MODULE 4: Chapter 5.1 Flashcards
What bacterium causes anthrax disease?
Bacillus anthracis
What is one component of the anthrax toxin?
Protective antigen
What role does protective antigen play in the anthrax toxin mechanism?
Binds to receptors on target cells and forms a pore for toxin entry
What technique was used to purify protective antigen for biochemical studies?
Column chromatography
What analytical method was used to determine the structure of the pre-pore complex of protective antigen?
X-ray crystallography
What method was used to determine the pore-forming structure of protective antigen?
Cryo-electron microscopy
How does the conformational change of protective antigen affect its function?
It transforms from a soluble form to a pore-forming structure
What is the importance of understanding the structure of anthrax protective antigen?
It aids in the development of vaccines and therapies against anthrax
What is the proteome?
The entire constellation of proteins in an organism
What are the three primary approaches used to determine protein structure?
- X-ray crystallography
- NMR spectroscopy
- Cryo-electron microscopy
What is the initial step in the protein purification process?
Preparing a cell extract
Which biochemical assay was historically significant for detecting luciferase?
The luciferin-luciferase reaction
What is the purpose of a biochemical assay in protein purification?
To uniquely identify a protein of interest among others in the sample
What are the commonly used methods for cell disruption in protein purification?
- Sonication
- Shearing
- Mild detergents
What is the function of preparative centrifugation in protein purification?
To separate cellular components into fractions
What does the term ‘high-throughput protein biochemical methods’ refer to?
Methods used in proteomics to analyze many proteins simultaneously
Fill in the blank: The primary challenge of protein purification is to exploit the _______ that distinguish a particular protein from others.
[chemical and physical properties]
True or False: Humans have about 20,000 protein-coding genes.
True
What happens during the luciferase reaction in the presence of ATP?
It oxidizes D-luciferin to produce oxyluciferin and generates visible light
What factors influence sedimentation during centrifugation?
- Density of the solvent
- Size and shape of solute molecules
What is the result of centrifugation at high speeds?
Particles of different sizes or densities sediment at different rates
What do the four fractions obtained from a eukaryotic cell extract typically contain?
- Nuclei
- Mitochondria
- Plasma membrane components
- Cytosol
What are the four fractions obtained from a eukaryotic cell extract during preparative centrifugation?
- Nuclei
- Mitochondria
- Components of the plasma membrane
- Cytosol
What is specific activity in the context of protein purification?
The total amount or activity of the target protein divided by the total amount of protein in the fraction.
How is the total amount of protein estimated in a sample?
By measuring the sample absorbance at 280 nm (A280).
What factors influence the A280 value?
The composition of the protein, particularly the number of aromatic residues.
What are the colorimetric assays used for more precise total protein concentration measurements?
- Bradford assay
- Lowry assay
What is the principle behind salting out in protein purification?
Adding increasing amounts of a saturated salt solution to the protein solution to exploit differences in solubility.
What is the common salt used in the salting out technique?
Ammonium sulfate.
What technique is used to remove ammonium sulfate after precipitation?
Dialysis.
What is the purpose of dialysis in protein purification?
To leave the protein in a buffer of the proper ionic strength and pH.
What is column chromatography?
A protein purification method that separates proteins based on differential physical or chemical interactions with a solid matrix.
What are the main types of column chromatography?
- Gel filtration chromatography
- Ion-exchange chromatography
- Affinity chromatography
What does gel filtration chromatography separate proteins based on?
Size.
In gel filtration chromatography, which proteins elute first?
Large proteins.
How can molecular mass be estimated using gel filtration chromatography?
By comparing the elution profile of the target protein to proteins with known molecular masses.
What is high-performance liquid chromatography (HPLC)?
A high-resolution version of gravity-based gel filtration chromatography.
What is the principle behind ion-exchange chromatography?
Exploiting charge differences between proteins.
Name two commonly used ion-exchange matrices.
- DEAE cellulose (anion-exchange)
- CMC (cation-exchange)
What is affinity chromatography based on?
Specific binding properties of the target protein.
What is required for a protein to be purified using affinity chromatography?
The target protein must have a binding function that can be exploited.
What is a common example of an affinity column used in modern biochemistry labs?
Ni2⁺ chelating column for purifying His-tagged proteins.
What is an example of a ligand used in affinity chromatography?
An antibody for isolating antigenic proteins.
Fill in the blank: The target protein’s specific binding to a ligand during affinity chromatography allows for _______.
[higher protein purity]
What happens to nonspecific proteins during affinity chromatography?
They flow through the column without interacting.
What are recombinant proteins that contain histidine residues called?
His-tagged proteins
His-tags strongly coordinate divalent metals.
What is the most commonly used column chromatography approach in modern biochemistry labs?
Affinity chromatography of His-tagged proteins
This approach bypasses many trial-and-error steps in developing purification protocols.
What is the effect of protein purification steps on specific activity?
Increases ∼3- to 30-fold with each step
Specific activity is based on the units of target protein activity relative to total protein.
What is a critical consideration in designing a protein purification protocol aside from high purity?
Overall yield of purified protein
This determines how much material can be obtained for biochemical experimentation.
What is the purpose of gel electrophoresis in protein purification?
To approximate the molecular mass of a protein and check for multiple polypeptide chains
It provides an estimation of the relative purity of the target protein.
What technique separates proteins based on charge and size?
Polyacrylamide gel electrophoresis (PAGE)
The method uses a molecular sieve made of polyacrylamide gel.
What is SDS-PAGE and how does it affect proteins?
SDS-PAGE uses sodium dodecyl sulfate to give proteins a net negative charge, denaturing them
Migration through the matrix is unaffected by the original shape of the protein.
What happens to small proteins during SDS-PAGE compared to large proteins?
Small proteins migrate faster through the gel than large proteins
This is due to their ability to maneuver through the gel matrix more easily.
What can be determined by comparing the migration distance of an unknown protein in SDS-PAGE?
The apparent molecular mass of the protein
This is done by comparing it to known molecular-mass markers.
What is the role of β-mercaptoethanol in protein samples?
It breaks disulfide bonds in proteins
This is crucial for denaturing proteins during SDS-PAGE.
What is the difference between denaturing SDS-PAGE and native PAGE?
Denaturing SDS-PAGE disrupts noncovalent interactions and denatures proteins, while native PAGE preserves protein structure
Native PAGE reveals information on charge or conformation under specific conditions.
What dye is commonly used to visualize proteins after SDS-PAGE?
Coomassie Brilliant Blue G-250
It forms ionic interactions with basic groups in proteins.
What does isoelectric focusing separate proteins based on?
Charge as a function of pH
This technique determines the pI where proteins have no net charge.
What is the purpose of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)?
To separate proteins based on both isoelectric point (pI) and molecular mass
This method can identify changes in the proteome under different conditions.
What is two-dimensional differential in-gel electrophoresis (2-D DIGE)?
A method that uses fluorescent dyes to distinguish protein samples on the same 2-D PAGE gel
It allows for the detection of proteins that differ in abundance, charge, or molecular mass.
What indicates the abundance of proteins in 2-D DIGE?
The intensity of fluorescence emission
More abundant proteins exhibit a more intense fluorescent signal.
What is the purpose of labeling proteins with Cy5 and Cy3 in the analysis of cancer cells?
To differentiate between treated and untreated proteins using fluorescence.
Cy5 labels proteins from treated cells, while Cy3 labels proteins from untreated cells.
In the 2-D DIGE analysis, what color indicates a protein unique to one sample?
A single fluorescent color.
Proteins common to both samples appear as shades of yellow.
What does a yellow spot in a 2-D DIGE analysis represent?
A mixture of red and green fluorescence from common proteins.
Yellow indicates proteins present in both treated and untreated samples.
How many total protein spots were detected in the 2-D DIGE analysis mentioned?
Approximately 1300 protein spots.
Out of these, 1250 were green, 27 red, and 21 blue.
What is the first step in purifying a protein that binds to a pharmaceutical drug?
Develop a specific detection method, such as an enzyme activity or binding assay.
The detection method can utilize radioactively labeled or fluorescently labeled drugs.
What technique can be used to disrupt cell membranes during protein extraction?
Sonication.
This technique uses ultrasonic waves for cell homogenization.
What is the principle behind centrifugation in protein purification?
Separates particles based on size or density by spinning samples at high speeds.
It helps in isolating specific cellular components.
What does ‘specific activity’ refer to in protein analysis?
The total activity of a target protein divided by the total amount of protein in the sample.
Fill in the blank: The technique that separates proteins based on differential physical or chemical interactions with a solid gel matrix is called _______.
column chromatography.
What is the purpose of elution buffer in ion-exchange chromatography?
To displace the bound protein using a competing ion.
This allows for the purification of the target protein.
True or False: Gel filtration chromatography is also known as size-exclusion chromatography.
True.
What is the role of sodium dodecyl sulfate (SDS) in SDS-PAGE?
To give proteins a net negative charge.
This allows proteins to be separated based on size during electrophoresis.
What distinguishes native PAGE from SDS-PAGE?
Native PAGE uses nondenaturing conditions, while SDS-PAGE denatures proteins.
What does isoelectric focusing accomplish in protein separation?
Separates proteins based on charge as a function of pH.
What does 2-D DIGE enable researchers to detect?
Proteins that differ in abundance, charge, or molecular mass.
Fill in the blank: The process of separating proteins on the basis of both pI and molecular mass is known as _______.
two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).
What is the main advantage of high-performance liquid chromatography (HPLC) over traditional gel filtration chromatography?
It uses high pressure for better resolution.
