MOL100 Lecture 5: Regulation and Analysis of Proteins

Flashcards covering protein regulation mechanisms, including synthesis, degradation via lysosomes and proteasomes, allosteric interactions, phosphorylation, and proteolytic cleavage. The cards also cover protein analysis techniques such as centrifugation, electrophoresis (SDS-PAGE), liquid chromatography (gel filtration, ion exchange, affinity), immunoblotting, mass spectrometry, and methods for conformation determination (X-ray crystallography, NMR).

What are the three principal ways to regulate protein function?

By changing protein levels (synthesis or degradation), protein activity (chemical modifications), or protein localization.

At what levels is protein synthesis regulated?

Both transcriptional and posttranscriptional levels, including mRNA stability and translation rate.

What are the main purposes of protein degradation?

Removal of toxic and non-functional proteins, and adjusting cellular activity to changes in environment or during cell division.

Which level of protein regulation is protein degradation considered?

Posttranslational level.

What are the two main ways for protein degradation in cells?

Lysosomes and the proteasome.

What do lysosomes primarily degrade?

Proteins and other macromolecules taken up from outside the cell, and non-functional organelles.

What is the catalytic core of the proteasome called?

The 20S proteasome.

What is the complete proteasome complex, including both regulatory caps and the catalytic core, called?

The 26S proteasome.

How does the proteasome identify proteins for degradation?

A chain of ubiquitin molecules, called polyubiquitination, marks them.

What enzymes are responsible for the final step of binding ubiquitin to target proteins?

E3 ligases.

Describe the journey of a polyubiquitinated protein into the proteasome for degradation.

The polyubiquitinated protein is recognized by the 19S cap complex, ubiquitin chains are removed, the protein is unfolded using ATP, and then moved to the 20S catalytic core where it is cleaved into small peptides.

How is specificity in protein degradation achieved by the proteasome?

Through the use of many different E3 ligases, each targeting only a few specific proteins, often after they have been modified (e.g., by phosphorylation).

What do allosteric interactions change in a protein?

The shape of a protein by altering its tertiary and quaternary structure.

What causes allosteric interactions?

The non-covalent binding of a ligand to a protein, where binding at one site changes the binding properties of another site.

Provide an example of cooperativity in allosteric interaction.

Oxygen binding by hemoglobin, where the binding of one oxygen molecule stabilizes the other three oxygen-binding sites.

How do Cadherins demonstrate allosteric regulation?

As calcium-dependent cell adhesion molecules, Cadherins can only bind to Cadherins on adjacent cells after Ca2+ binding changes their conformation.

How do GTPases regulate protein activity through allosteric interaction?

They are active when bound to GTP and become inactive upon hydrolyzing GTP to GDP; activity is restored when GDP is replaced with GTP.

What are the enzymes that catalyze the binding and removal of phosphate groups, respectively?

Kinases catalyze the binding of phosphate groups, and phosphatases remove them.

What are some effects of phosphorylation on protein activity?

It can affect protein activity directly, change localization, create new binding sites, or mark proteins for degradation (ubiquitination).

How does proteolytic cleavage regulate protein activity?

By irreversibly cleaving off an inhibitory peptide, activating the protein (e.g., digestive enzymes synthesized in the pancreas and activated in the intestine).

What are the three main features of proteins that allow their purification?

Size (mass), electrical charge, and affinity to another molecule.

How does centrifugation separate proteins?

According to their mass and density, by applying a high gravitational force to speed up sedimentation.

What is the difference between differential centrifugation and density gradient centrifugation?

Differential centrifugation is a preparative technique separating materials based on their sedimentation speed, while density gradient centrifugation separates particles based on their buoyant density within a sucrose gradient.

How does electrophoresis separate molecules?

In an electric field according to their mass and charge.

How does SDS-PAGE overcome the problems of charge and conformation in protein electrophoresis?

SDS denatures proteins, giving them an unfolded conformation, and coats them with a strong negative charge proportional to their length, allowing separation primarily by mass.

What are three types of liquid chromatography used for protein separation?

Gel filtration chromatography, ion exchange chromatography, and affinity chromatography.

How does gel filtration chromatography separate proteins?

According to their mass; smaller proteins enter porous beads and move slower, while larger proteins bypass the beads and elute faster.

How does ion exchange chromatography separate proteins?

According to their charge, using beads with charged molecules on their surface, where proteins with opposite charges bind and can be eluted with high salt concentration.

How does affinity chromatography separate proteins?

By their specific binding properties, using beads coupled with a molecule (e.g., an antibody) that binds specifically to the protein of interest.

What is the process of immunoblotting (Western blotting) used for?

To detect a specific protein after SDS-PAGE by transferring proteins to a thin membrane, then incubating with a specific antibody for visualization.

What does mass spectrometry analyze in proteins?

Fragments of proteins based on their mass-to-charge ratio to determine their identity (primary structure) by comparison with predicted peptides in the genome.

What is proteomics?

The study of all proteins (the proteome) of a cell, an organelle, a tissue, or an entire organism.

Which methods are used to determine the conformation (tertiary and quaternary structure) of proteins?

X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.