Module 4 - Protein Biochemistry and Hemoglobin Structures

Question 1

Describe the firefly enzyme and how it is purified

Answer 1

luciferase converts D-luciferin to oxyluciferin and produces light (which is used as the biochemical assay)

adding luciferin to protein extracts can determine the presence of luciferase based on the production of light

Question 2

How can cells be broken open for purification?

Answer 2

sonication (sound waves), shearing (grinding), or mild detergents

Question 3

How does centrifugation help purify proteins?

Answer 3

separates macromolecules on the basis of density and the amount of centrifugal force and time

Question 4

What is specific activity?

Answer 4

the total amount of target protein / the total amount of protein in the fraction

Question 5

What is the basic principle of column chromatography? How does the amount of protein concentration and target protein activity compare over the fractions collected?

Answer 5

separates proteins based on physical or chemical interactions with a solid gel matrix

fractions 1-6 contain 99% of the cell protein, but fractions 7-9 contain the most target protein activity

Question 6

Describe gel filtration

Answer 6

porous carbohydrate beads separate proteins based on size

larger proteins pass through first and smaller get caught up in the beads

Question 7

Describe ion exchange

Answer 7

proteins are separated. by charge depending on the charge of the buffer

anion exchange resins: DEAE cellulose, + beads catch anionic targets

cation exchange resins: carboxymethylcellulose, - beads catch cationic targets

a competing ion (same charge as the target) are used to elute the target proteins after other proteins have been eluted out

Question 8

Describe affinity chromatography

Answer 8

specific binding properties of the target proteins are used to separate it from other proteins without a binding site

target proteins are then eluted using a competing ligand

Question 9

How can multiple methods of protein purification be quantified?

Answer 9

specific activity can be calculated from:
total units of activity / total protein concentration

fold change of specific activity can be calculated from:
one specific activity / previous specific activity

Question 10

Describe SDS-PAGE

Answer 10

sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE)

Separates proteins based on charge and size. SDS binds to proteins giving them a charge proportional to mass. Smaller proteins migrate toward anode (+) and larger stay towards the cathode (-).

Low percentage gels are better for large proteins (better separation in 5%) but sacrifice the small proteins, while high percentage gels are better for small proteins (better separation in 15%) but sacrifice the large proteins.

Question 11

How can an SDS-PAGE chromatography be analyzed?

Answer 11

First elute contains the most concentration of the target protein

Question 12

What is the blue dye in SDS-PAGE?

Answer 12

coomassie blue

Question 13

What is isoelectric focusing?

Answer 13

uses isoelectric point (pI) to separate proteins. proteins migrate toward the opposite charge until they reach the pH gradient where they have no net charge (the pH = pI)

ex:
positive proteins migrate toward the cathode until they reach the pH that = their pI (they are deprotonated and become neutral)

negative proteins migrate toward the anode until they reach the pH that = their pI (they are protonated and become neutral)

Question 14

What is 2-D SDS PAGE?

Answer 14

Use isoelectric focusing followed by SDS PAGE.

L to R is low pI to high pI.
Top to Bottom is high mass to low mass.

Question 15

What is DIGE? Give an example.

Answer 15

differential in-gel electrophoresis (DIGE) is detection of proteins that differ in charge or mass between two closely related samples

breast cancer protein sample that is untreated (green) and treated (red) produces a SDS PAGE that has mostly yellow spots (present in both) but one green spot (unique to untreated sample) and one red spot (unique to treated sample)

Question 16

Describe Edman Degradation

Answer 16

Peptide is labeled at the N terminus with PITC followed by acid hydrolysis to yield a PTH amino acid derivative. These derivatives can be identified by HPLC against standards. Process repeated for entire peptide chain.

For large proteins, trypsin and chymotrypsin can perform differential protease cleavage to break the protein into fragments that are analyzed using Edman Degradation and then can be pieced back together.

Question 17

Describe mass spectrometry

Answer 17

Peptides undergo electrospray ionization to get individual peptide ions. Masses can be determined using tandem mass spectrometry, and then the masses can be used to infer amino acid sequences.

Question 18

Describe solid phase synthesis

Answer 18

To put together 2 amino acids, one of them has a Fmoc at the N terminal and a resin at the C terminal, while the other has Fmoc at the N terminal and DCC at the C terminal. Deblocking of the resin amino acid removes Fmoc and then they are combined (removing DCC) to form a chain of the two amino acids.
This is repeated for the entire chain (adding to the N terminus every time).
At the end, the final protecting groups are removed.

Question 19

Describe x-ray crystallogrpahy. What are its limitations?

Answer 19

Beam of x-rays is directed at a protein crystal, multiple images are collected and computationally analyzed to form a map of the electron density that then is used to generate a molecular model based on the location of atomic nuclei.

must be able to be crystallized, can’t show protein dynamics (crystals)

Question 20

Describe NMR spectroscopy. What are its limitations?

Answer 20

uses intrinsec magnetic properties of primarily H, N and C to determine relative locations of atoms.

must be relatively small (too big do not spin enough), need high protein concentrations, but are able to show protein dynamics and unfolding

Question 21

What is cryoelectron microscopy?

Answer 21

Electron beam produces two dimensional projections that can be put together using computer algorithms into a 3D model

must be cooled (“cryo”) to prevent damage from the electron beam radiation

Question 22

What are the 5 classes of proteins and their basic functions?

Answer 22

metabolic enzymes - lower activation energy of reactions without changing equilibrium or standard energy change

structural proteins - form filaments that provide cell structure and facilitate movement

transport proteins - form selective pores to permit or transport polar metabolites across the nonpolar membranes

cell signaling proteins - respond to changes in environment and relay information about changes to other proteins

genomic caretaker proteins - maintain genetic info in DNA and RNA

Question 23

What is an example of each of the 5 types of proteins and their basic function?

Answer 23

metabolic enzymes - malate dehydrogenase oxidizes malate to form oxaloacetate using NAD+

structural proteins - actin and tubulin control cell shape and migration

transport proteins - Ca 2+ ATPase transporter protein uses ATP hydrolysis to pump Ca 2+ ions across cell membrane against their concentration gradient

cell signaling proteins - erythropoietin receptors have two subunits that associate to form a single hormone binding site

genomic caretaker proteins - RecBCD binds to DNA, unwinds it, and cleaves a strand to facilitate repair or recombination

Question 24

Why does old meat turn dark?

Answer 24

fresh meat has Fe 2+ in hemoglobin, but it oxidizes to Fe 3+ resulting in a brown color

Question 25

What are the key differences between myoglobin and hemoglobin? What is the same for both?

Answer 25

myoglobin - monomer, storage of O2

hemoglobin - tetramer, transport of O2

both use heme to bind oxygen, and each subunit has eight a helices

Question 26

What happens molecularly when oxygen binds?

Answer 26

the proximal histidine (and the F helix it is attached to) moves toward the heme group in response to binding and coordinates with the Fe 2+

the distal histidine forms a hydrogen bond with the oxygen and stabilizes its interaction with the heme group

this produces the R/oxy state where the heme is planar instead of puckered, and the F helix/proximal histidine are closer to the heme

Question 27

What state is hemoglobin in when oxygen is bound or unbound?

Answer 27

R (relaxed) is the state when oxygen is bound

T (tight) is the state when oxygen is not bound

Question 28

What is the general equation for any protein and ligand binding? What is the expression for Keq, Ka, and Kd? What does a larger Ka or Kd indicate?

Answer 28

P+L = PL

Keq = [PL] / [P][L]

Ka (association constant) = same as Keq
- higher indicates more affinity

Kd (dissociation constant) = [P][L]/[PL]
- higher indicates lower affinity

Question 29

What is theta? How is it calculated in general and in terms of oxygen binding?
How is it calculated and what does it represent when = 0.5?

Answer 29

fractional saturation

occupied binding sites / total binding sites

[PL] / [PL] + [P]
or
[L] / [L] + Kd
or
[MbO2] / [MbO2] + [Mb]

when [L] = Kd, theta is 0.5
half binding sites are occupied when the concentration of ligand = the dissociation constant

Question 30

What do the curves look like for saturation vs. partial pressure of myoglobin and hemoglobin? What does this indicate?

Answer 30

myoglobin - hyperbolic
hemoglobin - sigmoidal, indicates cooperative binding

Question 31

Changes in one subunit cause changes in the others because they interact with each other through…

Answer 31

noncovalent contacts at the interfaces

T to R state involves breaking a Tyr-Asp hydrogen bond and forming a Asp-Asn hydrogen bond

Question 32

What are the two proposed models of cooperative binding?

Answer 32

concerted: as more ligands bing, the equilibrium shifts to the R state so that the other subunits spend more time in the R state and are more likely to bind oxygen. Ex: 1 ligand means more time is spend in T state, 2 means time is equally spend between R and T, while 3 ligands means most time is spend in R.

sequential: ligand binding to one subunit causes the neighboring subunits to also change conformation