Exam 1 Study guide Flashcards
Identify the four building blocks of cells and the macromolecule that each building block is used to create
The four building blocks of cells and their macromolecules are:
Sugars -> polysaccharides
amino acids -> protein
nucleic acids -> nucleotides
fatty acids -> lipids
Are macromolecules produced by hydrolysis or condensation reactions? You need to know the differences between these two types of reactions.
Macromolecules are made from condensation reactions with the removal of water. The difference between condensation and hydrolysis is that hydrolysis uses the input of water to break bonds while condensation reactions use the removal of water to create a bond.
Why do cells couple hydrolysis and condensation reactions?
Sometimes they couple reactions to use the energy from one reaction to initiate another one. Hydrolysis reactions are energetically favorable and can “power” condensation reactions which are energetically unfavorable.
Need to know the general structure of an amino acid and understand how the structure of an amino acid changes in water at pH7
(Hints for non polar: seeing aromatic rings, seeing only carbon and hydrogen, equal distribution of e)
Non polar:
-Glycine (Gly/G)
-Alanine (Ala/A)
-Valine (Val/V)
-Cysteine (Cys/C)
-Proline (Pro/P)
-Leucine (Leu/L)
-isoleucine (Ile/I)
-Methionine (Met/M)
-Tryptophan (Trp/W)
-Phenylalanine (Phe/F)
(Hints for polar: seeing an oxygen and nitrogen)
Polar:
-Serine (Ser/S)
-Threonine (Thr/T)
-Tyrosine (Tyr/Y)
-Asparagine (Asn/N)
-Glutamine (Gln/Q)
(Hints for positive charge: basic amino acids, accept a proton)
+Charge:
-Lysine (Lys/K)
-Arginine (Arg/R)
-HIstidine (His/H)
(Hints for negative charge: acidic amino acids, donated protons)
-Charge
-Aspartic Acid (Asp/D)
-Glutamic Acid (Glu/E)
Need to know the following terms and how they relate to protein structure: amino acids, N-terminus, C-terminus, and side chain/R group.
Amino acids are the subunits used to build protein. N-terminus is the first amino acid used to make protein. C-terminus is the last amino acid in the polypeptide chain. Side chain/R-groups are what makes the protein have different chemical properties.
Need to understand differences between polar, nonpolar, acidic (negative), and basic (positive) amino acids. Which can interact with water? Which can form hydrogen bonds? Which can form electrostatic interactions? Which are involved in hydrophobic forces? Note: you may want to review the Lecture 1 Background Information file to answer these questions.
Polar: Polar molecules are electronegative molecules with a partial charge. They are able to form hydrogen bonds with other molecules. They are also hydrophilic because of it.
nonpolar: non polar molecules do not form any hydrogen bonds with other particles and have an equal electron distribution. Because they are unable to form hydrogen bonds, non polar molecules are hydrophobic.
Charged molecules: charged amino acids mostly do electrostatic reactions. These include: aspartic acid, glutamic acid, Lysine, arginine, and histidine.
What is the role of covalent bonds, noncovalent bonds and hydrophobic forces in protein structure?
Covalent bonds are what build proteins with amino acids by creating bonds through condensation reaction. Also applies for a growing DNA strand.
Noncovalent bonds between polar amino acids influence three dimensional shape of proteins. (happening with side chains of amino acids)
Hydrophobic forces play a critical role in determining shape of protein; non polar amino acids cluster in core. Non polar amino (nonpolar side chains) acids create a hydrophobic core by clustering together, trying to avoid contact with water.
Why are alpha helices and beta sheets common structures in proteins?
Alpha helices and beta sheets form the secondary structures in proteins. They are common folding patterns because they rely on hydrogen bonds using N-H and C=O groups in polypeptide backbone (amino acid side chains are not involved in these folding patterns).
Describe the four different levels of protein structure.
Primary structure: only includes the amino acid sequence
Secondary structure: N-H and C=O start forming bonds to make alpha helixes and beta sheets.
Tertiary structure: Side chains start to get involved by bonding a collection of secondary structures in protein.
Quaternary structure: non covalently binding different polypeptide chains. More than one polypeptide chain connect together (for complex functions)
What are protein domains?
They are a segment of amino acids that can fold into stable structures. Form different structures in protein for different functions. ex: transcription factor
What are disulfide bonds? What is purpose of disulfide bonds?
Disulfide bonds are covalent bonds between cysteines that stabilize protein structure or combine different polypeptide chains. Solidifies tertiary structure. If a protein is outside the cell, it is reinforced with covalent bonds (disulfide bonds).
What is the association between protein shape/structure and protein function?
Different structures of a protein make proteins with unique functions. Shape dictates function. Function is also dependent on the ability the protein has to physically interact with other molecules.
Explain how a binding site of protein is able to form a stable interaction with a ligand.
the binding site of a protein consists of a cavity with amino acid side chains that bind to the ligand using non-covalent interactions. The amino acids in the cavity are polar which make them able to make noncovlent bonds like hydrogen bonds. Binding sites often use electrostatic interactions and hydrogen bonds to selectively bind to one specific ligand.
What is protein denaturation? Why does it occur (what is the molecular mechanism)?
Protein denaturation is when a protein unfolds and has reduced protein activity. This is because the weak covalent break between the proteins.
What causes protein denaturation? What happens when a cell has a large number of denatured proteins?
Protein denaturation can occur it is not an ideal environment for the protein. ex. heat, extreme pH, and chemicals. If a large number of proteins are denatured they can start to aggregate, which can diminish cell function and cause disease.
What are chaperones? Why are chaperones important for cellular function?
Chaperones are proteins that help refold denatured proteins or not properly folded proteins by binding to them. They go around the cell to check for unfolded proteins.
Where do chaperones bind on proteins?
They bind to the exposed nonpolar (hydrophobic) regions of the protein.
Identify two different strategies chaperones can use to help with protein folding.
- chaperone proteins bind to the partially unfolded protein to promote correct folding by extending it again and again to give it a chance to refold.
- chaperone protein forms a isolation chamber where a protein can be refolded without aggregating to other proteins.
What are heat shock proteins (Hsp)?
They are chaperone proteins that refold proteins that have been denatured
Why are heat shock proteins expressed when a cell is exposed to stress (like an increase in temperature)?
Hsp70 and Hsp60
Describe the differences between Hsp70 and Hsp60.
Hsp70 uses the first strategy of extending sections of a partially unfolded protein (of ezposed nonpolar amino acids) and gives it a chance of refolding correctly. It prevents protein from aggregating with unfinished protein. Speeds up folding process and makes it more efficient. Hsp60 uses the second strategy of isolating the protein so it can refold without aggregating to other proteins. The inside of the protein has a wall of hydrophobic side chains so that the protein unfolds and then switches to hydrophilic side chains to refold the protein, where its then ready to release.
Describe how Hsp70 and Hsp60 work together.
Hsp7o is considered to be the first line of defense and if that doesn’t work in refolding the protein correctly Hsp70 passes the protein to Hsp60 for another try at refolding it.
How does a cell regulate protein activity?
Protein activity is regulated at different steps of gene expression:
Transcriptional regulation- regulates if gene is transcribed
Post-transcription regulation- regulates if mRNA is translated
Post-transcriptional regulation- regulates if protein is active
protein degradation- regulates if protein is degraded.
What is protein phosphorylation? What is the role of a kinase and phosphatase in protein phosphorylation? Why does phosphorylation influence protein activity?
Protein phosphorylation is the attachment of a phosphate group to amino acid side chain.
Kinase: adds phosphate group to protein
Phosphatase: removes phosphate group to protein
Phosphorylation influences protein activity because it can either decrease or increase activity (always changes protein function)
What happens to proteins that cannot fold properly?
Proteins that cannot fold properly after going through Hsp70 and Hsp60 will be degraded by the proteasome.
What is a proteasome? What is ubiquitin?
A proteasome is s multiprotein complex that degrades damaged proteins within a cell. “trash compactor of cells” it consists of a cap and central cylinder, which is where peptide bonds are broken into individual amino acids. Ubiquitin is a small protein that is attached to other proteins. The c-terminus gets connected to side chain of another amino acid and essentially tags the protein.
Describe the relationship between the proteasome and ubiquitin.
The ubiquitin tags proteins that are destined to be degraded and are sent to the proteasome.Three steps to protein degradation:
1. a ubiquitin is added to the protein
2. tagged protein for degradation enters through the proteasome cap
3. protein is degraded in proteasome cylinder and released (ubiquitin is cleaved off to be recycled)
Describe the functions of the E1, E2, and E3 enzymes in protein degradation.
These are activating enzymes which add specificity to the targeted protein.
E1: gets ubiquitin ready to be added to targeted protein
-takes free ubiquitin and forms a covalent bond.
E2: ubiquitin on E1 is transferred to E2 which adds more specificity.
E3: E2 then adds to E3 where it is specific enough to target a specific substrate.
How does a specific protein get tagged for degradation?
A protein gets tagged through the process of ubiquitylation. This is where ubiquitin gets added to the target protein with the help of ubiquitin-activating enzymes (E1, E2, and E3). Firstly ubiquitin gets added to the E1 enzyme and then gets transferred to the E2 enzyme and where it lastly binds to E3 ligase and the ubiquitin ligase adds ubiquitin to target protein. (done multiple times)
What is the difference between a whole cell lysate and cell lysate/cell homogenate?
-A whole cell lysate is a single cell suspension of desired cells (just a collection of cells)
-cell homogenate is the rupture of membrane to release all the components of the cell.
What is cell fractionation and why is it useful for scientific research?
Cell fractionation is when you spin the sample at different speeds to segregate different components of the cell, so the heaviest component (nucleus) will pellet first. It is useful in scientific research because it allows you to separate components of cells and do further research on them.
Describe how a GST tag and immunoprecipitation can be used to purify a specific protein from a cell lysate?
They can be used to purify a protein by creating a fusion protein to tag the protein and be able to “pluck” the protein out of the cell lysate. The tag that is used in cell fusion is the GST tag and is put through a column that has glutathione bounded to a medium so that GST can bind to it and the fusion protein becomes immobile. Whatever protein does not have the GST tag will flow through the column. After everything has gone through the column, free glutathione is added so that it binds to GST and free itself from the anchored medium.
What is Coomassie Blue? Why is it used in scientific research?
Coomassie blue is a stain that is used in SDS-PAGE to stain all proteins that are present. It is useful in scientific research because it helps visualize the protein and can show if your’e protein has been purified.
Describe the purpose of SDS and BME when performing SDS-PAGE.
SDS is a detergent that denatures proteins and coats the protein with a negative charge. BME is a reducing agent that eliminates disulfide bridges. This causes the protein to return to its primary structure which is important to perform the gel.
What is the purpose of a western blot? What is being analyzed when a western blot is used?
the western blot uses antibodies to detect specific protein on membrane. Western blot measures the amount of a specific protein.
Need to know the steps to perform a western blot.
- Use SDS-PAGE to separate the protein mixture in the sample
- Transfer the proteins from the gel to a membrane by using a strong electrical charge (antibodies can’t bind to proteins on gel)
- Primary binds to the target protein
- Secondary antibody binds to the primary antibody and makes the protein visual. (enzyme that sends signal)
Describe the purpose of SDS-PAGE, the membrane, and antibodies in a western blot.
The SDS-PAGE is to separate the proteins by size before transferring it to the membrane to see the amount of that protein. The membrane is so that the antibodies are able to bind to the proteins since they are unable to do so on the gel. Antibodies are to bind to a specific protein of interest and only show that protein on the western blot.
What is the difference between a primary and secondary antibody?
a primary antibody binds to the specific protein of interest on the membrane and the secondary antibody binds to the primary antibody to make the protein visible.
