Passage
Many lysosomal proteins are transported to the lysosome by the cation-independent mannose 6-phosphate receptor (CI-MPR). CI-MPR is a transmembrane protein found in the Golgi apparatus and vesicles. It contains 15 globular domains folded similarly on the luminal side of the membrane, each of which has up to 3 glycosylation sites and 6−12 cysteine residues that aid in folding.The ability of CI-MPR to recognize multiple different ligands was studied by fluorescent microarray analysis. Microarray analysis was performed by cross-linking candidate ligands such as plasminogen, insulin-like growth factor 2 (IGF-II), and mannose 6-phosphate (M6P), to glass slides. The slides were then incubated with full-length CI-MPR or recombinantly expressed CI-MPR domains, followed by incubation with an anti−CI-MPR antibody. A fluorescently labeled antibody that binds the first antibody was then added, and the relative fluorescence levels of each candidate ligand were compared. Higher fluorescence levels correspond to greater binding.Experiment 1Microarray analysis was carried out on slides containing varying levels of candidate ligands, and binding affinities of each CI-MPR domain for its primary ligand were estimated. Table 1 shows the results.Experiment 2Domains 3, 5, and 9 bind carbohydrates. Amino acids in these domains were mutated to determine which are required for binding. Based on this experiment, scientists concluded that Arg, Glu, and Tyr are required for carbohydrate binding by domains 3, 5, and 9 of CI-MPR.Table 1
Adapted from Bohnsack RN, Song X, Olson LJ, et al. Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem. 2009;284(50):35215-26.
-Residues in the binding pockets of domains 3, 5, and 9 interact with carbohydrates through hydrogen bonds. In Experiment 2, tyrosine residues were mutated to remove hydrogen bonding while minimizing changes in the shape of the active site. Tyrosine residues were most likely mutated to:

Question

Passage
Many lysosomal proteins are transported to the lysosome by the cation-independent mannose 6-phosphate receptor (CI-MPR).  CI-MPR is a transmembrane protein found in the Golgi apparatus and vesicles.  It contains 15 globular domains folded similarly on the luminal side of the membrane, each of which has up to 3 glycosylation sites and 6−12 cysteine residues that aid in folding.The ability of CI-MPR to recognize multiple different ligands was studied by fluorescent microarray analysis.  Microarray analysis was performed by cross-linking candidate ligands such as plasminogen, insulin-like growth factor 2 (IGF-II), and mannose 6-phosphate (M6P), to glass slides.  The slides were then incubated with full-length CI-MPR or recombinantly expressed CI-MPR domains, followed by incubation with an anti−CI-MPR antibody.  A fluorescently labeled antibody that binds the first antibody was then added, and the relative fluorescence levels of each candidate ligand were compared.  Higher fluorescence levels correspond to greater binding.Experiment 1Microarray analysis was carried out on slides containing varying levels of candidate ligands, and binding affinities of each CI-MPR domain for its primary ligand were estimated.  Table 1 shows the results.Experiment 2Domains 3, 5, and 9 bind carbohydrates.  Amino acids in these domains were mutated to determine which are required for binding.  Based on this experiment, scientists concluded that Arg, Glu, and Tyr are required for carbohydrate binding by domains 3, 5, and 9 of CI-MPR.Table 1
  Adapted from Bohnsack RN, Song X, Olson LJ, et al. Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem. 2009;284(50):35215-26.
-Residues in the binding pockets of domains 3, 5, and 9 interact with carbohydrates through hydrogen bonds.  In Experiment 2, tyrosine residues were mutated to remove hydrogen bonding while minimizing changes in the shape of the active site.  Tyrosine residues were most likely mutated to:

Quizplus · Accepted Answer

11ecba2d_0e5d_68b0_a3bf_f39961ef206b_MD0008_00 Amino acids can be divided into different groups based on the properties of their side chains, with many amino acids falling into more than one group. For example, tyrosine is predominantly aromatic but also has some polar characteristics. The polar hydroxyl group on the aromatic ring allows tyrosine to hydrogen bond with other polar molecules such as carbohydrates. Amino acids that are similar in some ways and different in others can be substituted for each other to preserve some protein properties and change others. Like tyrosine, phenylalanine (Phe) is aromatic. It is identical to tyrosine except that it has a hydrogen atom where tyrosine has a hydroxyl group. The lack of a hydroxyl group prevents phenylalanine from forming hydrogen bonds, but its otherwise identical structure allows it to occupy similar spaces as tyrosine. It is the best substitute for tyrosine to remove hydrogen bonding without significantly altering the shape of the active site. (Choice A) Threonine (Thr) can hydrogen bond and is not aromatic. The desired mutation requires an aromatic amino acid that cannot hydrogen bond. (Choice C) Alanine (Ala) is not aromatic and is much smaller than tyrosine. Although its substitution for tyrosine would remove hydrogen bonding, it would likely significantly alter the shape of the binding pocket. (Choice D) Histidine (His) is aromatic but polar, and may even be charged in some instances. Its substitution for tyrosine would not remove the ability to hydrogen bond. Educational objective: Amino acids can be divided into different groups, with some amino acids falling into more than one group. Substituting one aromatic amino acid for another can preserve aromatic properties. Substituting a polar amino acid for a nonpolar amino acid can abolish interactions between proteins and ligands.

Passage Many Lysosomal Proteins Are Transported to the Lysosome by the the Cation-Independent