Material for Exam #1

Question 1

What are the 3 fundamental principals of biochemistry according to Dr. Capp?

Answer 1

1) Equilibrium is everything
2) Structure determines function
3) Nothing is wasted

Question 2

What is the ΔG equation for a system at standard conditions, and at non-standard conditions?

Answer 2

Standard Conditions: ΔG^0 = -RTln(Keq)

Non-Standard Conditions: ΔG = ΔG^0 + RTln(Q)

Q = [products]/[reactants] not at equilibrium

Question 3

What is the ΔG for ATP going to ADP + Pi

Answer 3

ΔG = -30.5 KJ/mol

Question 4

What are the 5 types of bonds?

Answer 4

Covalent Bonds
Ionic Bonds
Hydrogen Bonds
Dipole-Dipole Interactions
London Dispersion Forces

Question 5

What are the 2 terms for a molecule with both polar and non-polar components?

Answer 5

Amphiphillic/Amphipathic

Question 6

What is a micelle?

Answer 6

A shape formed by amphipathic molecules where the non-polar heads aggregate together away from the polar solvent and the polar ends are facing the solvent (like a cell membrane)

Question 7

What are the two definitions for an acid?

Answer 7

Donates an H+ - Bronstead-Lowry definition

Accepts an electron pair - Lewis definition

Question 8

What are the two definitions for a base?

Answer 8

Accepts an H+ - Bronstead-Lowry definition

Donates an electron pair - Lewis definition

Question 9

What pH conditions lead to a buffering effect, and in what pH range?

Answer 9

When the pH = pKa, there is a buffering effect +/- 1 pH value

Question 10

What is the pH of blood?

Answer 10

pH = 7.4

Question 11

What is the pKa combo for bicarbonate?

Answer 11

pKa = 6.35

Question 12

What is the pKa of the amino group on an amino acid?

Answer 12

pKa = 9.5

Question 13

What is the pKa of the carboxyl on an amino acid?

Answer 13

pKa = 2.2

Question 14

What is the stereoisomer for all amino acids?

Answer 14

“L” stereoisomer

Question 15

What are the small, non-polar amino acids?

Answer 15

GAVIL

glycine, alanine, valine, isoleucine, leucine

Question 16

What are the weird, non-polar amino acids?

Answer 16

MP

methionine, proline

Question 17

What are the large, non-polar amino acids?

Answer 17

TTP

tryptophan, tyrosine, phenylalanine

Question 18

What are the alcohol and thiol amino acids?

Answer 18

STC

serine, threonine, cystine

Question 19

What are the polar and acidic amino acids?

Answer 19

AAGG

aspartic acid, asparagine, glutamic acid, glutamine

Question 20

What are the base and buffer amino acids?

Answer 20

HAL

histidine, arginine, lysine

Question 21

How many amino acids are generally in a protein?

Answer 21

Generally, there are between 100 and 1,000 amino acids in a protein

Question 22

What part of an amino acid is flexible?

Answer 22

The alpha carbon is flexible

Question 23

What are 3 examples of secondary structure? What forces are responsible for secondary structure?

Answer 23

Alpha helices
Beta sheets
Loops

H-bonding between the amine and carboxyl groups in the backbone are responsible for secondary structure

Question 24

What kind of turn is in an alpha helix, how many amino acids per turn, is the C or N on top, and where do the side chains point?

Answer 24

An alpha helix has the C on top and is a right handed turn with 3.6 amino acids per turn and side chains pointing down and out from the helix

Question 25

Where are the H-bonds in a beta sheet, what are two types of ways a beta sheet can be organized, and where do the R groups point?

Answer 25

The H-bonds in a beta sheet form between the rows, it can be parallel or anti-parallel, and the R groups are normal to the surface of the sheet

Question 26

What are two kinds of loops and what are their internal structures like? Where do loops usually show up on a protein?

Answer 26

Turns: internal H-bonds made up of 3-5 amino acids Random Coil: very long loop, around 20 amino acids, and have no internal structure Loops tend to show up on the outside of a protein

Question 27

What are two examples of super secondary structure?

Answer 27

Coiled-Coil: 2 alpha helices wrapped around each other Helix-Turn-Helix: two alpha helices joined by a turn

Question 28

How can the tertiary structure of a protein be characterized?

Answer 28

Alpha, beta, or alpha beta depending on the presence of alpha helices or beta sheets

Question 29

What 3 types of interactions seal tertiary structure?

Answer 29

1) Salt bridges (amino acid side chains with stable charges forming ionic bonds) 2) Disulfide Bonds (proteins to be exported from cell) 3) Metal ions (stable cations like Zn+2 or Ca+2)

Question 30

What are 3 ways of destroying tertiary structure?

Answer 30

1) Changing temperature 2) Changing pH 3) Overcoming the hydrophobic effect (done by detergents which are amphipathic, or chaotropic agents which disrupt water and H-bonding)

Question 31

What are the general steps in the protein folding model?

Answer 31

``` Random coil Nucleation Hydrophobic Collapse Molten Globule Native Protein ```

Question 32

When protein folding goes wrong, the clumps are called _____ and when they are outside of a cell they are called _____ which are long _____.

Answer 32

The clumps are called amaloyd fibers and when they are outside of the cell, they are called amaloyd plaques which are long beta sheets

Question 33

Where does a small ligand bind to a protein?

Answer 33

The protein's binding pocket

Question 34

What are 4 things a bound ligand can do?

Answer 34

1) Cause a conformational change 2) Catalyze a reaction 3) Regulate protein function 4) Do nothing (just hold the ligand)

Question 35

What are the units of Kd, and what do high and low Kd values mean?

Answer 35

Kd is measured in "molar" and a low Kd means good binding (high protein affinity for ligand)

Question 36

What is Y (and Fo) and what are 2 equations for it?

Answer 36

Y and Fo are fraction occupancy, meaning the fraction of protein bound to ligand. ``` Y = [P:L] / [P] + [P:L] Y = [L] / Kd+[L] ```

Question 37

When is [L] = Kd?

Answer 37

[L] = Kd at 50% occupancy

Question 38

What are two ways of visualizing protein structure and roughly, how do they work?

Answer 38

X-ray crystallography: electron clouds interact with x-rays and must be in crystal form which might not be regular structure NMR (nuclear magnetic resonance): the way an atom responds to a magnetic field is effected by the atoms around it and this has hard math so best for small proteins

Question 39

What kind of cooperativity does hemoglobin display?

Answer 39

Hemoglobin displays positive cooperativity

Question 40

What are the two states hemoglobin, and which one lets hemoglobin have oxygen bound to it and which one does not? What part of hemoglobin moves to change states?

Answer 40

R (relaxed state) - oxygen can bind T (taught state) -oxygen cannot bind (stabilized by pH dependent salt bridges) The movement of residues at the alpha-beta interfaces account for these changes

Question 41

What doe the Bohr effect say about hemoglobin's oxygen affinity? How does this translate to oxygen loading and unloading in the body?

Answer 41

Hemoglobin's affinity for oxygen increases as pH increases. This means it has a higher affinity in basic conditions. In the capillaries of the body, the pH is acidic so oxygen is unloaded. In the lungs, the pH is more basic so it causes oxygen to bind to hemoglobin.

Question 42

What are the two models of allosterism, and how are they different?

Answer 42

Symmetry model: the allosteric protein has R and T states that are always in equilibrium and a ligand can bind to either one. R and T subunits are never there at the same time Sequential model: a ligand binds to the allosteric protein which causes a conformational change in that subunit as well as the other subunits

Question 43

What are antibodies made up of?

Answer 43

4 polypeptide chains: 2 heavy chains and 2 light chains with 2 identical binding sites

Question 44

How does SDS page work?

Answer 44

SDS denatures proteins and makes them negative so they can be separated just by molecular weight (not charge or shape) The polyacrylamide gel is a medium that has little holes that helps sort the proteins

Question 45

What is the best stain to use for gels?

Answer 45

Coomassie Blue

Question 46

How does a western blot work and what does it show?

Answer 46

A western blot separates proteins in a gel using SDS-PAGE and then proteins are transferred from the gel to nitrocellulose. The nitrocellulose is then incubated with a primary antibody and then a secondary antibody (that is linked to a detection mechanism). It very specifically shows your protein.

Question 47

What are the two ways you can express a protein, and what are some benefits and drawbacks of these methods?

Answer 47

Endogenous expression: express in the protein's native environment (likely get stable protein and my discover related proteins, may see any post translational modifications, not that much protein) Exogenous expression: use another organism to harvest protein (usually get a lot of protein and perhaps avoid interfering proteins)

Question 48

What are some methods to quantify a protein?

Answer 48

Absorbance at 280nm (tyrosine and tryptophan) Bradford assay with coomasie blue (red and positive with no protein, blue and negative with protein and then absorbance at 600 nm) Activity assay SDS-PAGE followed by coomassie blue staining SDS-PAGE followed by western blot (identifies presence of protein in impure samples)

Question 49

What are the three general protein isolation methods?

Answer 49

1) Salting out (ammonium sulfate) 2) Chromatography 3) Density gradient centrifugation

Question 50

What are the 4 types of chromatography?

Answer 50

Ion exchange chromatography (cation and anion sticking or not sticking to column) Hydrophobic interaction chromatography (load with low salt and elute with high salt) Affinity chromatography (relies on protein-ligan interaction) Gel filtration chromatography (separates based on size)