Lecture 8

Question 1

Factors determining 2 structure:

Answer 1

• primary structure can sometimes predict secondary structures
• certain amino acids are prone to specific reactions or none (proline)

Question 2

Amino acids are alpha helix formers:

Answer 2

leucine and methionine

Question 3

Amino acids are alpha helix terminators:

Answer 3

proline, where no hydrogens are available or bonding and structure resists rotation of the right-handed helix

Question 4

Anfinsen Folding Experiment:

Answer 4

1. An RNAse is the native protein (folded)
2. The RNAse is treated with BME (reduces -S-) and urea (breaks H-bonds)
3. Denatured protein
4. Removed denaturing agents
5. Native protein returns

Question 5

Anfinsen Folding Experiment:

Removing urea and then oxidizing:

Answer 5

native molecule returns

Question 6

Anfinsen Folding Experiment:

Oxidizing and then removing urea:

Answer 6

molecules with randomly formed disulfide bonds

Question 7

Conclusion of the Anfinsen Follding Experiment:

Answer 7

Demonstrated that amino acid sequence was sufficient to direct folding

Question 8

Denaturing of proteins:

Answer 8

changing temperature, pH, or adding solvents like alcohol.urea

Question 9

Favorability of protein folding:

Answer 9

thermodynamically unfavorable, entropy decreases, but enthalpy increases because of favorable interactions in water

Question 10

Protein folding depends on 3 factors:

Answer 10

1. unfavorable confirmation change, which favors the unfolded state
2. favorable enthalpy contribution arising from intramolecular noncovalent interactions
3. favorable entropy change of the solvent arising from the burying of hydrophobic groups
   2, 3 > 1 = spontaneous

Question 11

Conclusions of protein folding:

Answer 11

• not a random process as there are too many different possibilities
• small sections fold as they are translated
• mediated by Chaperones
• stabilized by disulfide bonds, but they aren’t usually in intracellular proteins as this is a reducing environment

Question 12

Chaperones:

Answer 12

proteins that help in correct folding

Question 13

Examples of chaperones:

Answer 13

heat shock proteins (HSP 60 and HSP70)

Question 14

Function of Hsp 70:

Answer 14

• has a high affinity for unfolded proteins
• ATP is hydrolyzed, causing a conformation change and enclosing polypeptide in unfolded state (inhibits incorrect interactions)
• ATP is dephosphorylated and releases polypeptide

Question 15

Function of Hsp 60 (Chaperonin):

Answer 15

1. Groel binds to unfolded proteins via hydrophobic residues
2. ATP and Groes causes a conformation change that hides hydrophobic residues and forces the protein into hydrophilic chamber and induce folding
3. ATP hydrolysis releases Groes and the folded protein

Question 16

Properties that cause protein stability:

Answer 16

disulfide bonds, ions, and cofactors (prosthetic groups)

Question 17

Examples of prosthetic groups:

Answer 17

“zinc finger” and heme group

Question 18

Result of improper protein folding:

Answer 18

disease

Question 19

Quatenary structure:

Answer 19

subunits held together by patches of noncovalent bonds to form the whole protein complex

Question 20

Homodimer:

Answer 20

two identical subunits

Question 21

Heterodimer:

Answer 21

two different subunits

Question 22

Example of a heterodimer:

Answer 22

hemoglobin consists of two alpha globin and two beta globin subunits for a total of four subunits

Question 23

Protein isolation:

Answer 23

a protein must be separated from the other cellular components before its function and structure can be further studied

Question 24

Homogenization:

Answer 24

to lyse the cell membrane

Question 25

Methods of homogenization:

Answer 25

* sonification: break cells with high-frequency sound * detergent lysis: use a mild detergent to make holes in the plasma membrane * force: force cells through a small hole using high pressure * mortar/pestle: shear cells between a close-fitting rotation plunger and the thick walls of a glass vessel

Question 26

Production of homogenization:

Answer 26

homogenate/ cell extract

Question 27

Differential centrifugation:

Answer 27

separates cellular components by mass to determine the richest source of the protein 1. after forming a homogenate, the mixture is centrifuged in a step-by-step fashion of indcreasing centrifugal force 2. At each step, the supernatant is removed form the pellet that is formed, and then subjected to further centrifugation

Question 28

Zonal centrifugation:

Answer 28

* separates matters according to density * often provides a better resolution of separation of particles than differential 1. sucrose is centrifuged to create a gradient 2. sample is layered on top of gradient 3. particles settle accoridng to mass 4. colect fractions and do assay

Question 29

Fractioning by Precipitation:

Answer 29

adding a small amount of salt helps a protein become soluble, but adding a lot can make it precipitate out of solution

Question 30

Protein purification by Column Chromatography:

Answer 30

solid and liquid phases are collected out the bottom of the tube and measured at absorbance 280 nm

Question 31

Size Exclusion Chromatography:

Answer 31

separates macromolecules by size and/or shape differences * larger molecules migrate faster * smaller molecules migrate through channels within beads

Question 32

Ion-exchange Chromatography:

Answer 32

separates mainly by charge differences between proteins 1. protein binds the column based on opposite charge 2. column is washed to remove other proteins 3. release by eluting the protein with a pH of the buffer

Question 33

Affinity Chromatography:

Answer 33

separates molecules according to their affinity to bind to specific ligands (DNA-DBP, Enzymes-Substrates) 1. beads can be covalently linked to the ligant that the target protein will bind 2. results is highly specific purifiation of a single protein

Question 34

Example of Affinity Chromatography:

Answer 34

1. glucose-binding protein attaches to glucose residues (G) on beads 2. glucose-binding proteins are released on addition of glucose