BIOL 213 Exam 2

What determines the value of the isoelectric point (pl)?

The pKa values of every ionizable group and the side chains.

How does phosphorylation affect the isoeletric point? Why?

By adding a phosphate group, which is charged negatively, to a protein we, obviously lower its charge, which shifts the isoelectric point to a lower value.

What is the net charge when pH > pl? What about pH < pl? For each charge, what electrode are they attracted to?

When pH > pl: Negative net charge, and attracted to the anode (+).

When pH < pl: Positive net charge, and attracted to the cathode (-).

What does the single amino acid difference (Glu → Val) cause at pH 8.6 between sickle-cell and normal red blood cells?

The normal red blood cells at pH 8.6 are more attracted to the anode than the sickle-cell, because glu has two more negative charges at that pH.

After homogenization is carefully conducted, what is in the homogenate?

The organelles and the cytosolic material.

What is pelleted at the lowest speed during centrifugation? Why?

Whole cells, nuclei, and cytoskeleton. The largest and densest items are sorted out first in centrifugation. These are the largest, so it doesn’t need to go very fast to have them separate.

Between each stage of differential centrifugation, what is being carried over?

The supernatant of the previous stage.

What is the main difference between velocity and equilibrium centrifugation?

Velocity separates based on size and shape, so the sedimentation speed is what creates the bands. Equilibrium centrifugation separates based on buoyant density.

What separates proteins in ion-exchange chromatography? What happens to proteins of opposite charges to the beads?

Charge. They are slowed down if the beads have opposite charges. That’s what makes this good to use to separate proteins by charge.

If size is not a factor in trying to determine the difference between two proteins, but their pl’s are different, what would be the best form of chromatography to use in discovering more about it?

Ion-exchange chromatography

Explain gel-filtration chromatography.

Separates proteins by size and shape by having beads with tiny pores in them that the proteins can enter. If they’re small enough they can fit inside, and if they’re large enough they go past and elute.

Explain affinity chromatography.

Separates proteins based on how they bind. The matrix is coupled to a specific ligand (like an antibody or enzyme), such that the proteins that would bind this ligand are slowed and those that don’t elute first.

What eluting behavior is most similar between ion-exchange chromatography and affinity chromatography?

Both have proteins capable of being eluted by changing pH or salt concentration.

Why do all proteins in an SDS-PAGE gravitate towards the anode?

Because the detergent coats them in a uniform negative charge.

What is the reasoning for the uniform negative charge in SDS-PAGE?

In order to make them all uniform and gravitate towards the anode. This allows us to separate them purely on molecular weight.

What is the role of mercaptoethanol?

It’s a reducing agent that breaks disulfide linkages in polypeptide chains.

How does isoelectric focusing separate proteins?

Through the use of a pH gradient. Each protein migrates until it reaches the pH equal to its pl, at which point it has no net charge and ceases movement. It will attempt to balance this if the pH changes.

In what order do you do the electrophoresis techniques when creating two-dimensional gels?

First isoelectric focusing (IEF), then SDS-PAGE.

What is conserved more in evolution: the 3D fold of a protein or its amino acid sequence? What does that say about its family?

The 3D fold of a protein is much more strongly preserved. It says that proteins can be within the same structural family even if they don’t have the same amino acid sequence.

How are novel proteins created?

By rearranging/reconstructing modular protein domains into new shapes.

Approximately how thick is the plasma membrane bilayer?

Approximately 5 nanometers.

What are the three main amphipathic lipid classes?

Phospholipids, sterols (like cholsterol), and glycolipids.

How do the amphipathic lipid classes protect their hydrophobic tails?

They spontaneously assemble into bilayers, then close into sealed spherical compartments called liposomes.

Why do triacylglycerols form large oil droplets in water? What are they missing?

Because they are entirely hydrophobic. They lack a polar head group (no hydrophilic head).

Why is flip-flop lipid movement energetically unfavorable?

Because you would need to move the hydrophilic polar head through the hydrophobic portion of the membrane to get to the other side.

What are the four main determinants of membrane fluidity?

Tail saturation, temperature, cholesterol, and tail length.

How does cholsterol influence fluidity?

It packs the spaces between phospholipids to increase rigidity. So when it becomes too hot and the membrane becomes too rigid, it secretes cholsterol to fill in the gaps to stiffen them.

Not including the use of cholsterol, how does the cell adapt to the cold?

They create lipids with shorter, more unsaturated tails to increase fluidity.

What is the purpose of the endoplasmic reticulum?

To synthesize new phospholipids and insert them into the cytosolic leaflet.

What is the purpose of scramblase? Where is it?

It randomly flips phospholipids between leaflets to ensure symmetric growth. It exists in the endoplasmic reticulum.

What is the purpose of flippase? Where is it?

It specifically moves the lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the cytosolic leaflet, establishing membrane asymmetry.

What are the three kinds of integral membrane proteins?

Transmembrane, monolayer- associated, and lipid-linked.

• Transmembrane extends through the bilayer

• Monolayer-associated is attached to the cytosolic half by alpha helix

• Lipid-linked are covalently attached to a lipid molecule within the bilayer

What is the main kind of peripheral membrane protein?

Protein-attached proteins. They’re bound to other membrane proteins by non-covalent interactions.

Most transmembrane segments are what?

Made of ~20 residue hydrophobic alpha helices.

What is a detergent?

Small, amphipathic molecules used to solubilize membrane proteins into lipid-protein detergent micelles.

What is the main difference between SDS (Strong Ionic) detergents and Triton X-100 (Mild Nonionic) detergents?

SDS unfolds proteins and disrupts their native structure, while Triton X-100 solubilizes them without unfolding, preserving their native structure and function.

What is bacteriorhodopsin?

A retinal containing, light-driven proton pump.

What are the four ways proteins can be restricted in mobility?

1. Cell Cortex - Protein meshwork that tethers proteins

2. Extracellular Matrix - Tethered to proteins outside the cell

3. Cell-cell adhesion - binding of proteins on one cell surface to another

4. Diffusion Barriers - Tight junctions prevent proteins from diffusing past junction

What are the two main variables that determine whether or not something can pass through membrane?

Size and polarity. Small, nonpolar molecules can pass through the easiest, whilst larch polar ones struggle. Ions also completely can’t enter due to their charge.

What are the main differences between channels and transporters?

Channels are gated, aqueous pores, are fast, and can only participate in passive transport. Transporters use conformation shifts/alternating access, are slower, and can be active (pumps) or passive (uniporters)

What is the one thing that transporters can do that channels simply cannot?

Perform active transport against a gradient.

What kind of gradient does Na+ have both concentration and voltage-wise?

A steep inward gradient. Both voltage and concentration make it want to go into the cell.

What kind of gradient does K+ have, both concentration and voltage-wise?

Concentration pulls K+ out, voltage pulls it in. Makes it balanced at rest.

What ion do plant cells, fungi, and bacteria mainly use for active import?

H+ ions.

Describe the general order of a sodium-potassium pump’s process.

1. 3 Na+ bind from cytosolic side

2. Pump gets phosphorylated to open up to outside cell

3. 3 Na+ released extracellularly

4. 2 K+ bind from extracellular side

5. Cell dephosphorylates to return to original position

6. 2 K+ are released inside cytosol

What does the apical surface in the gut lining do?

It houses a Na+-driven glucose symport. Pumping of Na+ in powers import of glucose uphill.

What does the basolateral surface in the gut lining do?

It houses a passive glucose uniporter that helps glucose exit into the blood.

Why is Na+ excluded from the K+ channel despite being the smaller ion?

The Na+ ion is too small to favorably interact with the oxygen molecules that normally interact with the hydration shell of K+, making dehydration cost too much for the smaller ion.

What is the general order of the active potential pathway?

1. Depolarization: Stimulus reaches threshold, causing voltage-gated Na+ channels open

2. Peak: Na+ channels inactive (make it so the signal only goes one way)

3. Repolarization: Voltage-gated K+ channels open; K+ flows out

4. Synapse: Action potential reaches terminal → Voltage-gated Ca²+ channels open → Ca²+ influx triggers neurotransmitter release

5. Inhibition: Cl- channels open, driving Vm towards -75 mV which stops the membrane below the threshold

What are the main two stages of chemiosmosis?

1. High-energy electrons pump H+ out to create a gradient

2. H+ flows back through ATP synthase to drive ATP production

What are uncouplers?

They’re amphipathic weak acids that carry H+ ions across the membrane, causing the gradient to collapse. This ceases ATP synthesis, but still allows electron transport. Excess energy is dissipated as heat.

What does regulated uncoupling in brown fat do?

Provides non-shivering thermogenesis.

Where does the citric acid cycle occur within the cell?

Inside the mitochondrial matrix.

Where are the cristae within the cell? And what do they do?

They sit on the inner membrane of the mitochondria. And they maximize surface area for cell energy production.

What is the difference between photosystem I and photosystem II?

PS I uses light to reduce NADP+ to NADPH. PH II uses light to split water, releasing O2.

What do plants do when it’s dark?

They take up O2 and release CO2. Their mitochondria are the only things working, as respiration is the only thing happening.