3. Proteins

Question 1

(3.1) How do cytoskeletal proteins differ from motor proteins?

Answer 1

Cytoskeletal proteins tend to be fibrous with repeating domains

Motor proteins tend to have ATPase activity and binding heads.

Both types of protein function in cellular motility

Question 2

(3.1) True or false: motor proteins are not enzymes.

Answer 2

False. An enzyme is a protein or RNA molecule with catalytic activity, which motor proteins do have.

Motor function is generally considered nonenzymic, but the ATPase functionality of motor proteins indicates that these molecules do have catalytic activity.

Question 3

(3.1) What could permit a binding protein involved in sequestration to have a low affinity for its substrate and still have a high percentage of substrate bound.

Answer 3

If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all substrates will be bound despite a low affinity.

Question 4

(3.1) What are the three main classes of cell adhesion molecules? What type of adhesion does each class form?

Answer 4

Cadherin: two cells of the same or similar type using calcium
Integrin: one cell to proteins in the extracellular matrix
Selectin: one cell to carbohydrates, usually on the surface of other cells.

Question 5

(3.1) When an antibody binds to its antigen, what are the three possible outcomes of this action?

Answer 5

(1) neutralization of the pathogen or toxin
(2) opsonization (marking) of the antigen for destruction, or

(3) agglutination or creation of insoluble antigen–antibody complexes that can be phagocytized and digested by macrophages

Question 6

(3.1) What type of bonds hold the heavy chain and light chain of immunoglobulins together?

Answer 6

disulfide linkages and noncovalent bonds.

Question 7

(3.1) What is a binding protein and what is an example?

Answer 7

Binding proteins bind a specific substrate, either to sequester it in the body or hold its concentration at steady state.

Example is hemoglobin

Question 8

(3.1) What important enzyme do motor proteins have?

Answer 8

They have ATPase that power movement.

examples include muscle, cilia, flagella

Question 9

(3.1) What are different types of non-enzyme proteins?

Answer 9

1. Structural proteins (i.e. collagen).
2. Motor proteins (i.e. myosin)
3. Binding proteins (i.e. hemoglobin).
4. Cell adhesion molecules (Cadherins, integrins, selectins).
5. Antibodies

Question 10

(3.1) True or false: both actin and myosin are motor proteins

Answer 10

False

Actin is a structural protein
Myosin is a motor protein (has the ATPase and completes the power stroke)

Question 11

(3.2) What type of transport is facilitated diffusion?

Answer 11

It is a type of passive transport.

The molecules move down a concentration gradient through a pore in the membrane created by a protein. These molecules that are being transported would otherwise be impermeable to the membrane because they would be large, polar, or charged.

Question 12

(3.2) What are the three types of ion channels?

Answer 12

1. Ungated channels
2. Voltage gated channels
3. Ligand gated channels

Question 13

(3.2) How do ungated channels work?

Answer 13

They are always open and unregulated. The books says potassium channels are an example of these.

Question 14

(3.2) How do voltage gated channels work?

Answer 14

They are open within a range of membrane potentials. The membrane potential regulates their functioning. The depolarization of the membrane may cause a conformational change that allows it to quickly open and close. Sodium and potassium voltage gated channels are an example of these.

Question 15

(3.2) How do ligand-gated channels work?

Answer 15

Open in the presence of a specific binding substance, usually a hormone or neurotransmitter.

The Km and vmax parameters that apply to enzymes are also applicable to transporters such as ion channels in membranes.

Question 16

(3.2) How do enzyme-linked receptors work?

Answer 16

Enzyme-linked receptors participate in cell signaling through extracellular ligand binding and initiation of second messenger cascades.

Question 17

(3.2) What are the three primary protein domains of a enzyme linked receptor?

Answer 17

1. membrane spanning domain
2. The ligand binding domain.
3. The catalytic domain

-The membrane spanning domain anchors the receptors in the cell membrane.
-The ligand-binding domain is stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain.
This often results in the initiation of a second messenger cascade.

Question 18

(3.2) What is an important physical part of the G-protein coupled receptor?

Answer 18

1. 7 membrane-spanning alpha helices
2. trimeric G protein

Question 19

(3.2) What are the three main types of G-proteins and what do they do?

Answer 19

1. G_s- stimulates adenylate cyclase, which increases levels of cAMP
2. G_i- inhibits adenylate cyclase, which decreases levels of cAMP in the cell
3. G_q- activates phospholipase C, which terminal product IP3 can open calcium channels in the endoplasmic reticulum, increasing calcium levels in the cell.

Question 20

(3.2) Walk me through the steps of how G-protein receptors work?

Answer 20

1. Ligand binds to GPCR (G-protein coupled receptor)
2. GPCR undergoes a confirmation change.
3. Because of the change, the alpha subunit exchanges GDP for GTP, causing the alpha subunit to disassociate
4. The alpha subunit will then target and regulate a target protein, which can be enzymes or ion channels.
5. Target protein relays signal via a secondary messenger
6. GTP is hydrolyzed to GDP, everything goes back to normal, the ligand leaves, and the alpha reattaches to the beta and gamma subunit

Question 21

(3.2) Contrast enzyme-linked receptors with G protein-coupled Receptors.

Answer 21

Enzyme linked receptors: have auto activity, and enzymatic activity.
G-protein Coupled Receptors- have two protein complexes, dissociate upon activation, and form a trimer.
Shared Characteristics: Have an extracellular domain, a transmembrane domain, and a ligand binding domain.

Question 22

(3.2) What type of ion channel is active at all times?

Answer 22

Ungated channels are always open

Question 23

(3.2) How do transport kinetics differ from enzyme kinetics?

Answer 23

Transport kinetics display both Km and vmax values. They also can be cooperative, like some binding proteins. However, transporters do not have analogous Keq values for reactions because there is no catalysis.

Question 24

(3.3) How does electrophoresis work?

Answer 24

Electrophoresis uses a gel matrix to observe the migration of proteins in response to an electric field.

Proteins are separated in general by charge and size.

They move from cathode (negative side) to the anode (positively charged). Keep in mind this is an electrolytic cell.

Question 25

**(3.3)** What is the benefit of **native PAGE electrophoresis** and how does it work?

Answer 25

**Useful**: comparing molecular *size or charge* of proteins with already known proteins. Maintains proteins shape, and can *potentially recover protein*.

Question 26

**(3.3)** What is the benefit of **SDS-PAGE electrophoresis** and how does it work?

Answer 26

**Benefit**- you can *accurately determine size* without worrying about the charge as a cofounder. Drawback: all proteins are denatured, so they are *not recoverable*.

Question 27

**(3.3)** What is **isoelectric focusing electrophoresis**?

Answer 27

Isoelectric focusing separates protein by their **isoelectric point** (pI); the protein migrates toward an electrode until it reaches a region of the gel where pH= pI of the protein.

Question 28

**(3.3)** How is the *medium* in isoelectric focusing?

Answer 28

The *medium has a pH gradient* (there's acidic gel at the positive anode, and basic gel at the negative cathode, and neutral in the middle)

Question 29

**(3.3)** How does chromatography work?

Answer 29

Chromatography *separates protein* mixtures on the basis of their affinity for a *stationary phase or a mobile phase*. **Key Concept**: all chromatography is about the affinity of a substance for the mobile and stationary phases, except for size-exclusion chromatography. The concept is: the more similar the compound is to its surroundings (by polarity, charge, and so on) the more it will stick to and move slowly through its surroundings.

Question 30

**(3.3)** How does **column chromatography** work?

Answer 30

**Column chromatography** uses beads of a polar compound, like silica or alumina (stationary phase), with a nonpolar solvent (mobile phase). As the solution flows through the column, both size and polarity have a role in determining how quickly the compound moves through the polar silica or alumina beads. The solvent polarity, pH, or salinity can easily be changed to help elute the protein of interest

Question 31

**(3.3)** How does **ion-exchange chromatography work**?

Answer 31

Ion-exchange chromatography uses a **charged column** and a variably salient eluent The beads in the column are coated with charged substances, so they *attract or bind compound that have opposite charges*

Question 32

**(3.3)** How does **size exclusion chromatography work**?

Answer 32

Size exclusion chromatography relies on **porous beads**. *Larger molecules elute first because they are not trapped in the small pores*. Small compounds are slowed down and retained longer Common approach is in protein purification is to us an ion-exchange column followed by a size exclusion column

Question 33

**(3.3)** How does *affinity chromatography work*?

Answer 33

**Affinity chromatography** uses a *bound receptor* or *ligand* and an eluent with free ligand or a receptor for the protein of interest

Question 34

**(3.3)** What separation methods can be used to *isolate a protein on the basis of isoelectric point*? (x2)

Answer 34

**Isoelectric focusing** and **ion-exchange chromatography** both separate proteins based on charge The charge of a protein in any given environment is determined by its isoelectric point (pI).

Question 35

**(3.3)** What are the *relative benefits* of native PAGE compared to SDS-PAGE?

Answer 35

**Native PAGE** allows a *complete protein to be recovered after analysis*; it also more accurately *determines the relative globular size of proteins*. **SDS-PAGE** can be used to eliminate conflation from mass-to-charge ratios. SDS really *examines the size of the molecule*

Question 36

**(3.3)** What are two potential *drawbacks* of **affinity chromatography**?

Answer 36

1. The protein of interest *may not elute* from the column because its *affinity is too high* 2. It may be *permanently bound* to the free receptor in the eluent.

Question 37

**(3.3)** **True or False**: in size-exclusion chromatography, the largest molecules elute first.

Answer 37

**True**. The small pores in size-exclusion chromatography trap smaller particles, retaining them in the column.

Question 38

**(3.4)** What is the primary way of determining protein structure?

Answer 38

X-ray crystallography. However NMR can be used.

Question 39

**(3.4)** How does x-ray crystallography work?

Answer 39

It measures electron density on an extremely high-resolution scale and can also be used for nucleic acids.

Question 40

**(3.4)** How can you determine an amino acid sequence?

Answer 40

You must degrade it sequentailly, such as using the Edman Degradation technique

Question 41

**(3.4)** What is the Edman Degradation technique?

Answer 41

It is used to evaluate protein amino acid sequence. uses cleavage to sequence proteins of up to 50 to 70 amino acids. It remove the n-terminal amino acid of the protein, which can be analyzed via mass spectroscopy.

Question 42

**(3.4)** What are some methods used to evaluate the activity of a protein?

Answer 42

BCA Assay Lowry Reagent Assay Bradford Protein Assay

Question 43

**(3.4)** Why are proteins analyzed after isolation?

Answer 43

Protein isolation is generally only the first step in an analysis. The protein identity must be confirmed by amino acid analysis or activity. With unknown proteins, classification of their features is generally desired.

Question 44

**(3.4)** What factors would cause an activity assay to display lower activity than expected after concentration determination?

Answer 44

Contamination of the sample with detergent or SDS could yield an artificially increased protein level, leading to lower activity than expected (because the protein concentration was calculated as higher than its actual value). Alternatively, the enzyme could have been denatured during isolation and analysis.

Question 45

**(3.4)** True or False: The Edman degradation processes from the carboxyl c-terminus.

Answer 45

False. The Edman degradation proceeds from the amino (N-) terminus.