Conceptual Exam 1 Flashcards
1
Q
Polar AA’s?
A
STCNQY
2
Q
Positively charged AA’s @ 7?
A
RK
3
Q
What forms disulfide bonds?
A
C
4
Q
Peptide bonds flow in what order?
A
N —–> C terminus
4
Q
Negatively charged AA’s @ 7?
A
ED
5
Q
What is the free energy equation?
A
_____________
5
Q
What is change in G?
A
Free energy
6
Q
What is change in H?
A
enthalpy
7
Q
What is change in S?
A
Entropy
8
Q
Is higher or lower entropy favorable? Enthalpy?
A
Higher; Lower
9
Q
Is a negative or positive free energy more favorable? Negative or positive spontaneous?
A
Negative; negative
10
Q
Describe the hydrophobic effect.
A
Polar regions interact with H_2O. Nonpolar cluster to form hydrophobic core.
11
Q
Write out the Henderson-Hasselbach equation.
A
________________________
12
Q
What are the AA’s with titratable side chains? pKa’s?
A
HERCKDY; 6, 4,12, 8, 10, 4,10
13
Q
Draw out a titration graph for histidine.
A
___________________________
14
Q
What does it mean when pka=pH?
A
50% of AA is deprotonated/protonated
15
Q
What is the pI?
A
Isoelectric point where net charge = 0
16
Q
What is the equivalence point?
A
pH=pI
17
Q
How can proteins be separated?
A
- Size
- Function
- Charge
18
Q
What does a low pI tell us?
A
There are negative side chains.
18
Q
What AAs absorb UV light in order of lowest absorbance to highest absorbance?
A
F, Y, W
19
Q
What is the elution order of cation exchange? What is the gel? Resin charge?
A
- Negative
- Neutral
- Positive
CM
Negative
20
Q
What is the elution order of anion exchange? What is the gel? Resin charge?
A
- Positive
- Neutral
- Negative
DEAE
Positive
21
Q
What are the steps of affinity chromatography?
A
- Loading
- Separation
- Elution
22
What is size exclusion chromatography? Elution order?
Separates based on size, large to small
23
What is affinity chromatography?
Separates based on binding affinity
24
What do you use in the elution step of AC?
ATP or salt
25
What is recombinant DNA? Example?
Artificial DNA with unnatural sequence combinations, tagged histidines that help anchor proteins
26
Most effective type of separation technique?
Affinity chromatography
27
How do you find the most effective purification system in a table?
How much specific activity increases
28
Two types of PAGE?
SDS and native page
29
What does denatures mean?
removes 2, 3, and 4 degree structure
30
What does SDS do and how?
Denatures proteins by giving them uniform charge which separates by size
31
What does BME do? What is it used for?
Reduces disulfide bonds on cysteine residues, finding disulfide bonds
32
Reducing SDS? Non-reducing?
SDS + BME; SDS only
33
What does native page do? Allows for determination of?
Separates proteins without denaturation; mass, size, and charge
34
Where are larger/smaller proteins on PAGE?
Larger top; smaller bottom
35
Homo vs hetero oligomers?
Hetero have different molecules and homo have the same molecules
36
Without BME can you determine whether something is a hetero or homo oligimer?
NO
37
What does IEF PAGE do?
Separates proteins based on their isoelectric point (where PH charge = 0).
38
How many levels of protein structure?
4
39
What is primary structure?
Amino acid sequence joined together by peptide bonds
39
What is secondary structure?
Helix, beta sheets, turns, coils with hydrogen bonds
40
What is tertiary structure?
3-D folds with hydrophobic interaction, disulfide bonds, H-bonds, and ionic bonds
41
What is quaternary structure?
Interaction of many polypeptide subunits
42
What does entropy pertain to? Enthalpy?
Hydrophobic; aqueous
43
What drives folding (2)?
1. Negative free energy
2. Entropy
44
What is the hydrophobic effect and why does entropy increase?
Clusters of lipids cause H_2O molecules to be disordered
45
What are Van der Waal interactions?
Very weak interactions between two atoms
46
Draw graph of van der Waal interaction.
__________________________
47
Draw the graph of the Leonard Jones potential.
____________________________
47
What is the Leonard Jones potential?
2 interacting atoms repel when too close, attract at moderate distance, and DO NOT interact when too far away
48
Write out Coulomb's Law.
_____________________________________
49
What is Coulomb's Law?
Electrostatic interactions affect energy of system
50
What is E in Coulomb's Law? Example in H_2O and core of protein?
Dielectric constant; 80 and 2
51
Draw out the Ramachandran Plot and where alpha helices and betas live.
______________________________________
52
What two AA are not favored in alpha helices? Why?
Gly and Pro; steric hindrance
53
For every turn, how many residues and length in alpha helices? Beta?
3.6; 5.4
2; 7
54
What is the hydrogen bonding pattern of alpha helices?
i to i+4
55
Intramolecular or intermolecular bonding in alpha helices?
Intra
56
When are proteins exposed? Buried?
Polar; nonpolar
57
Two types of B-sheets? Describe differences.
Antiparallel has alternating NC terminus pattern, colinear H bonding so favorable and parallel same NC pattern
58
Which two AA involved in B-turns?
Gly and Pro
59
What type of AA do B-sheets favor?
Room for bulky so F, W, Y
60
What are B-turns?
connect two segments of B-sheets
61
What is a domain? Importance?
Independent folding unit that could undergo movement at a single entity
Important because a single protein can have multiple functions.
62
What is an active site?
Where substance binds
63
Is folding spontaneous?
YES
63
What is usually lost after protein denatured?
Function
63
Once denaturation happens, is it always reversible?
NO
64
What is critical for the function of RNase?
disulfide bonds
64
Draw out the energy funnel? Label entropy, energy, native percent, and three stages of protein.
_________________________________________
65
What did Anfinsen discover? Order of experiment?
He discovered that RNase is 100% recoverable after denaturation.
Steps: Urea, reducing agent (BME), removes urea, adds oxygen
66
What is urea, BME, oxygen?
strong denaturant; reducing agent; oxidant (reforms disulfide bonds)
67
Two ways to determine charge in protein structure?
Tryptophan fluorescence and circular dichroism
68
What does tryptophan fluorescence do? Draw graph.
Wavelength shifts when denatured
69
What does circular dichroism do? Draw graph.
Polarized light bends based on 2/3 structure
70
What is aggregation?
Process in which oxidized, misfolded, or damaged proteins bind together forming large mass without original function
71
What are chaperones?
Proteins that assist folding of other proteins and prevent misfolding
72
Equation of ligand and protein interaction.
____________________
73
What is Kd?
Dissociation constant, ligand concentration @ which 50% of binding sites are occupied
74
Draw out protein and ligand interaction graph.
__________________________
75
Write Fraction bound equation and change in G equation.
_____________________
76
What does a low KD tell us?
High affinity so more spontaneous
77
What does a high KD tell us?
Low affinity so less spontaneous
78
Write out Beer's Law.
_______________________
