Chapter 3- Nonenzymatic Protein Function and Protein Analysis

Question 1

motif

Answer 1

repetitive organization of secondary structural elements together

Question 2

primary structural proteins in body

Answer 2

collagen, elastin, keratin, actin, and tubulin

Question 3

collagen

Answer 3

trihelical fiber (3 a-helices woven together to form a secondary helix). provides strength and flexibility—ex: makes up most of extracellular matrix of connective tissue

Question 4

elastin

Answer 4

extracellular matrix of connective tissue. stretch and recoil like a spring.

Question 5

kertains

Answer 5

intermediate filament proteins found in epithelial cells. mechanical integrity of cell and also function as regulatory protein.—– primary protein in hair and nails

Question 6

actin

Answer 6

protein that makes up microfilaments and thin filaments in myofibrils. MOST ABUNDANT PROTEIN IN EUKARYOTIC CELLS.

Question 7

Tubulin

Answer 7

protein that makes up microtubules. provide structure, chromosome separation in mitosis, and intracellular transport with kinesin and dynein. has polarity.

Question 8

kinesins and dyneins

Answer 8

motor proteins associated with microtubules. have 2 heads, one is always attached to tubulin at all times. important for vesicle transport in the cell.

Question 9

kinesins

Answer 9

play key roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis. bring vesicles toward positive end of microtubule (ex: toward synaptic terminal in neurons)

Question 10

dyneins

Answer 10

involved in sliding movement of cilia and flagella. bring vesicles toward negative end of microtubules (ex: back to soma in neurons).

Question 11

examples of binding proteins

Answer 11

hemoglobin, Ca2+ binding proteins, DNA-binding proteins.

Question 12

Cell adhesion molecules (CAMs)

Answer 12

proteins found on the surface of most cells that aid in the binding of the cell to the extracellular matrix. all integral membrane proteins.

Question 13

3 types of adhesion molecules

Answer 13

1. cadherins
2. integrins
3. selectins

Question 14

cadherins

Answer 14

group of glycoproteins that mediate Ca2+ dependent cell adhesion. hold similar types of cells together (ex: epithelial cells)

Question 15

integrins

Answer 15

group of proteins that all have two membrane-spanning chains called a and B. important for binding and communication w/ extracellular matrix. cellular signaling (promoting division or apoptosis). also stabilize clots

Question 16

selectins

Answer 16

bind to carbohydrate molecules that project from other cell surfaces. weakest bonds formed by the CAMs . expressed on white blood cells and endothelial cells that line blood vessels.

Question 17

3 ourcomes when antibodies bind to their targets (antigens)

Answer 17

1. neutralize antigen
2. opsonization (marking pathogen for destruction by other white blood cells immediately
3. agglutinating (clumping together) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages.

Question 18

3 types of ion channels

Answer 18

1. ungated (unregulated)
2. voltage-gated (regulated by membrane potential change)
3. ligand-gated (binding specific substance or ligand to channel for it to open or close)

Question 19

enzyme-linked receptors

Answer 19

3 primary protein domains.

1. membrane-spanning domain (anchors the receptor in the cell membrane)
2. ligand-binding domain (stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain)
3. catalytic domain (once activated, typically initiates a second messenger cascade)

Question 20

G-protein coupled receptors

Answer 20

integral membrane proteins involved in signal transduction

Question 21

heterotrimeric G protein

Answer 21

used by G-protein coupled receptors to transmit signals to an effector in the cell. 3 subunits (a, B, gamma)

Question 22

3 main types of G proteins

Answer 22

1. Gs stimulates adenylate cyclase- increases levels of cAMP
2. Gi inhibits adenylate cyclase- decreases levels of cAMP
3. Gq activates phospholipase C- cleaves phospholipid from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3. IP3 can open Ca2+ channels in ER increasing Ca2+ levels in the cell.

Question 23

inactive form of G protein

Answer 23

a subunit binds to GDP in complex with B and gamma

Question 24

active form of G protein

Answer 24

ligand binds to GPCR, receptor becomes active, GDP is replaced with GTP, a-subunit is able to dissociate from B and gamma subunits, a-sub alters activity of adenylate cyclase. then GTP becomes dephosphorylated to GDP then B and gamma-sub rebind to a-sub rendering G protein inactive.

Question 25

how are proteins/ biomolecules isolated from body tissues or cell cultures

Answer 25

lysis and homogenization (crushing, grinding, blending) tissue of interest into an evenly mixed solution, then centrifugation will isolate proteins

Question 26

most common isolation techniques

Answer 26

electrophores and chromatography

Question 27

electrophoresis

Answer 27

subjecting compounds to an electric field, which moves according to their net charge and size.

Question 28

PAGE

Answer 28

method for analyzing proteins in their native states. limited to mass-to-charge and mass-to-size ratios. most useful to compare the molecular size or charge of proteins known to be similar in size from other analytic methods.

Question 29

SDS-PAGE

Answer 29

separates proteins based on mass alone. denatures the protein.

Question 30

Isoelectric point (pI)

Answer 30

pH at which protein or amino acid is electrically neutral, with an equal number of positive and negative charges.

Question 31

Isoelectric focusing

Answer 31

separates amino acids on basis of pI. mixture of proteins placed in a gel with a pH gradient (acidic at positive anode, basic at negative cathode, and neutral in the middle) protein stops moving when pH = pI

Question 32

chromatography

Answer 32

variety of techniques that require the homogenized protein mixture to be fractionated through a porous matrix. isolated proteins are available immediately for identification and quantification. CONCEPT: the more similar the compound is to its surroundings the mre it will stick to and move slowly through its surroundings. (better for large amounts of proteins)

Question 33

process of chromatography

Answer 33

1. place sample onto solid medium called stationary phase/ adsorbent
2. let sample run through mobile phase into the stationary phase (elute)
   - retention time: amount of time compound spends in stationary phase
   - partitioning: varying retention times for each compound in solution results in separation of components within the stationary phase

Question 34

column chromatography

Answer 34

Used to separate and collect other macromolecules besides proteins such as nucleic acids. column filled with silica/ alumina beads and gravity moves solvent down. both size and polarity matter. less polar = faster it’ll go through.

Question 35

ion-exchange chromatography

Answer 35

beads in column are coated with charged substances, so they attract or bind compounds that have an opposite charge. at the end a salt gradient is used to elute the charged molecules that have stuck to the column.

Question 36

size-exclusion chromatography

Answer 36

beads used in the column contain tiny pores of varying size. this allows the larger ones to move faster around the beads and the smaller ones to move slower

Question 37

affinity chromatography

Answer 37

customized columns to bind any protein of interest by creating a column with a high affinity for that protein or specific antibody to the protein. the protein of interest is retained in the column. protein can be removed with elution (may not come out or protein used to elute it may stick to the protein of interest)

Question 38

2 ways to determine protein structure

Answer 38

first the protein must be isolated

1. X-ray crystallography (after isolation it must be crystallized. most reliable and common method. measures electron density on extremely high resolution scale and can also be used for nucleic acids)
2. nuclear magnetic resonance (NMR) spectroscopy

Question 39

Edman degradation

Answer 39

to determine primary structure. uses cleavage to sequence proteins of up to 50 to 70 amino acids. selectively and sequentially removes N-terminal aa of the protein, which can be analyzed via mass spectroscopy.

Question 40

4 ways to determine concentration

Answer 40

1. UV spectroscopy
2. bicinchoninic acid (BCA) assay
3. Lowry reagent assay
4. Bradford protein assay (most common b/c its simple and reliable)

Question 41

Bradford protein assay

Answer 41

mixes a protein in solution with Coomassie Brilliant Blue dye.
protonated (acidic) dye- brown/green
deprotonated (basic) dye- blue — created by interactions with proteins in solution

Question 42

what type of receptor are hormones likely to act on

Answer 42

enzyme-linked and G protein-coupled receptors