Ch 3 - Nonenzymatic Protein Function and Protein Analysis

Question 1

What do structural proteins compose? What are the most common?

Answer 1

• compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix
• collagen, elastin, keratin, actin, and tubulin
• generally fibrous in nature

Question 2

What are the motor proteins?

Answer 2

• have one or more heads capable of force generation through a conformational change
• have catalytic activity, acting as ATPases to power movement

Question 3

What are the most common applications of motor proteins?

Answer 3

• muscle contraction, vesicle movement within cells, and cell motility
• myosin, kinesin, and dynein

Question 4

What to binding proteins bind?

Answer 4

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

Question 5

What do cell adhesion molecules (CAM) allow?

Answer 5

cells to bind to other cells or surfaces

Question 6

What is the difference between cadherins, integrins, and selectins?

Answer 6

• cadherins: calcium dependent glycoproteins that hold similar cells together
• integrins: have 2 membrane-spanning chains and permit cells to adhere to proteins in the ECM (some also have signaling capabilities)
• selectins: allow cells to adhere to carbohydrates on the surface of other cells and are most commonly used in the immune system

Question 7

How are antibodies used by the immune system?

Answer 7

• or immunoglobulins, Ig

- used to target a specific antigen, which may be a protein on the surface of a pathogen (invading organism) or a toxin

Question 8

What are the 2 regions within immunoglobulins?

Answer 8

• a constant region and a variable region

- variable responsible for antigen binding

Question 9

What chains form antibodies and what holds them together?

Answer 9

• 2 identical heavy chains and 2 identical light chains form a single antibody
• they are held together by disulfide linkages and non covalent interactions

Question 10

What are ion channels used for?

Answer 10

used for regulating ion flow in or out of the cell

Question 11

What are the 3 types of ion channels?

Answer 11

• ungated: always open
• voltage-gated: open within range of membrane potentials
• ligand-gated: open in the presence of a specific binding substance, usually a hormone or neurotransmitter

Question 12

What role to enzyme-linked receptors play in cell signaling?

Answer 12

through extracellular ligand binding and initiation of second messenger cascades

Question 13

What re G protein-coupled receptors?

Answer 13

• have a membrane-bound protein associated with a trimeric G protein and they also initiate the second messenger systems

Question 14

What engages G protein?

Answer 14

ligand binding

Question 15

What is electrophoresis?

Answer 15

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

Question 16

What is the difference between Native PAGE, SDS PAGE, and isoelectric focusing?

Answer 16

• native: maintains the proteins shape, but results are difficult to compare because the mass-to-charge ratio differs for each protein
• sds: denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel
• isoelectric: separates proteins by their pI; the protein migrates toward an electrode until it reaches a region of the gel where pH = pI of the protein

Question 17

What is chromatography?

Answer 17

separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase
- all are about the affinity of a substance for the mobile and stationary phases except for size-exclusion

Question 18

What is the difference between column, ion-exchange, size-exclusion, and affinity chromatography?

Answer 18

• column: uses beads of a polar compound (stationary phase) with a non polar solvent (mobile)
• ion exchange: uses a charged column and a variably saline eluent
• size: relies on porous beads (larger molecules elute first because they are not trapped in the small pores)
• affinity: uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest

Question 19

What primarily determines protein structure/

Answer 19

Xray crystallography after the protein is isolated, although NMR can also be used

Question 20

How can amino acid composition be determined?

Answer 20

by simply hydrolysis, but amino acid sequencing requires sequential degradation, such as Edman degradation (proceeds from the amino N terminus)

Question 21

How are activity levels of enzymatic samples determined?

Answer 21

by following the process of a known reaction, often accompanied by a color change

Question 22

How are protein concentrations determined colorimetrically?

Answer 22

either by UV spectroscopy or through color change reaction
- BCA assay, Lowry reagent assay, Bradford protein assay (most common) each test for protein and have advantages/disadvantages

Question 23

What are the functions of the heterotrimeric G proteins?

Answer 23

• Gs: stimulates
• Gi: inhibits
• Gq: activates phospholipase C

Question 24

How do cytoskeletal proteins differ from motor proteins?

Answer 24

• cyto: tend to be fibrous with repeating domains
• motor: tend to have ATPase activity and binding heads
• both have protein functions in cellular motility

Question 25

Are motor proteins considered enzymes?

Answer 25

• yes
• an enzyme is a protein or RNA molecule with catalytic activity, which motor proteins have
• motor function is generally considered nonenzymatic, but the ATPase functionality of motor proteins indicates that these molecules do have catalytic activity

Question 26

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 26

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

Question 27

When an antibody binds to its antigen, what are the 3 possible outcomes of this interaction?

Answer 27

• antigen-antibody interactions can result in neutralization of the pathogen or toxin,
• opsonization (marking) of the antigen for destruction,
• or creation of insoluble antigen-antibody complexes that can be phagocytized and digested by macrophages (agglutination)

Question 28

What are the similarities and differences between enzyme-linked receptors and G protein coupled receptors?

Answer 28

• enzyme: autoactivity and enzymatic activity
• G: 2 protein complex, dissociation upon activation, trimer
• both: extracellular domain, transmembrane domain, ligand bonding

Question 29

What are the disadvantages of affinity chromatography?

Answer 29

• the protein if interest may not elute from the column because its affinity is too high or it may be permanently bound to the free receptor in the eluent

Question 30

Why are proteins analyzed after isolation?

Answer 30

• protein identity must be confirmed by amino acid analysis or activity
• with unknown proteins, classification of their features is generally desired

Question 31

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

Answer 31

• 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 32

What determines the overall pI of a protein?q

Answer 32

the relative number of acidic and basic amino acids

Question 33

What are UV spectroscopy best used with?

Answer 33

conjugated systems of double bonds