Posttranslational Modifications

What assists in protein folding during or after synthesis?

Chaperones or chaperonins assist in protein folding.

What is protein folding?

The process by which a polypeptide coils and folds into its three-dimensional shape, often assisted by chaperones.

What are post-translational modifications?

Changes to proteins that occur after translation and are essential to their activity.

When does protein folding occur for secreted or membrane proteins?

Folding occurs as the protein is "stuffed" into the rough ER during translation.

What determines the primary structure of a protein?

The amino acid sequence, which is determined indirectly by the DNA sequence.

What are the two types of secondary structure in proteins?

Alpha helix and beta-pleated sheet.

What causes secondary structure in proteins?

Hydrogen bonding between the backbones (amino and carboxyl groups) of amino acids.

What determines the tertiary structure of a protein?

Random interactions between R groups of amino acids.

What is quaternary structure in proteins?

A structure formed when more than one polypeptide chain comes together to function as one unit.

What is an example of a protein with quaternary structure?

Hemoglobin, which has four different polypeptide chains.

Do all proteins have quaternary structure?

No, only proteins composed of multiple polypeptide chains have quaternary structure.

What assists in protein folding?

Folding can be assisted by chaperones.

What is the first post-translational modification?

Folding is the first one - the protein folds spontaneously to the correct structure.

What proteins assist with folding?

Folding may be assisted by other proteins called 'chaperones' or "chaperonins".

When does folding occur for secreted or membrane proteins?

Folding happens as the protein is "stuffed" into the rough ER during translation.

What is the primary structure of a protein?

The amino acid sequence of the protein determined indirectly by the DNA sequence.

What forms can the secondary structure take?

The alpha helix or beta-pleated sheet.

What causes secondary structure formation?

Interactions (hydrogen bonds) between the backbones of the amino acids (amino/carboxyl groups).

What is quaternary structure?

The result of more than one polypeptide chain coming together to function as one protein unit.

What is proteolytic cleavage?

A posttranslational modification involving the cutting of the peptide chain.

What is commonly removed during proteolytic cleavage?

The initial methionine.

What else can be removed from a protein after translation?

Signal peptides.

What recognizes the signal peptide during protein targeting?

The signal recognition particle (SRP).

Where does the SRP direct the protein after recognizing the signal peptide?

Into the rough endoplasmic reticulum (ER).

Where are proteins made on rough ER ribosomes typically sent?

Through the endomembrane system (ER to Golgi to final destination).

What determines where a protein will go after synthesis?

Signal sequences translated as part of the protein.

Where are signal peptides located in the protein?

Near the amino end of the protein.

What does the signal peptide on rough ER ribosomal made proteins do?

Signals that the protein should be inserted into the ER.

What happens to the signal sequence once the protein reaches its destination?

It is usually cleaved.

What types of destinations can proteins be targeted to by signal sequences?

Mitochondria, chloroplasts, nucleus, or extracellular space.

What is the function of the Golgi apparatus in protein processing?

It acts as the "UPS center" of the cell to package and ship proteins.

What is proteolytic activation of proteins?

A mechanism where inactive proteins are activated by cleavage of a pro-domain.

What are proproteins?

Inactive precursor proteins that are activated by proteolytic cleavage.

What is an example of a proprotein?

Caspase.

What are preproproteins?

Proteins with both a signal sequence and a pro-domain that require two cleavages for activation.

What is an example of a preproprotein?

Insulin.

Why is proteolytic activation useful?

It allows proteins to be made in advance and stored for rapid activation when needed.

When are procaspases cleaved to become active?

Only when apoptosis is triggered.

What happens to the pro-domain during activation?

It is cleaved off to activate the protein.

How is proteolytic cleavage detected?

The easiest way is through western blotting.

What does western blotting detect in proteolytic cleavage?

It measures the size of protein bands to see if cleavage has occurred.

What is an example of proteolytic cleavage detection using western blot?

Cleavage of PARP from 116K to around 85K during apoptosis.

What is phosphorylation?

The addition of a phosphate group, which is negatively charged and affects protein conformation.

What enzymes carry out phosphorylation?

Kinases.

What types of kinases exist?

Serine/threonine kinases and tyrosine kinases.

What do dual specificity kinases do?

They can phosphorylate serine, threonine, and tyrosine residues.

How does phosphorylation regulate proteins?

It causes conformational changes and can activate or repress protein activity.

Is phosphorylation reversible?

Yes, it is a rapid and reversible form of regulation.

How can phosphorylation affect protein interactions?

It can change the ability of proteins to associate with others.

What signaling pathway involves phosphorylation?

MAP kinase and other signaling pathways.

How is kinase activity detected?

By western blotting with phosphor-specific antibodies.

What do phosphor-specific antibodies detect in western blotting?

They detect only the phosphorylated forms of the protein of interest.

What is an example of a protein detected by phosphor-specific antibodies?

Phosphorylated ERK (pERK).

What is used as a control in western blotting for kinase activity?

An antibody that detects the total amount of the protein (e.g., ERK1/2 detection).

What is a second method for detecting kinase activity?

Using an in vitro kinase assay.

What does an in vitro kinase assay involve?

Testing the activity of a kinase protein using a purified substrate.

What is needed to perform an in vitro kinase assay?

A kinase protein to test and a purified substrate.

What does phosphorylated JNK do in an in vitro kinase assay?

It adds a radioactive phosphate group from ATP to Jun, its target.

How can the phosphorylation of Jun be detected in an in vitro kinase assay?

By detecting the radioactivity from the labeled phosphate group added to Jun.

What is the second method to check for phosphorylation activity?

A kinase assay in vitro.

What is needed to perform a kinase assay in vitro?

A kinase protein and a purified substrate, along with [32P]-ATP.

What is the purpose of using [32P]-ATP in a kinase assay?

To label the phosphate group, making it detectable after the reaction.

How is JNK activity measured in a kinase assay?

By isolating JNK from treated or untreated cells and adding it to JUN with [32P]-ATP.

What is the next step after mixing the reaction in a kinase assay?

The reaction is run on a gel and exposed to film.

What indicates activation of JNK in a kinase assay?

A band appearing at the size of JUN in the lane with JNK from UV-treated cells.

What does the appearance of the JUN band mean in the kinase assay?

It means that JNK was activated by UV light and was able to add a phosphate group to JUN.

What is glycosylation?

The addition of sugar residues to proteins.

Where does glycosylation begin and end in the cell?

It begins in the ER and is completed in the Golgi apparatus.

What role do sugars play in glycosylation?

They may play a role in cell-cell recognition.

In which type of cells does glycosylation occur?

Only in eukaryotic cells.

Why is glycosylation important for protein expression systems?

Insect cells can glycosylate proteins, unlike bacteria, making them useful for expressing proteins for in vitro assays.

What are homodimers and heterodimers?

Homodimers are dimers formed by identical polypeptides, while heterodimers are formed by different polypeptides.

How do protein-protein interactions contribute to quaternary structure?

They help form complex protein structures by associating polypeptides together.

What is an example of a transcription factor where homodimers are inactive?

The Jun-Jun homodimer.

What is an example of a transcription factor where heterodimers are active?

The Fos-Jun heterodimer.

How can you detect protein-protein interactions?

GST pull-down assays.

What does GST stand for?

Glutathione S-transferase.

What does GST bind to?

Glutathione.

How do you create a GST-tagged protein?

Clone the gene into a vector containing GST before the multiple cloning site.

What is the purpose of tagging a protein with GST?

To detect or purify proteins that interact with it.

What is mixed with the GST-tagged protein to detect interactions?

Cell lysate containing potential binding targets.

What beads are used in GST pull-down assays?

Glutathione-sepharose beads.

What is the next step after adding glutathione-sepharose beads?

Centrifuge to pull down the GST-tagged protein and any bound proteins.

How are interacting proteins detected after the pull-down?

Run on a gel and analyze by western blotting.

How do you tag a protein with GST using a bacterial expression vector?

Use a vector with a GST sequence before the MCS and insert your gene of interest into the MCS.

What happens when a gene is cloned into a GST vector?

The expressed protein will be fused to GST.

Why is GST placed before the MCS in the vector? Where is GST added to the proteins?

So the GST tag will be at the N-terminus of the expressed protein.

What promoter drives expression in GST-tagged bacterial vectors?

The lac promoter.

What induces expression in a lac promoter system?

IPTG.

Why won't bacteria express the protein without IPTG?

Because the lac promoter is not active until induced.

How can you detect protein-protein interactions? (Method 2)

Immunoprecipitation (IP)

What is the first step in immunoprecipitation?

Lyse cells or tissues to release proteins.

What is added to the lysate in immunoprecipitation?

An antibody specific to one protein of interest.

What is the purpose of the antibody in IP?

It binds the target protein, which may have other proteins attached.

What is added after the antibody in IP?

Sepharose beads linked to Protein A or Protein G.

What do Protein A or Protein G bind to?

Antibodies.

What does centrifugation do in immunoprecipitation?

Pellets the beads, bringing down the antibody-protein complex and any associated proteins.

What is done after centrifugation in IP?

Western blotting to detect interacting proteins.

What is ubiquitination?

A posttranslational modification that tags proteins for degradation.

What type of residue does ubiquitin attach to?

Lysine residues.