Midterm 1 BIOL 201

What is the purpose and process of SDS-PAGE?

Purpose:

Identify proteins that are known to have a certain molecular weight

Process:

1. SDS

• Negative charged detergent that unfolds proteins at high temps. by breaking non-covalent bonds

• Coats denatured protein in negative charges to migrate to positive end of the gel

2. Beta-mercaptoethanol

• Break covalent disulfide bonds

3. Put the samples in their lanes with the and compare with the ladder (standard gels)

How are proteins separated in SDS-PAGE? What is the order of elution?

Separated:

By mass

Elution:

Top: Heavy proteins / less negative

• Bigger/folded proteins move through the small holes of the gel harder and slower

Bottom: Lightest proteins / more negative

• Smaller/unfolded proteins move through the small holes of the gel easier and faster

Top arrow: Large proteins

Bottom arrow: Small proteins

Why do you heat the protein sample before loading them onto the gel?

To make sure the protein is deatured

What would happen if you didn’t heat the protein? How would it affect migration of the protein?

the protein will not be fully denatured, resulting in incomplete linearization and failed coating with SDS

Samples of a monomeric proteins were treated with SDS at different temperatures (20, 30, 40, 50, 60, 70, 80, 90, 100 degrees C). Assign the temperature to the lane and explain why?

Lanes 4-9:

• There are no proteins on the bottom, they’re only at the top

• Those are all big proteins meaning they are unfolded so they are denatured from the hot temperatures (unfolded is big, folded is small)

Lanes 1-2:

• There are only two bands

• The top band are large proteins because they are denatured

• The bottom band are small proteins that aren’t completely unfolded

Lane 3:

• The only lane with a protein gradient

• Best temperature for the proteins

What is the purpose and process for 2D separation?

Purpose:

Separate proteins based on net charge and molecular weight

Process:

1. Isoelectric focusing

• Proteins are run through a gel strip with a pH gradient (positive end - 2, 4, 6. 8. 10, 12, 14 - negative end)

• The proteins move through the gel when electric current is applied and stop on the gel when they reach a net neutral charge

2. SDS-PAGE

• Immobilized protein samples on the pH gradient strip are treated with SDS (denature, break non-covalent bonds at high temp., coat in negative charges), and beta-mercaptoethanol (break covalent disulfide bonds)

• Samples are subjected to electrophoresis

How are proteins separated in 2D separation? What is the order of elution?

IEF:

• pH = pI

• Positive end = low pI

• Negative end = high pI

SDS:

Top: Heavy proteins / less negative

• Bigger/folded proteins move through the small holes of the gel harder and slower

Bottom: Lightest proteins / more negative

• Smaller/unfolded proteins move through the small holes of the gel easier and faster

What is the purpose and process for Native-PAGE separation?

Purpose:

Analyze proteins in their folded form

• Based on changes in their function

  • Change in net charge

  • Protein misfolding and aggregation

Process:

• 
  

How are proteins separated in Native-PAGE? What is the order of elution?

(Histone acetylation & Amyloid Beta Plaques)

Histone acetylation

• Histone acetylation removes the positive charge so the histones are more negative

• The more acetylation there is, the more negative histones are, they will be on the bottom where the positive end is

Top: More positive / less acetylation

Bottom: Less positive / more acetylation

Amyloid Beta Plaques

• Misfolded amyloid proteins aggregate

• Small proteins go through the gel holes easier so they are on the bottom

Top: Big proteins / high aggregation

Bottom: Small proteins / low aggregation

What is the process of size-exclusion chromatography? What are the stationary and mobile phases? What is the order of elution?

Process:

Separate proteins by size and shape

Stationary:

Beads

Mobile:

Liquid solvent

Elution:

Top: Small proteins

• Take longer travelling through the beads

Bottom: Big proteins

A

Green is on the bottom because those are larger proteins (they elute faster), so they are collected first which is the first peak

What is the process of ion-exchange chromatography? What are the stationary and mobile phases? What is the order of elution?

Process:

Separate based on differences in the sign and magnitude of the net electric charges of a protein at a given pH

Stationary:

Anion exchanger / anion collector: Beads are positive

Cation exchanger / cation collectors: Beads are negative

Mobile:

Positive or negative proteins

Elution:

Depends if anion or cation exchanger

If anion exchanger (collecting negative, stationary is positive):

Top: Positive proteins

Bottom: Negative proteins

How do you release the proteins from the column that have less charges in ion-exchange chromatography?

Use a low-salt elution buffer to neutralize proteins with less charges

How do you release the proteins from the column that have more charges in ion-exchange chromatography?

Use a high-salt elution buffer to neutralize proteins with more charges

How can you change the interaction of ion chromatography?

Change ionic strength or pH

A

You are collecting negative charges so anion exchanger

What is the process of affinity chromatography? What are the stationary and mobile phases? What is the order of elution?

Process:

• Protein interacts with the ligand on the bead and is stuck

• The rest of the proteins are eluted

• “Flush” protein of interest with free ligand, different pH, or a different salt concentration

Stationary:

Beads with ligands

Mobile:

Protein sample

Wash with free ligand, different pH, or a different salt concentration

Elution:

Top: Proteins that are stuck (proteins of interest)

Bottom: Excess proteins

Name examples of ligands

Antibodies

Metal ions

Enzymes

Substrates

B, C

Free ligands

Alter pH or salt concentration of elution buffer

C

B

Histone acetylation removes their positive charge so the histone is more negative

Usually, the positive end of the gel is on the bottom so the negative histone will be on the bottom

What’s the difference between gel electrophoresis and column chromatography?

Gel electrophoresis:

• Separate proteins for detection

Column chromatography:

• Separate proteins for purification

Which amino acids have the same skeletal shape?

Asn (N), Asp (D), Leu (L)

Gln (Q), Glu (E)

Cys (C), Ser (S)

Thr (T), Val (V)

Which amino acid has a functional group that ionizes completely in pH 2-9?

Arginine (R)

pH 2-9 < pka 12.5 ∴ protonate N to be positive

Arg has a unit charge so it is ionized completely in the entire pH range

Which amino acid has a functional group that has a unit charge and a fractional charge in pH 2-9?

Aspartate (D)

pH 1.7 - 5.7 ± pka 3.7 ∴ fractional charge

pH 5.8 > pka 3.7 ∴ deprotonate O to be negative

Lysine (K)

pH 8.5 - 12. 5 ± pka 10.5 ∴ fractional charge

pH 2 - 8.4 < pka 10.5 protonate N to be positive

Which amino acid has a functional group that has no charge and a fractional charge in pH 2-9?

Tyrosine (Y)

pH 8.5 - 12.5 ± pka 10.5 ∴ fractional charge

pH 2 - 8.4 < pka 10.5 protonate O to be neutral

Cysteine (C)

pH 6.2 - 10.2 ± pka 8.2 ∴ fractional charge

pH 2 - 6.1 < pka 8.2 protonate S to be neutral

Which amino acid has a functional group that has a unit charge, no charge and a fractional charge in pH 2-9?

Histidine (H)

pH 4 - 8 ± pka 6 ∴ fractional charge

pH 2 - 3.9 < pka 6 protonate N to be positive

pH 8.1 - 9 > pka 6 deprotonate N to be neutral

Glutamate (E)

pH 2.1 - 6.1 ± pka 4.1 ∴ fractional charge

pH 2 < pka 4.1 protonate O to be neutral

pH 6.2 - 9 > pka 4.1 deprotonate O to be negative

Which amino acid has a functional group that has no charge in pH 2-9?

Proline (P)

Doesnt’ have a functional group so it can’t have a charge

Serine (S)

pH 2 - 9 < pka 13.5 protonate O to be neutral

Which amino acids are basic?

Histidine (H)

Arginine (R)

Lysine (K)

Which amino acids are acidic?

Glutamate (E)

Aspartate (D)

What amino acid is prevalent in alpha-helices and why?

Alanine

It’s small and uncharged, it won’t hinder folding

Which amino acids are less common in alpha-helices and why?

Asparagine (N) and Tyrosine (Y)

Side chains are too bulky, it will break the helix

Which amino acids are at the beginning and end of alpha-helices and why?

Glycine

It’s small so it is flexible around its alpha-carbon. If it was in the alpha-helix, it would destabilize it. But it can participate in the turns

What amino acid is the least common in an alpha-helix and why?

Proline

It can’t Hbond because it doesn’t have a hydrogen on its amide nitrogen

It’s side chain links back to the amino group and it’s rigid. This would disrupt right-handed helical conformation

What is the distance of a beta-sheet two residue repeat?

0.7 nm

Which is more stable, antiparallel or parallel beta-strands and why?

Antiparallel is more stable

The Hbonds are straighter because of better alignment of the donor and acceptor atoms

It has shorter loops N → C

What is the problem at the end of alpha-helices? What could be done to prevent this problem?

There are 8 atoms unpaired

• The first four N-H groups and last four C=O groups lack intra-helical hydrogen bonds

Make the alpha-helix longer

• The longer it is, there are more Hbonds in the middle of the helix to compensate the 4 lost Hbonds

What is the spacing for Hbonds in alpha-helices?

Hbonds form between the carbonyl oxygen (C=O) of one amino acid residue and the amide hydrogen (N−H) of a residue located four units away in the polypeptide chain

Which direction does a right-handed helix and left-handed helix rotate?

Right-handed helix: clockwise

Left-handed helix: counter clockwise

How many residues are there per turn in an alpha-helix?

3.6 residues/turn

What is the problem of the margins of beta-sheets? How can you prevent this?

There are atoms on the margins that aren’t bonded

Make the sheet wider to compensate the atoms that are unbonded

Why do beta-sheets not have a dipole?

The N-H bonds are perpendicular to the strand

They have a local dipole from the N-H bonds but the adjacent residue also has the same N-H bond, but in the opposite direction. So the two dipoles cancel each other out W

Why do alpha-helices have a dipole?

The N-H bonds are parallel along the axis and they point to the same direction

The N-terminal is positive

The C-terminal is negative

Where do they side chains stick out for beta-sheets and alpha-helices?

Beta-sheets: Alternate sticking up and down on the bend

Alpha-helices: Stick out side

Which is more stable, a right or left-handed helix and why?

Right-handed helix is more stable

Left-handed helix is less stable because of steric hinderance of the R-groups

What types of bonds are in a beta-sheet in the x,y,z dimensions?

x: covalent bonds

y: Hbonds

z: IDID interactions

What type of turns do antiparallel and parallel sheets use?

Antiparallel: Tight turns

• It bends and has less residues

Parallel: Wide turns

• It loops and has many residues

What do turns do and why are they important?

Turns connect alpha-helices and beta-strands. They allow the peptide chain to fold back on itself

• Help with the compact 3D shape

How many atoms are in the same plane in an alpha-helix?

6 atoms in the same plane. Those 6 atoms restrict rotation

What is the formula for the number of Hbonds a beta-sheet can form between strands?

#hbonds between strands = # gaps x # aa

What is the formula for the number of Hbonds a beta-sheet can form on the edges of the sheet?

#hbonds on the margins = 2 edges x # aa

Where are loops seen on a folded polypeptide and why?

On the surface since they have hydrophilic residues

How is protein folding spontaneous? Use only Δ fold terms

ΔG fold = (more negative) ΔH fold - (positive)TΔS fold

How is protein folding spontaneous? Use only ΔG terms

ΔG fold = (more negative) ΔG water - (positive) ΔG polypeptide

How is protein folding spontaneous? Use only Δ folding, water, and polypeptide terms

ΔG fold = (negative ΔH water - more negative TΔS water) - (negative ΔH poly. - more positive TΔS poly.)

TΔS poly - TΔS water = TΔS fold

very positive - negative = positive

but now you have to consider ΔH fold which is very negative

ΔH fold - TΔS fold = ΔG fold

very negative - positive = negative

In terms of water, what is ΔS when the protein is unfolded vs. when the protein is folded?

Unfolded:

-ΔS because water shields the non-polar residues so freedom of movement is restricted

Folded:

+ΔS because water is free to move, it’s bulk water

In terms of the polypeptide, what is ΔS when the protein is unfolded vs. when the protein is folded?

Unfolded:

+ΔS because the protein is free to move, it’s floppy

Folded:

-ΔS because the protein is compact, it’s restricted to move

What do catalysts do?

Lower Ea by stabilizing the transition state so reactions can happen faster

Break down reactions into several steps

Does adding a catalyst change delta G?

No, it only lowers the Ea

It doesn’t change the stability of the products

What is the minimum number of steps a reaction can have form a catalyst?

3 steps

Because in a catalytic reaction, there is always an enzyme-substrate complex and an enzyme-product

A 2-step reaction will have how many intermediates and how many transition states?

1 intermediate, 2 transition states

A 3-step reaction will have how many intermediates and how many transition states?

2 intermediate, 3 transition states

What are the outcomes if you use a base as a nucleophile?

From substrate to intermediate: Good transition state - small hill so it’s not bad

From intermediate to products: Bad leaving group - big hill

Ea is lowered by a little bit

What are the outcomes if you use an acid as a nucleophile?

From substrate to intermediate: Bad transition state - big hill

From intermediate to products: Good leaving group - small hill

Ea is lowered by a little bit

What are the three active site residues aka the catalytic triad?

His-12, Lys-41, His-119

The RNAse acid/base catalysis is a 6 step reaction:

How many transition states are there?

How many intermediates are there?

6 transition states (6 peaks)

5 intermediates (5 low points)

Why are enzymes good catalyts?

1. Breaks it down into many intermediates

2. It works on the energy of the central intermediate

3. It makes it go over small hills on either side of the central intermediate

Draw and compare the delta G double daggar from an uncatalzed, base catalyzed, acid catalyzed, and enzyme catalyzed reaction

Explain the stability for the 5 intermediates

1. ES (#2, second most stable)

2. High energy intermediate (#4, least stable)

   1. No resonance stability

   2. More steric hinderance

   3. Accumulation of charges

   4. But Lys-41 does lower the Ea

3. Central intermediate is more stable (#3 most stable)

   1. Resonance

   2. Less steric hinderacne

   3. Less charges

   4. Ring structure destabilizes because of freedom of motion is restricted

4. High energy intermediate (#4, least stable)

   1. No resonance stability

   2. More steric hinderance

   3. Accumulation of charges

   4. But Lys-41 does lower the Ea

5. EP (#1, most stable)

Enzymes need to be careful in binding specific to what? How does the enzyme know?

Binding something specific to the transition state, not to the substrate

So enzymes have a specific geometry to distinguish between transition state and substrate

What would happen if the enzyme binds to the substrate more strongly than binding to the transition state?

It would increase Delta G double daggar so the reaction slows down

• Es to Transition state

What does Lys-41 do?

It binds to the high energy intermediates’ transition states to lower the Ea

What does His-12 and His-119 do?

Cyclization step: His-12 acts as a base, His-119 acts as an acid

Hydrolysis step: His-12 acts as an acid, His-119 acts as a base

Summarize the RNAse mechanism

1. His-12 deprotonates the 2’OH

2. 2’OH nuc attacks phosphate

3. His-119 protonates phosphate oxygen

4. 5’oxygen gets protonated and makes an alcohol leaving group

5. Product 1 leaves, water enters

6. His-119 deprotonates water

7. Hydroxide anion nuc attack electrophile forming P-O

8. His-12 protonates 2’oxygen and makes an alcohol leaving group

9. Product 2 leaves

What are the specificities in the RNase mechanism?

1. Enzyme-substrate binding geometry

• Pyrimidine-binding pocket

2. RNA has the 2’-OH group where the oxygen will act as a nucleophile

• DNA doesn’t have the oxygen

3. Location of His-12 forms only 3’phosphate products are formed