Glycosylation Sites and Cysteine Mutagenesis Quiz

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What is a potential issue with inserting 'unnatural' glycosylation sites?

They are more reactive than natural glycosylation sites
They may not be recognized properly by the glycosylation machinery (correct)
They require a longer spacer region than native sites
They always result in successful glycosylation

Why might N-glycosylation not occur if the glycosylation site is too close to the membrane?

Excessive interaction with membrane proteins
Inhibition of enzyme activity near the membrane
Lack of proper recognition by the glycosylation machinery (correct)
Increased reactivity near the membrane

What characteristic makes cysteinyl residues suitable for site-directed mutagenesis?

Inability to be incorporated into proteins
High reactivity due to free thiol groups (correct)
Resistance to labeling with maleimide derivative
Low reactivity and stability in proteins

Why might endogenous cysteines need to be knocked out before introducing new cysteines by mutagenesis?

To avoid unwanted cross-reactions with existing cysteines (B) Signup and view all the answers

How can cysteine mutagenesis be used in protein engineering?

Introducing cysteines at specific sites for labeling (A) Signup and view all the answers

Which of the following techniques is used to identify the location of a protein tag or loop?

Using fluorescent antibodies against the epitope under intact and permeabilized conditions (A) Signup and view all the answers

What is the purpose of introducing a glycosylation site (Asn-X-Ser/Thr motif) into a protein?

To determine if a loop is extracellular by detecting glycosylation (A) Signup and view all the answers

How can the presence of glycosylation at an introduced site be detected?

By observing a shift in the protein's mobility on SDS-PAGE and a change in molecular weight after N-glycanase treatment (A) Signup and view all the answers

What is the purpose of site-directed mutagenesis in the context of determining the topology of a membrane protein?

To introduce cysteine residues for labeling with thiol-specific reagents (B) Signup and view all the answers

What is the purpose of using thiol-specific reagents in the study of membrane protein topology?

To label cysteine residues introduced by site-directed mutagenesis and determine their accessibility (B) Signup and view all the answers

Which of the following statements about N-glycanase is correct?

N-glycanase cleaves N-linked oligosaccharides from glycosylated proteins (B) Signup and view all the answers

In cysteine mutagenesis, what is the primary purpose of replacing naturally occurring cysteine residues with alanine residues?

To obtain a cysteine-less construct that is typically still functional (B) Signup and view all the answers

Which of the following statements accurately describes the use of membrane-permeant and membrane-impermeant maleimide reagents in cysteine mutagenesis?

Both membrane-permeant and membrane-impermeant maleimides react with extracellular cysteine residues, but only membrane-permeant maleimides react with intracellular cysteine residues. (D) Signup and view all the answers

What is a potential limitation of cysteine mutagenesis that should be considered when interpreting the results?

The cysteine residues may be unreactive due to their location in tightly folded regions of the protein, making them inaccessible to the reagents. (B) Signup and view all the answers

In the context of glycoprotein engineering, what is the significance of introducing cysteine residues at specific locations?

It allows for the site-specific introduction of N-glycosylation sites by creating a consensus sequence for glycosylation. (D) Signup and view all the answers

Which of the following statements is true regarding the use of cysteine mutagenesis in combination with thiol-specific reagents?

It can be used to determine the topology of transmembrane proteins by identifying extracellular and intracellular cysteine residues. (C) Signup and view all the answers

In the context of site-directed mutagenesis, what is the purpose of introducing cysteine residues at specific locations within a protein?

To facilitate the binding of thiol-specific reagents for structural and functional studies of the protein. (B) Signup and view all the answers

Potential issue with unnatural glycosylation sites

Unnatural glycosylation sites might not be recognized by the cellular glycosylation machinery.

N-glycosylation hindered by membrane proximity

If the glycosylation site is too close to the cell membrane, the glycosylation machinery may not access it.

Cysteine suitability for mutagenesis

Cysteine residues are good for site-directed mutagenesis due to their highly reactive free thiol groups.

Knocking out endogenous cysteines

Before introducing new cysteines, existing cysteines are sometimes removed to avoid unwanted reactions.