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Module 3: Protein

Terms in this set (76)

Five components of an amino acid
1. alpha carbon
2. amino group
3. carboxyl group
4. hydrogen
5. side chain (R group)
At physiological pH the amino and carboxyl group are
ionized
Side chains give the amino acid its unique properties (T or F)
true
Rotation from the alpha carbon to the N-H group is called __________. Rotation from the alpha carbon to the C=O group is called __________.
phi ; psi
What are the four non-covalent interactions?
1. van der waal
2. hydrogen bonds
3. electrostatic attractions
4. hydrophobic phorces
Which amino acids would be found in the hydrophobic core of a protein?
main: phenylalanine, leucine, valine, tryptophan
and other nonpolar amino acids
Which amino acids have a polar (hydrophilic) side chain?
main: arginine, glutamine, histidine
and other nonpolar acids
Linus Pauling Study
that when a folded protein was denatured using a solvent (like SDS or urea), it would unfold but then when solvent was removed, it would refold back into original conformation. This means that the amino acid sequence contains all of the information needed to specify the three dimensional shape of a protein.
___________ _________________ assist in protein folding
molecular chaperones
a-helices and B-sheets are both made up of ___________ bonds in the ____________ _______________
hydrogen bonds between atoms in the polypeptide back bone not as a result of interactions in the side chains
____________ found in keratin meanwhile ________ found in fibronin protein
alpha Helix ; Beta sheets
Can proteins have both types of secondary structures?
Yes, could have antiparallel or parallel B sheets and alpha helices (could be coiled-coil)
What interactions cause the formation of tertiary structures?
weak non-covalent interactions
What interactions are used to form the primary structure of a protein?
covalent peptide bonds
Is amino acid sequence or structure more highly conserved in a protein family?
structure
What makes humans more complex (similar genome size to plants)?
due to more combinations in domains. domain shuffling.
Define domain shuffling
creation of a new gene through the accidental joining of DNA sequences that encode different domains - makes humans more complex
Define quaternary structure
When two or more polypeptides get together
Define homodimer and heterodimer
Homodimer: protein composed of two identical proteins bound together through non-covalent interactions
Heterodimer: protein composed of two different polypeptide chains
Globular proteins are usually ____________. Filamentous proteins are usually ________________.
enzymes ; structural
Functions of intrinsically disordered regions of proteins.
1. binding sites for other proteins
2. easily modified for signal transduction
3. holds two interacting protein domains together
4. forms a diffusion barrier as a random coil meshwork
What is the purpose of disulfide bonds?
to stabilize protein structure
Define assembly factors
Portions of polypeptide that guide construction of a protein but are not found in the final tertiary structure
Stable β-sheet aggregates can form from many proteins, forming intertwined cross-beta strands that have the potential to kill cells or damage tissues. Which of the following is NOT true regarding these aggregates?
A. They form almost exclusively in the cells of the nervous system. - we see this predominantly in nervous system is because brain cells cannot replace themselves. But it is not EXCLUSIVE to the nervous system. You do get aggregates around the body - weird folding.
B. Different types of such aggregates can form from the same protein.
C. Their formation is associated with conditions such as Parkinson's disease.
D. They can form spontaneously, but also can be triggered to form by an infection with the same aggregate.
E. Some healthy cells form these aggregates to store their secretory proteins.
What does flow cytometry measure?
Protein abundance
Where would a positive, positive cell type be found in a flow cytometry plot?
top right hand side
What is the difference between flow cytometry and FACS?
In both, cells are analyzed. However, with FACS, cells are collected after being analyzed. With the cells obtained from FACS, we can prepare a primary culture.
What gives the cell a charge in FACS? What does this charge enable them to do?
depending whether the cell is fluorescently labeled or not. Charge with a positive/negative charge will be deflected by an electric field into collection tubes according to charge.
How do we attain cells for primary cultures?
cells can be used from FACS
Define replicative cell senescence and culture shock.
- Most vertebrate cells stop dividing after a finite number of cell divisions due to the silencing of telomerase.
- while in a dish, vertebrate cells stop dividing due to this. An innate protective mechanism to stop a cell from undergoing too many rounds of mitosis.
Silencing of telomerase causes _________
replicative cell senescence
How can we immortalize cell lines? Are these the same as transformed cell lines, why?
by adding telomerase or cancer promoting oncogenes that overcome culture shock. not the same because these are cell lines generated from cancer cells, not normally seen in bodies.
Two steps for purifying proteins
1. disruption of plasma membrane - osmotic shock, ultrasonic vibration, blender - make sure organelles are intact
2. separation of homogenate through ultracentrifugation
Velocity versus equilibrium sedimentation
velocity - sucrose gradient 5-20% and components are separated through size and shape, moving down their gradient based on size.

equilibrium - create a steep density gradient of sucrose or cesium chloride. components are separated by their buoyant density. cellular components move until reaching a position in which density of solution = to its own density. can isolate isotopes.

both will have distinct bands
What are the three types of column chromatography?
1. charge (ion-exchange)
2. size (gel-filtration)
3. ability to bind particular small molecule (affinity chromatography)
What are recognition tags used for?
Modifying the protein and adding something to the end for column chromatography. Epitote tag, Histidine (binds to metals), Glutathione-S-transferase (use glutathione to purify)
Disadvantages to genetically altering proteins through affinity chromatography?
we might have altered its function, shape = function.
What does SDS-PAGE do? What are the reagents involved and functions?
SDS-Page will separate based off size. If bands are bigger, there is more of that protein present.

SDS - masks the intrinsic charge of protein and unfolds or denatures the protein

B-mercaptoethanol - used as a reducing reagent to break any S-S linkages

Coomassie blue dye - stains protein bands to visualize
What does 2D Polyacrylamide Gel Electrophoresis do? What are the reagents/steps involved?
Separates based on charge and size.

First step: separate by charge through isoelectric focusing.
- do not use SDS
- use ß- mercaptoethanol and urea to break disulfide bonds.
-create a gradient pH in which protein will move until it reaches isoelectric point (pH where no net charge)

Step two: original gel soaked in SDS so that proteins can be separated by size
Describe the western blot procedure
1. add nitro cellular or nylon membrane and apply strong current, proteins will move onto a membrane and stick to this
2. Proteins identified by using an antibody labeled with radioactive isotope or a fluorescent due
How does one ensure that an enhancement seen following a treatment as observed by a Western blot is not due to a discrepancy in the same amount of protein added to each well?
Through the presence of a housekeeping gene. i.e. beta actin and GAPDHase - this is called a loading control (like a positive control but for western blotting)
When is a loading control used?
Western Blots
What is Quantitative Reverse Transcriptase Polymerase Chain Reaction? What does it test for?
RNA is converted into DNA via reverse transcriptase. Then, the cDNA is amplified by PCR using primers for specific sequences of interest.

Tests for presence of RNA virus like COVID or HIV.
What are microarrays? What are they used for?
a collection of microscopic DNA spots attached to a solid surface.

used to analyze thousands of mRNA produced from a cell simultaneously.
What is chromatin immunoprecipitation? What is the purpose of this procedure?
Proteins are covalently cross linked to DNA in living cells - cells are broken open and DNA sheared into small fragments. Antibodies directed against the transcription regulator are added to purify the DNA that is cross linked to the protein. DNA is sequenced using PCR - precise location fo each DNA is determined by comparing that to the whole genome sequence. All sites occupied by transcription regulator in the cell sample can be mapped across cell genome.
What are some drawbacks of co-immunoprecipitation?
- weaker signals from low affinity proteins are not detected
- antibody selection is critical and target protein prediction needs to be correct
- antibodies with high affinity are hard to isolate
What are some drawbacks to western blotting?
- requires precision in every step for proper identification
- can only be performed if primary antibodies against the protein of interest are available and are not cheap
How is the function of a protein determined?
By the molecule it binds to - ligand. This can be determined by its shape.
A ligand is...
Binding site is...
a substance bound by protein
region of the protein associate with ligand
Do hydrophobic atoms participate in binding with the ligand?
Yes, they form the framework of the protein - the 3D structure that helps create the binding site.
What are two ways in which conformation determines reactivity?
1. by restricting access of water to the ligand binding site - water decreases the strength of noncovalent interactions

2. neighboring amino acids side chains can alter reactivity - many repelling side chains clustered can attract or increase affinity for an opposing charge
What part of the protein structure is most likely to be unchanged in evolving organisms?
binding sites
What are the three types of protein interactions? Which one is most common?
1. surface-string
2. helix-helix interactions - forming a coiled-coil
3. surface-surface (precise matching) - involving pairing of ß sheets
What is an example of a surface-string reaction?
antibodies - loop, and the antigen has the surface
Antibodies tightly bind to a target molecule to do two things, what are they?
1. inactivate the molecule
2. mark it for destruction
what is an antigen?
a ligand - portion of a foreign molecule recognized by antibody
What is Ka? Units?
[AB] / [A] + [B} or Kon / Koff

units - L/mol
What does a high Ka mean? A low Ka?
high Ka = higher binding strength
lower Ka = lower binding strength
What is the formula for Km?
Km = 0.5(Vmax)
What does a low Km mean?
the tighter the enzyme binds to the substrate. means that an enzyme reaches maximum catalytic rate at a lower concentration of substrate.
What are four ways in which enzymes could create a conformational change (step 2)?
1. position two reactants together in proper orientation
2. charge groups alter electron distribution in substrate
3. alteration of substrate shape that distorts bonds in the substrate
4. forming transient covalent bonds with substrate
What are the four enzyme steps?
1. substrate binds to the active site
2. conformational change occurs at the active sites
3. change reduces the activation energy (transition state)
4. new products are released
How do enzymes stabilize the transition states? (step 3)
substrate molecule must pass through a series of intermediate states of altered geometry and electron distribution. enzymes have a greater affinity for the transition state (most unstable intermediate) and once they bind, they reduce the free energy therefore lowering the activation energies
What are coenzymes and what are they used for?
enzymes that frequently require a small molecule or metal atom in an enzyme's active site - help to assist with catalytic function. Heme is an examble
What are two mechanisms cells use to increase rate of reactions?
1. multienzyme complexes - using a tether (intrinsically disordered region)
2. intracellular membrane compartments
What are the six types of enzymatic regulation?
① gene regulation
② confinement to subcellular compartments
③ confinement to protein scaffold
④ covalent modifications
⑤ targeted protein destruction
⑥ feedback inhibition
What is a regulatory site used for? Is this the same as a binding site?
No - regulatory site used for a regulatory molecule and causes a conformational change that either decreases or increases the affinity for the substrate at the active site.

Binding site does not do this -affinity
When is Hemoglobin in a tense conformation? relaxed conformation?
T = low affinity for oxygen
R = high affinity for oxygen
What are the four processes phosphorylation/dephosphorylation is involved in
1. assembly and dissasembly of protein complexes
2. transmembrane signal transduction
3. intracellular signal amplification
4. cell cycle control
What is the name of the enzyme that adds a phosphate group to a protein? How about the name of an enzyme that removes a phosphate?
kinase ; phosphotase
What amino acids contain a hydroxyl group?
serine, threonine, tyrosine
activation of tyrosine receptors is caused by ______________. This causes a __________ _________________ _____________.
kinase ; signal transduction cascade
What does a GAP do? What does the name stand for?
Turns off activity - GTPase-activating protein induces the protein to hydrolyze GTP to GDP.
What does GEF do? What does it stand for?
Guanine nucleotide exchange factor causes the protein to release GDP which is replaced by a new GTP.
What does ubiquitination do? What does it bind to?
It is a covalent modification that targets proteins for proteasome degradation

Ubiquitin molecules are covalently linked to side chain of a lysine amino acid
What are scaffold proteins?
proteins with binding sites fro multiple proteins that link specific sets of interacting proteins and position them at specific locations in the cell
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