Module 4 - Protein Biochemistry and Hemoglobin Structures Flashcards
Describe the firefly enzyme and how it is purified
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
How can cells be broken open for purification?
sonication (sound waves), shearing (grinding), or mild detergents
How does centrifugation help purify proteins?
separates macromolecules on the basis of density and the amount of centrifugal force and time
What is specific activity?
the total amount of target protein / the total amount of protein in the fraction
What is the basic principle of column chromatography? How does the amount of protein concentration and target protein activity compare over the fractions collected?
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
Describe gel filtration
porous carbohydrate beads separate proteins based on size
larger proteins pass through first and smaller get caught up in the beads
Describe ion exchange
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
Describe affinity chromatography
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
How can multiple methods of protein purification be quantified?
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
Describe SDS-PAGE
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.
How can an SDS-PAGE chromatography be analyzed?
First elute contains the most concentration of the target protein
What is the blue dye in SDS-PAGE?
coomassie blue
What is isoelectric focusing?
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)
What is 2-D SDS PAGE?
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.
What is DIGE? Give an example.
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)
Describe Edman Degradation
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.
Describe mass spectrometry
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.
Describe solid phase synthesis
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.
Describe x-ray crystallogrpahy. What are its limitations?
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)
Describe NMR spectroscopy. What are its limitations?
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
What is cryoelectron microscopy?
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
What are the 5 classes of proteins and their basic functions?
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
What is an example of each of the 5 types of proteins and their basic function?
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
Why does old meat turn dark?
fresh meat has Fe 2+ in hemoglobin, but it oxidizes to Fe 3+ resulting in a brown color
What are the key differences between myoglobin and hemoglobin? What is the same for both?
myoglobin - monomer, storage of O2
hemoglobin - tetramer, transport of O2
both use heme to bind oxygen, and each subunit has eight a helices
What happens molecularly when oxygen binds?
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
What state is hemoglobin in when oxygen is bound or unbound?
R (relaxed) is the state when oxygen is bound
T (tight) is the state when oxygen is not bound
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?
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
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?
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
What do the curves look like for saturation vs. partial pressure of myoglobin and hemoglobin? What does this indicate?
myoglobin - hyperbolic
hemoglobin - sigmoidal, indicates cooperative binding
Changes in one subunit cause changes in the others because they interact with each other through…
noncovalent contacts at the interfaces
T to R state involves breaking a Tyr-Asp hydrogen bond and forming a Asp-Asn hydrogen bond
What are the two proposed models of cooperative binding?
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
