Part 3 Flashcards
What length of amino acids allows for a stable protein domain that is not too short and not too long?
50-300 amino acids
-longer polypeptides can fold into independent domains. Different domains are stable structures that fold independently and often have different functions
What are protein domains?
independent, stable structures
they are usually composed of a continuous sequence of amino acids
Give one example of a protein that contains multiple domains and explain its structure.
Yeast Gal4 transcription factor. This helps turn on transcription to turn on expression of the galactose gene.
- activation domain: used to turn on activation of the gene by recruiting factors
- DNA-binding domain: recognizes the specific sequence next to the binding site
* 2 motifs found are the helix loop helix and the zinc finger** - DNA-binding site
Can you swap domains in a protein?
Yes you can, they are completely independent but work together in the protein
What are antibodies?
They are produced and used by the immune system to identify and neutralize foreign objects such as bacteria and viruses
What are antibodies?
They are produced and used by the immune system to identify and neutralize foreign objects such as bacteria and viruses
What is an antigen?
ANTIBODY GENERATOR
-foreign substance that elicits antibody production
What is an example of circulating antibodies in our immune system?
IgG: immunoglobulin G molecules
Explain the structure of an antibody.
Y-shaped
- 4 chains: 2 identical light chains and 2 identical heavy chains that are held together by DISULFIDE bonds
- 12 independent domains total
- the variable domains determine antigen affinity
Where is the antigen-binding region in an antibody located?
it is formed at the interface between the VL and VH domains
What is an epitope?
region of antigen that is recognized by the antibody
Describe the protein-binding specificity of antibodies.
- complementary interaction of antigen and antibody fits like a glove
- antibodies can distinguish among proteins that differ by only a single amino acid
- strong antibody-antigen binding is important for many molecular bio techniques
What is an example of a protein enzyme that undergoes a conformational change?
hexokinase
- with glucose, its structure closes in
- conformational changes can often affect protein function
T/F. DNA binding proteins can be specific or non specific.
True
Compare specific and non-specific DNA binding proteins
- specific: recognize specific DNA sequences primarily via specific pattern of chemical groups on the sides of bps in the major groove
- non-specific: bind any sequence; independent of sequence and structure; often relies on the polyanionic backbone. Ex: histones and ssb protein
Compare specific and non-specific DNA binding proteins
- specific: recognize specific DNA sequences primarily via specific pattern of chemical groups on the sides of bps in the major groove
- non-specific: bind any sequence; independent of sequence and structure; often relies on the polyanionic backbone. Ex: histones and ssb protein
What is a common theme for DNA binding proteins?
an alpha helix is inserted into the major groove
- “recognition helix”
- the H-bonding and non polar contact properties of side chains on the lambda repressor “recognition helix” are complementary to those of the specific base sequence recognized
Does the H-bonding with proteins affect the H-bonding in the DNA double helix?
no
What kind of proteins catalyze biochemical reactions?
Enzymes
-they provide an environment where a reaction can occur rapidly
Most catalysts are proteins. Some specific catalysts are RNA. T/F
True
ribozymes
A molecule that undergoes an enzyme-catalyzed reaction is a ______.
substrate
T/F. An enzyme converts a substrate to a product without changing itself.
True
Enzymes speed up reactions by factors of 10^6 or more. T/F
True
A barrier to a chemical reaction is a ____________. This is a high-energy arrangement of the reactants.
Transition state
T/F. Many chemical reactions do not proceed unaided at a measurable rate even though deltaG for the reaction is negative.
True
T/F. Many chemical reactions do not proceed unaided at a measurable rate even though deltaG for the reaction is negative.
True
How do enzymes speed up reactions?
They lower the activation energy. Most enzymes achieve this by having an active site such as a pocket or groove that is complementary in shape and interaction properties (H-bonds, non polar contacts) to the transition state of the reaction.
Post-translational modifications of proteins
What is one type?
- essential for regulating the structure and function of a protein
- covalent modification (group addition) to amino acid side chains
What are 4 types of covalent modifications that occur post-translationally in proteins?
- glycosylation
- phosphorylation
- acetylation
- methylation
Protein glycosylation
- covalent attachment of sugars/glycans to an amino acid chain
- Asparagine, serine, threonine
- generates “glycoproteins”
- very common in cell-surface membrane proteins and secreted proteins
structural and functional roles:
- proper protein folding
- stability (protection from degradation)
- cell-cell adhesion
Protein glycosylation
- covalent attachment of sugars/glycans to an amino acid chain
- Asparagine, serine, threonine
- generates “glycoproteins”
- very common in cell-surface membrane proteins and secreted proteins
structural and functional roles:
- proper protein folding
- stability (protection from degradation)
- cell-cell adhesion
What are 2 types of glycosylation?
- N-linked: to the amide nitrogen of asparagine side chains
2. O-linked to the hydroxyl oxygen of serine and threonine side chains
Protein phosphorylation
- covalent attachment of a phosphate group to the -OH group of Ser, Thr, Tyr by a protein kinase enzyme
- some functions turn protein activity on/off, signal transduction, and apoptosis (cell death)
What is phosphoregulation?
regulation of protein activity via kinase/phosphatase switch
- one of the most common ways to regulate protein function
- reversible protein switch
- phosphorylated protein can be active or inactive
Phosphorylation occurs by _____ and dephosphorylation occurs by_____.
phosphorylation: protein phosphatase (uses water) to remove P
dephosphorylation: protein kinase (uses ATP) to add on a P
Phosphorylation occurs by _____ and dephosphorylation occurs by_____.
phosphorylation: protein phosphatase (uses water) to remove P
dephosphorylation: protein kinase (uses ATP) to add on a P
Protein Acetylation
What is an acetyl group?
Which enzymes add on/take off acetyl groups?
lysine acetylation is an important histone protein modification that affects chromatin structure and can regulate gene expression. Genes don’t usually get expressed in tight, compact forms and lysine acetylation allows it to get looser.
An acetyl group is a carbonyl + methyl group
HAT: histone acetyl transferase
HDAC: histone deacetylase
Protein Acetylation
What is an acetyl group?
Which enzymes add on/take off acetyl groups?
lysine acetylation is an important histone protein modification that affects chromatin structure and can regulate gene expression. Genes don’t usually get expressed in tight, compact forms and lysine acetylation allows it to get looser.
An acetyl group is a carbonyl + methyl group
HAT: histone acetyl transferase
HDAC: histone deacetylase
Protein Methylation
- methyl groups are covalently attached to Arg or Lys via methyltransferase enzymes
- most studied in histone proteins as a way to regulate chromatin structure
What are 3 protein purification methods?
column chromatography
SDS-Page
Western Blot
What must occur first in order to study the function of a given protein?
The protein must be purified to explode their unique features such as size, shape, charge, etc.
Unlike nucleic acids, proteins do not have a consistent shape and size.
Column Chromatography
- separates mixtures of proteins based on mass, charge, and binding affinity
- glass or plastic cylindrical columns are filled with small bead that are modified to have different properties
- the nature of the beads determines the type of protein separation
1. Gel-filtration: size of protein
2. ion-exchange: charge of protein
2. affinity chromatography: specific protein
Which type of chromatography separates proteins based on size and shape?
Gel-Filtration
- each bead contains different sized pores
- small proteins can enter all of the pores and will migrate slower through the column to elute
Which type of chromatography separates proteins based on size and shape?
Gel-Filtration
- each bead contains different sized pores
- small proteins can enter all of the pores and will migrate slower through the column and take longer to elute
- larger proteins can access few or no pores in the column beads and elute faster
*the total volume of liquid required to elute a protein from a gel filtration column depends on its mass: the smaller the mass, the greater the elution volume
Which type of chromatography separates proteins by their charge (the sum of the charges on the amino acid side chains)?
Ion-exchange
- the beads are modified with positively charged or negatively charged chemical groups
- a salt buffer is used to elute proteins that bind to the charged beads
- low salt concentration: proteins that weakly bind are eluted
- high salt concentration: proteins that strongly bind are eluted
the salt masks/completes for the charges on the column
Affinity chromatography
- more specific type of protein purification, but requires knowledge about the protein
- separates proteins based on their affinity to bind to a particular ligand that is bond to the bead
-Immunoaffinity chromatography: uses a specific antibody for the target protein, attached to the column beads (highly specific)
- loaded with PH 7 buffer
- eluted with PH 3 buffer (your protein ends up being a bit denatured) This is because the protein may bind so tightly to the antibody that it must be denatured before it can be eluted
What method can be used in affinity chromatography so that your protein won’t be denatured?
you can add a short amino acid sequence “tag” to the N- or C-terminus
- a histidine “tag” causes a protein to bind tightly to Ni2+ attached to column beads
- epitope: a short peptide sequence (7-10aa) that is recognized by a particular antibody
- **it is important to choose an epitope/Ab system that is known to change its binding affinity from high to low with a simple change to the solution (pH, salt, etc)
What method can be used in affinity chromatography so that your protein won’t be denatured?
you can add a short amino acid sequence “tag” to the N- or C-terminus
- a histidine “tag” causes a protein to bind tightly to Ni2+ attached to column beads
- epitope: a short peptide sequence (7-10aa) that is recognized by a particular antibody
- **it is important to choose an epitope/Ab system that is known to change its binding affinity from high to low with a simple change to the solution (pH, salt, etc)
SDS Gel electrophoresis
Gives proteins a uniform charge and structure by removing 2, 3, 4 structures.
SDS coating forces the polypeptide chains into an extended conformation with a similar charge:mass ratio, eliminating the effects of differences in shape and charge. Only the mass of the chain determines migration rate in a SDS-gel.
SDS
strong ionic detergent that disrupts non-covalent interactions between amino acid residues and coats the polypeptide, giving it a uniform negative charge
(removes the structure basically)
B-mercaptoethanol
reducing agent the breaks disulfide bonds
SDS
strong ionic detergent that disrupts non-covalent interactions between amino acid residues and coats the polypeptide, giving it a uniform negative charge
**eliminates differences in shape and charge
***protein now moves based on a charge: mass ratio
(removes the structure basically)
B-mercaptoethanol
reducing agent the breaks disulfide bonds
SDS-Page Gel Electrophoresis
Page: stands for polyacrylamide gel. This is used for proteins because the pores are not too big to separate proteins properly
Western Blot/ Immunoblot
Done to find a specific protein
- proteins are negatively charged due to SDS electrophoresis
- transfer the separated proteins from a gel to a filter using an electric field
- incubate in a solution with an antibody against the protein of interest
- visualize the bound antibody- is attached to an enzyme that creates luminescence when it acts on a particular substrate
