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