Lecture 8

Question 1

T or F, secondary structure can sometimes be predicted by analyzing primary structure of given protein

Answer 1

T; certain amino acids are prone to be involved in one type or the other

Question 2

What are the alpha helix formers ?

Answer 2

Leucine and methionine

Question 3

What are the alpha helix terminators

Answer 3

Proline; due to the fact that there is no hydrogen available for bonding and structure resits rotation of right-handed helix –> kinks cause too much strain

Question 4

What was the Anfinsen Folding Experiment

Answer 4

1. ) Native protein (folded)
2. ) Treated with beta-ME (reduces –S) and urea (which breaks Hydrogen bonds)
3. ) Protein becomes denatured
4. ) He noticed when dentaureing agens are removed the native protein returns

– when urea is removed –> natural protein is returned; when denatured protein is oxidized it returns to its natural state

• overalln experiment reveal that the amino acid sequence was theoretically, sufficient to direct folding

Question 5

What are the 3 factors that the stability of the folded structure of globular proteins depends on?

Answer 5

1. The unfavorable conformational entropy change, which favors the unfolded state
2. The favorable enthalpy contribution arising from intramolecular noncovalent interactions
3. The favorable entropy change of the solvent arising from the burying of hydrophobic groups within the molecule –> think of how water is more ordered when protein is unfolded

Thus, factor 1 works against folding, whereas factors 2 and 3 favor folding. The overall consequence is that the folded structure corresponds to a free energy minimum for the polypeptide under physiological conditions. This is why the protein folds spontaneously.

– all of which leads to a negative change in free energy

Question 6

What are intramolecular noncovalent interactions?

Answer 6

• charge - charge, hydrogen bonds, Van der Waals, hydrophobic effect

Question 7

T or F, it is hypothesized that small sections of proteins fold as they are translated

Answer 7

T, newly made protein is folded in 1 minute

Question 8

T or F, protein folding is mediated by Chaperones

Answer 8

• T, they are proteins that help in correct folding

Question 9

T or F, disulfide bonds are weak covalent bonds that aren’t usually in intracellular proteins

Answer 9

F, these are covalent bonds that are very stable, but aren’t usually in intracellular proteins as this is a reducing environment

Question 10

T or F, there are two general classes of HSP: HSP60 and HSP70 are the most common

Answer 10

False, there are three general classes

Question 11

What is the function of a HSP (Heat Shock Protein)

Answer 11

• they isolate proteins and bind to their hydrophobic groups

Question 12

T or F, HSP with ATP has a low affinity for unfolded proteins and is found throughout the cell

Answer 12

• False, it has a high affinity for unfolded proteins

Question 13

How does HSP60 work ?

Answer 13

• GroEL binds to unfolded proteins via hydrophobic residues (because hydrophobic groups are exposed)
• ATP and GroES causes a conformation change that hides hydrophobic residues and forces the protein now into a hydrophilic chamber causing hydrophobic residues on the protein to hide and induces folding –> this is where the chaperone aspect happens

–> ES is the cap and when its added to GroEL ring it changes conformation

• After ATP hydrolysis releases GroES and the folded protein
• • Found mostly in mitochondria in eukaryotes, though can be in other locations

Question 14

What is the role of cofactors typically? What is another name for them?

Answer 14

• stabilizes active state of protein; these are often needed for active enzymes

– also called prosthetic groups

Question 15

What aside from cofactors stabilizes proteins?

Answer 15

• disulfide bonds –> once protein is completely folded that’s when they form

– ions

Question 16

T or F, you can predict which amino acids are interacting before 3-D structure

Answer 16

False, you can’t predict which ones are interacting until you see the 3-D structure

Question 17

What is the general description of Quaternary structure?

Answer 17

• most proteins are made up of more than one subunit

– subunits of polypeptide held together by patches of noncovalent bonds

Question 18

What is the difference between homodimers and heterodimers ?

Answer 18

• Homodimer: two identical subunits

- Heterodimer: two different subunits

Question 19

What is an example of a protein with quaternary structure?

Answer 19

• Hemoglobin

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

Question 20

What are typical characteristics to purify proteins by?

Answer 20

• Solubility
• Size
• Charge
• Binding affinity

Question 21

How do we extract protein?

Answer 21

• in order to release cell’s content we must first break (lyse) the cell membrane –> called homogenization
  1. sonication –> breake cells with high frequency sound

2. detergent lysis –> use a mild detergent to make holes in plasma membrane
3. force –> force cells through small hole using high pressure
4. mortar/pestle –> shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel

Question 22

How does differential centrifugation work?

Answer 22

• it is a way to separate cellular components by mass
• You fractionate the cell into components to see which is the richest in source of protein.
• The homogenate is formed and then an increasing centrifugal force is applied.

– at each step supernatant is removed from pellet that is formed and then subjected to further centrifugation

Question 23

What is seen at different G’s in supernatant vs pellet?

Answer 23

• • At 500g, the pellet will be mainly nuclear fraction
• • at 10,000g the pell will be mitochondrial fraction
• • at 100,000g the pellet will be microsomal fraction
• • and the final supernatant will be the cytoplasm filled with soluble proteins

Question 24

How does zonal centrifugation work ?

Answer 24

– centrifugation separates matters according to density (m/v)

– often provides a better resolution of separation of particles than differential

Question 25

What are the two common methods of causing detachment of proteins from their binder in protein analysis that allows “elution” of that compound ?

Answer 25

• You can salt out.
• This disrupts interactions between the desired protein and the solution and results in salting out (precipitation) of the protein.
• This is good because different proteins salt out at different concentrations –> can be used to further fractionate proteins

Question 26

What is interesting about the salt concentration and solubility trend?

Answer 26

• there is an optimal concentration where solubility of solution is maximized –> “salting in”

– however exceed this will cause “salting out” :

–> there’s so much salt that the salt interacts w/ water that there is no water left for protein to interact

Question 27

Describe column chromatogrpahy.

Answer 27

• type of purification that separates compounds based on ability to be displaced by a mobile phase.
• Consist of a solid and liquid phase.

– measure absorbance at 280 nm

Question 28

What is size exclusion chromatography ?

Answer 28

• macromolecules are separated by size and/or shape differences
• proteins elute according to their size –> first proteins to come out are large

– smallest proteins elute last –> spend the longest time inside the beads

Question 29

What is ion-exchange chromatography ?

Answer 29

• separates mainly by charge differences between proteins
• You have a resin molecule with a desired charge on the surface.
• Proteins with charges similar to those on the resin will elute first because they aren’t attracted to it.
• Those with opposite charge will be attracted to it an elute out. The elution can be done by salting out or altering pH

Question 30

Why does elution work by pH in ion-exchange chromatopgraphy

Answer 30

• when we change the pH the charges are changed

Question 31

What is the difference between cation and anion exchange column?

Answer 31

– cation column has negative charge on the resin and it binds cations

– whereas anion columns have positive charges on their resin and bind negative charges.

Question 32

How does affinity chromatography work?

Answer 32

• separates molecules according to their affinity to bind to specific ligands, i.e. antibodies, DNA

– If a compound has high affinity to ligand, it will stick to it and not elute out. You can remove those attached by adding more ligand, changing pH, or salting out.

– Adding more beads with ligands attached won’t make it elute it will just increase overall affinity. (so if ur adding more ligands they can’t be attached to beads)

Question 33

What other naturally occurring interactions can be used for Affinity Chromatography ?

Answer 33

• • antibody - antigen interactions
• • DNA – DNA binding proteins
• Enzymes – Substrates (or Inhibitors)