Lecture 6

Question 1

What does ProParam give?

Answer 1

obtain molecular mass, pI, number of each type of residue

Question 2

What does Blast give?

Answer 2

obtain related protein sequences.

Question 3

What is the sequence format computers use?

Answer 3

FASTA

Question 4

Ways to obtain a protein sample in molecular biology?

Answer 4

1. Clone the gene from genomic DNA or cDNA or synthesize the gene
2. Over express protein in bacterial or other host
3. In vitro expression
   (need: plasmid with cloned gene, RNA polymerase, NTPs, ribosomes, amino acids)

Question 5

Ways to obtain a protein sample in chemistry?

Answer 5

• Chemical synthesis.
• Has the advantage of not being restricted to the standard 20 amino acids.
• There is currently a length limit.

Question 6

Examples of proteins purified from natural sources?

Answer 6

Digestive enzymes (pepsin, chymotrypsin, trypsin)
α-keratin (Wool)
Collagen (gelatin)
Serum albumin (bovine serum albumin or BSA)
Hemoglobin from RBCs (Horse)
Myoglobin from muscle (whale)
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), first major step of carbon fixation – CO2  glucose (most abundant protein on earth)
Lysozyme
Ribonuclease A
Photosynthetic rxn center (photosynthetic bacteria)

Question 7

Eqn to determine possible potential polymers?

Answer 7

N^L
N = # of diff monomers
L = length of polymer (monomer units)

Question 8

How many possible potential polymers are there for dipeptide Ala + Gly?

Answer 8

N^L
2^2
= 4

Question 9

How to optimize rxn so polypeptide synthesis?

Answer 9

1. Protection group e.g. Fmoc
   - Prevents it from activating as a nucleophile
2. Leaving group e.g. DCCD
   - Inc electriphicity
3. Polystyrene bead support

Question 10

What is the direction of synthesis of polypeptides?

Answer 10

C -> N

Question 11

What are the 4 general steps in preparing pure, monodispersed protein

Answer 11

1. Fractionation
2. Chromatography
3. Concentration
4. Analyzing

Question 12

3 examples of 3 fractionations?

Answer 12

1. Ammonium sulfate cuts (salting-out, precipitations, Hofmeister series)
2. Organic extraction
3. Temperature

Question 13

3 examples of chromatography?

Answer 13

1. Ion exchange – separation by charge (anion exchange, cation exchange)
2. Affinity
3. Gel-filtration (size-exclusion)

Question 14

2 examples of concentrating?

Answer 14

1. AS precipitation, solubilize in smaller volume.

2. Ultra-filtration

Question 15

5 examples of analyzing techniques?

Answer 15

1. Concentration determination – BCA (Cu reduction), Bradford assay (Coomassie G-250), based on sequence calculate theoretical, use Beers eqn.
2. SDS-PAGE
3. N-terminal sequencing
4. Mass-spectrometry
5. Analytical gel-filtration / light scattering

Question 16

Draw protein solubulity vs. ssalt conc

Answer 16

N/A

Question 17

What are the 3 maintypes of ion exchange?

Answer 17

1. Ion exchange
2. Affinity/ HIC
3. Size exclusion

Question 18

Two types of ion exchange chromatography?

Answer 18

• Proteins with a net negative charge (pI below 7) will likely stick to an anion-exchange resin
  Example: Q-sepharose

0 Proteins with a net positive charge (pI above 7) will likely stick to an cation-exchange resin
Example: S-sepharose

Question 19

What is affinity chromatography, using His?

Answer 19

Engineer 6 His tag at N terminus to purify protein out of all other proteins in lysate
- waters replaced by the imidazole side chains of 2 histidine reisues in the 6His-tag. The protein is usually eluted off using imidazole to complete off His-tag

Question 20

What are the 2 protein fusion affinity tags?

Answer 20

1. Maltose Binding Protein (MBP)
   - Amylose column
2. Glutathion S-transferase (GST)
   - Glutathione column

Question 21

Fxn of dialysis?

Answer 21

For changing buffers or desalting.
The tubing comes in different “Molecular Weight Cut Off” (MWCO).
A good type of tubing is Spectra/POR.

Can also use gel-filtration / desalting column: Sephadex G-10

Question 22

What is SDS Page?

Answer 22

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Question 23

What is native blue electrophoresis?

Answer 23

Instead of SDS the anionic dye Coomassie Brilliant blue R250 is used for solubilization of proteins, in the absence of reducing agents.

Question 24

What is isoelectric focusing for?

Answer 24

Experimentally determines the isoelectric point (pI)

Question 25

Steps in isoelectric focusing?

Answer 25

1. Cast gel 2. Establish gradient 3. Add sample 4. Run gel

Question 26

What is 2-D electrophoresis?

Answer 26

~ 10 000 different proteins can be distinguished

Question 27

3 ways to concentrate protein?

Answer 27

1. Precipitation 2. Compressed gas ultra-filtration 3. Centrifugation ultra-filtration

Question 28

How can you use precipitation to concentration proteins?

Answer 28

You can use precipitation to concentrate proteins. You use concentrated ammonium sulfate to precipitate the protein and then you can resuspend the protein precipitate in a smaller volume of buffer. This sometimes does not work.

Question 29

How can you use compressed gas ultra filtration to concentration proteins?

Answer 29

Compressed gas is used to move liquid through a membrane with a molecular weight cut off (example, molecules smaller than 10 KiloDaltons (including water) will flow through the membrane, and the protein will concentrate. This device is called the Amicon Stir-Cell.

Question 30

How can you use centrifugation ultra-filtration to concentration proteins?

Answer 30

replace gas pressure with centrifugal force

Question 31

How to determine protein concentration by UV spec?

Answer 31

1.  Calculate theoretical extinction coefficient based on the amino acid sequence via the program ProtParam 2. Measure absorbance (UV-vis spectrometer) at 280nm, Tryptophan contributes the most to the absorbance at this wavelength

Question 32

What is the Beer-Lambert equation?

Answer 32

A / Eb = c ``` A = absorbance (unitless) E = extinction coefficient b = path length (1cm) c = concentration (m) ```

Question 33

How to determine protein concentration by chemical methods?

Answer 33

- The Bradford protein assay (Coomassie Dye-based Protein Assays) - Absorbance shift (465 nm to 595 nm) upon binding to protein - BCA protein assays (BCA = bicinchoninic acid) 562 nm, reduction of Copper, binding of BCA

Question 34

Issues with bradford protein assay?

Answer 34

1. Depend on a standard curve using BSA (bovine serum albumin) which is of course not the protein you are interested in. 2. There are potential effects from the other molecules in solution, buffers, detergents, etc..

Question 35

3 ways to access purity of protein sample?

Answer 35

1. Specific activity (activity/total protein) can be used to assess protein purity 2. Mass spectrometry 3. Electrophoresis - Coomassie Brilliant Blue (R-250) ~ 100 ng - Silver staining ~ 10 ng

Question 36

How to store proteins?

Answer 36

1. Avoid freeze/thaw – aliquot into usable volumes, eg. 250 μL. 2. Sometimes it is helpful to add compounds like glycerol to prevent protein damage that can occur during freezing. 3. Quick freezing in liquid nitrogen is sometimes used to help prevent problems with freezing.

Question 37

How is purifying membrane proteins different from soluble proteins?

Answer 37

Purified the same way as soluble proteins, but you first need to extract them from the membrane and you need to always have detergents/lipids present.

Question 38

What is the aggregation #?

Answer 38

of detergent molecules in the micelle