17 Questions
What is a potential issue with inserting 'unnatural' glycosylation sites?
They may not be recognized properly by the glycosylation machinery
Why might N-glycosylation not occur if the glycosylation site is too close to the membrane?
Lack of proper recognition by the glycosylation machinery
What characteristic makes cysteinyl residues suitable for site-directed mutagenesis?
High reactivity due to free thiol groups
Why might endogenous cysteines need to be knocked out before introducing new cysteines by mutagenesis?
To avoid unwanted cross-reactions with existing cysteines
How can cysteine mutagenesis be used in protein engineering?
Introducing cysteines at specific sites for labeling
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
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
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
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
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
Which of the following statements about N-glycanase is correct?
N-glycanase cleaves N-linked oligosaccharides from glycosylated proteins
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
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.
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.
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.
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.
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.
Test your knowledge on glycosylation sites and cysteine mutagenesis. Learn about the considerations for inserting and engineering glycosylation sites, and the challenges involved in modifying protein structures. Understand the importance of proper spacing and recognition in glycosylation processes.
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