent, suggests that at least one disulfide bond is present in the protein and that the disulfide bond(s) hold(s) two separate subunits of different masses together. This is the best response. It is a Data-based and Statistical Reasoning question because you are asked to interpret data in order to make a prediction.

Question 53

Solution: The correct answer is B. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because phosphodiester bonds link adjacent nucleotides in DNA. They do not contribute to the stabilization of protein structure. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall the types of interactions that stabilize protein structure.

Question 54

Solution: The correct answer is B. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because uncompetitive inhibitors do not alter the slope of the Lineweaver–Burk plot, which is equal K_M/V_max. It is a Scientific Reasoning and Problem Solving question because you must work with various models of enzyme inhibition to determine the correct answer.

Question 55

Solution: The correct answer is D. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because uncompetitive inhibitors bind their target enzymes only when the substrate is first bound to the enzyme. Since at higher substrate concentrations, the substrate–enzyme complex are more abundant, the uncompetitive inhibitor will work most effectively when the substrate concentration is the highest. Additionally, an increase in the inhibitor concentration results in increased enzyme binding and inhibition. It is a Scientific Reasoning and Problem Solving question because you must work with the scientific model of uncompetitive enzyme inhibition to determine the correct answer.

Question 56

Solution: The correct answer is A. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is A because transmembrane helices are made up of mostly unbroken stretches of hydrophobic amino acids. It is a Scientific Reasoning and Problem Solving question because you must evaluate the sequence of each peptide and identify the one that fit the description stated in the question.