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BIO315: Exam 1
Terms in this set (40)
Name a technique used to separate a complex mixture of proteins.
2D Gel Electrophoresis: separates proteins by pI (isoelectric point) and molecular weight
When trying to identify a particular protein you use a spectrophotometer. What ratio does that give you? Does this identify your protein?
Gives a mass: charge ratio; The charge may not be unique but the mass is, which will identify the protein
What is one downfall of use spectrophotometry to identify proteins?
Can't tell where it is in the cell
What is one technique one could use to identify where a certain protein is expressed in a cell?
by creating a fusion protein (expressing GFP for example); Add the gene expressing the specific protein into a plasmid with a GFP marker and grow up cells with the recombinant DNA; then use fluorescent spectroscopy to determine where in the cell the protein was being expressed
Draw a naturally occurring amino acid using a generic R group in the correct conformation.
Name all and draw out all of the hydrophobic amino acids.
A, I, L, M, V, P, CYSTEINE (C), F, W
Name and draw all of the uncharged polar amino acids.
GLYCINE (G), Q, N, T, S, Y
Name and draw the basic amino acids.
H, R, K
Name and draw the acidic amino acids.
Two unique characteristics about glycine.
-Nonpolar but can exist in hydrophilic environments
-high torsional flexibility so is often found in turns and loops
Two unique characteristics about cysteine.
-Thiol group is mainly hydrophobic but can participate in very weak dipole-dipole interactions
-Thiol group can form a disulfide (covalent) bond with another cysteine's thiol group (cystine) through an oxidation reaction
Four unique characteristics about proline.
-creates kinks in polypeptide chains
-disrupts alpha-helical secondary structure
-often found in tight turns
-can have trans and cis configurations
Is the cis or trans conformation for proline preferred?
trans; interconversion tends to be very slow but is also usually accompanied by a larger conformational change in the protein so that it could allow it to do its function
Draw the mechanism for peptide bond formation.
Refer to notes
Why are there only two types of secondary structure?
The phi and psi angles won't allow atoms to overlap so we get about (-135, 135) for beta strands and (-60, ~0) for alpha helices - because of steric hindrance
What interactions are present between parallel beta strands?
Hydrogen bonds between the carboxyl oxygen and hydrogen on the amino group - hydrogen bonds are slightly diagonal
What interactions are present between antiparallel beta strands?
Hydrogen bonds between the carboxyl oxygen and hydrogen on the amino group - hydrogen bonds are straight across; H bonds are closer together so a more stable structure
What interactions are present between alpha helical amino acids?
Hydrogen bonds between the carboxyl oxygen and hydrogen on the amino group
Are parallel or antiparallel beta sheets stronger?
antiparallel; hydrogen bonds are closer
What kind of amino acids prefer alpha helices? Beta strands?
Alpha helix - longer side chains because shorter side chains interfere with the hydrogen bonds with the backbone
Beta Strand - beta branched side chains
Draw a rakmachandran plot and describe where alpha helical and beta strands lie.
Upper left - beta strands
middle left - alpha helical
How many amino acids make up one turn of alpha helices?
Four amino acids
What trend to we see with amino acid groups on beta sheets?
Hydrophobic residues are on one side of the strand while hydrophilic residues are on the other side
Difference between Type I and Type II turn.
Type I: proline present
Type II: glycine present
Which of the following does thermo energy compare to?
-steric hindrance, dispersion, dipole-dipole, H bonds, salt bridges, disulfide bonds
dispersion (0.1-0.2 kcal/mol)
What is the stability difference between a folded and unfolded protein?
~10 kcal/mol more stable (a couple of H bonds)
What are two reasons proteins were become denatured? What helps with refolding?
1) thermal fluctuations and contact with other proteins
2) chaperone proteins help with refolding
Do the number of chaperone proteins increase or decrease with a rising temperature?
They increase to help assist with the denaturing of proteins
How do we know proteins fold on their own?
A protein can be denatured via beta-mercaptoethanol and urea (no activity)
Once Beta-Met and urea are removed, activity is reestablished.
Why might it be difficult for a protein to fold in a cell?
Pushes into other proteins and may not be able to fold properly.
Describe the mechanism for chaperonins.
-Top GroEL chamber opens has exposed hydrophobic patches
-Unfolded protein enters chamber via hydrophobic region sticking to hydrophobic patches
-7 ATP attach allowing GroEL, allowing GroES to bind
-Protein folds in cavity
-After 10-15 seconds 7 more ATP attach and cause conformational change that releases GroES
-Folded protein leaves GroEL
True or False. Chaperonins require ATP but Hsp70 does not.
False. They both require ATP
What are G-proteins?
guanosine nucleotide-binding proteins
What molecule is responsible for "shutting off" G proteins?
What are the two enzymes that affect G-proteins?
-GAP (GTPase Activating Protein)-accelerates hydrolysis of GTP (turns off GTP)
-GEF (Guanine nucleotide Exchange Factor) - accelerates release of GDP so GTP can bind quickly and active g-protein
What is an example of a g-protein that helps regulate the cell cycle?
Ras (regulated by GEF and GAP)
What GEF affects Ras? Does is activate or inactivate Ras?
SOS activates Ras by catalyzing the removal of GDP so GTP can enter
Which five motifs does ras contain?
1) P-loop (binds beta-phosphate)
2) switch I (binds gamma-phosphate through Thr)
3) Switch II (contains catalytic residue Gly)
4 and 5 are consensus sequences that make ras specific for guanine and not adenine
Where do most oncogenic mutations occur?
Why do eukaryotes have so much membrane?
Low volume: surface area ratio
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