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BIOC4331: Biochemistry I > Quiz 4 (Lectures 10-12) > Flashcards

Quiz 4 (Lectures 10-12) Flashcards

1
Q

Name the three components to protein purification.

A
  1. Solubilization (Making the protein soluble)
  2. Separation (Separating from other proteins)
  3. Protein detection
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2
Q

How are high yields of protein obtained?

A

Special cell lines (such as E. coli) are engineered to express the protein of interest.

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3
Q

What is one disadvantage of engineered cells used for overexpression?

A

They may not be capable of the required post-translational modifications essential to its function that are usually added under normal expression conditions.

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4
Q

What is the first step to solubilizing a protein?

A

Breaking open the cells containing the protein (cell lysis)

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5
Q

Cell lysis results in a ______.

A

cell lysate/crude extract

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6
Q

Name the two common ways to break open cells and explain them.

A
  1. Osmotic shock: If the cells don’t have a cell wall, they can be broken open by placing the cells in a hypotonic solution (one that has less dissolved salt than inside the cells). Water flows into the cells, and the increased pressure causes them to burst.
  2. Mechanical: Sonication (ultrasonic vibration) or French press (pushing cells using high pressure through a small hole) will break open the cells through mechanical stress.
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7
Q

Name and explain four factors in stabilizing proteins.

A
  1. pH: Proteins are easily denatured by changes in pH, so buffer are used to keep proteins in solution at a physiological pH.
  2. Temperature: Proteins are normally stored in a fridge or a cold room. They can also be frozen for long-term storage. For some proteins, glycerol needs to be added before freezing to prevent ice crystal damage.
  3. Inhibition of protein degradation: After cell lysis, proteases in the lysate can destroy the protein of interest. Protease inhibitors can be added to protect the protein.
  4. Detergents: Hydrophobic proteins need to be kept soluble through the addition of detergents.
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8
Q

What is the first step in protein separation?

A

Centrifugation

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9
Q

If a protein is in the soluble fraction, what happens when you centrifuge the mixture?

A

The insoluble cell debris can be centrifued to give an insoluble pellet, leaving the protein of interest in the supernatant.

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10
Q

If the protein of interest is in the subcellular fraction in the pellet, then it can be solubilized by what two things?

A

Concentrated salts (soluble protein) or detergents/organic solvents (membrane protein)

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11
Q

If the protein of interest is found in a particular subcellular fraction or organelle, this can be separated through what?

A

Differential centrifugation: Lysate is centrifuged at a speed in which denser material than the organelle of interest pellets out. The remaining lysate is decanted off, and then spun again at a speed that brings down the organelle of interest.

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12
Q

Describe isopycnic centrifugation.

A

The solution in the centrifuge tube varies in density (less dense at the top). Layering of different concentrations of solutions produces the gradient.

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13
Q

Describe the process of concentrating a protein.

A
  1. Force the protein to lose solubility (precipitate).
  2. The precipitated protein can be centifuged into an insoluble pellet.
  3. The buffer is removed.
  4. The protein is redissolved in a new buffer.
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14
Q

Name some common ways to precipitate proteins.

A
  1. Addition of highly concentrated salts (balances out charges on protein)
  2. Precipitation with strong acids
  3. Precipitation by organic solvents (acetone, ethanol)

(Disrupt interactions of proteins with water. May also denature the protein, so their functional activity may not be retained.)

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15
Q

Define salting in.

A

A technique used to keep proteins in solution

In order to dissolve, solution ions are needed to shield the charges on the proteins by acting as counter ions to these charges.

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16
Q

Define salting out.

A

A technique used to purify and concentrate proteins

Ammonium sulfate is commonly used to precipitate proteins from a large dilute volume (ammonium sulfate cut).

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17
Q

What is used in dialysis?

A

A semipermeable membrane that only allows small molecules to pass

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18
Q

Describe the process of dialysis to remove salt.

A

A dialysis bag is placed in a large volume of buffer. The salt ions will move out of the bag to equilibrate the salt concentration inside and outside the bag.

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19
Q

How is a dialysis membrane used in protein separation?

A

A dialysis membrane that separates the top of a centrifuge tube from the bottom can be used with centrifugation to allow smaller proteins to pass through the membrane, retaining the larger ones above the membrane.

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20
Q

Define chromatography.

A

A technique that separates/fractionates molecules based on physiochemical properties (chemical, mechanical, size, shape, etc.)

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21
Q

In chromatography, the mixture containing the protein of interest must be in solution. What is this called?

A

Mobile phase

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22
Q

How is the fractionation of the mixture accomplished in chromatography?

A

The solution is moved through a column of packed, porous, solid material (stationary phase).

Different components have unique mobilities through the column, and each elutes at different times from the bottom of the column (different retention times).

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23
Q

Theoretically, the longer the chromatography column, the better the separation. Why isn’t this the case?

A

Diffusional processes counter this by causing the expansion of the protein band on the column.

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24
Q

Describe ion exchange chromatography.

A

Ions that are electrostatically bound to a chemically inert column material of the opposite charge are exchanged for charged protein ions in solution.

Through a use of differing conditions, the protein of interest can be bound to the column and eluted off by adding other similarly charged ions that compete for binding to the column.

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25
Q

What are anion exchangers?

A

Column materials that are positively charged, so anions bind to them

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26
Q

What are cation exchangers?

A

Column materials that are negatively charged, so cations bind to them

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27
Q

Proteins are polyelectrolytes. What does that mean?

A

They have both positive and negative charges.

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28
Q

Cation exchange is best for ____ proteins, and anion exchange is best for ____ proteins.

A

basic; acidic

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29
Q

Proteins with ____ affinity for the ion exchanger move through the column faster than those with ____ affinity.

A

less; more

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30
Q

Gel filtration chromatography is also known as…

A

size exclusion chromatography.

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31
Q

The stationary phase in gel filtration chromatography consists of what?

A

Beads made of a hydrated spongy material with pores (gel) that span a narrow range of size

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32
Q

Explain the concept of gel filtration chromatography.

A

Large proteins won’t be able to enter the bead pores, so they’ll have to go through the void volume/the volume between the beads. They will travel through and elute first.

Very small proteins will be able to enter the beads through all the pores. They’ll have to travel through the void volume and the volume inside the beads, so they’ll elute last.

Proteins separate by size.

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33
Q

What is the advantage of affinity chromatography?

A

The other separation techniques are time-consuming, and material is often lost or destroyed along the way.

Affinity chromatography provides a stationary phase to which only the protein of interest will bind and can be reversibly eluted. This enables a one-step purification.

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34
Q

What are affinity tags in affinity chromatography? What’s the most common tag?

A

They can be added to the protein of interest, usually at the N or C-terminus, without affecting function.

The most common tag is a string of six His residues added to either terminus.

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35
Q

What’s the stationary phase in affinity chromatography?

A

A resin with Ni2+ bound (Nickel affinity)

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36
Q

What is used to compete off the protein and elute it from the column in affinity chromatography?

A

A solution of imidazole

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37
Q

In protein detection, as the protein is eluted off a column, it is collected as _____ by a _____.

A

fractions; fraction collector

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38
Q

Proteins are collected as fractions that generally __ mL.

A

1

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39
Q

How are fractions that contain proteins identified?

A

The UV absorbance of proteins is tracked.

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40
Q

Aromatic amino acid residues absorb wavelengths of ____ nm.

A

< 300

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41
Q

At 280 nm absorbance contributions are primarily from ___ and ___ residues.

A

Trp and Tyr

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42
Q

The peptide bonds making up the peptide backbone absorb strongly at ___ nm.

A

214

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43
Q

What is the absorbance formula?

A

Absorbance,A = log(Intensity of incident light, I0/Intensity of transmitted light, I)

44
Q

What is the Beer-Lambert Law? Define the variables.

A

A = εcl

A = Measured absorbance
ε = Molar extinction coefficient
c = Concentration (M)
l = Path length of sample in cell (cm)

45
Q

How do you calculate ε at 280 nm for a given protein?

A

ε = (#Trp x εTrp) + (#Tyrs x εTyr)

46
Q

What is the goal of protein purification?

A

Increase the purity of the protein at each step, losing as little of the protein of interest.

Total protein in your sample goes down throughout the procedure.

47
Q

What’s electrophoresis?

A

As proteins are charged, they can be separated by their differential mirgration in an electrical field.

48
Q

Why electrophoresis not used for preparative work?

A

It often has an adverse effect on the protein structure.

49
Q

Protein electrophoresis is carried out in gels made out of what?

A

Cross-linked polyacrylamide

This is known as polyacrylamide gel electrophoresis (PAGE).

50
Q

What function does the gel serve in electrophoresis?

A

The gel acts as a molecular sieve, slowing the migration of proteins based on their charge-to-mass ratio and their shape.

51
Q

The most common electrophretic method makes use of what?

A

The detergent sodium dodecyl sulfate (SDS)

52
Q

What does SDS do to a protein?

A

It denatures it.

~1 SDS molecule binds for each pair of amino acids. This large negative charge makes the intrinsic charge of the protein insignificant.

This makes the protein into a rod-like shape.

53
Q

What are the cleavage points of trypsin?

A

Lys, Arg

54
Q

What are the cleavage points of chymotrypsin?

A

Phe, Trp, Tyr

55
Q

What is the cleavage point of cyanogen bromide?

A

Met

56
Q

What does mass spectrometry do?

A

Accurately measures mass-to-charge (m/z) ratios for ions in the gas phase, including proteins

57
Q

What are the two common techniques for getting biomolecules into the gas phase without destroying them?

A
  1. Matrix-assisted laser desorption/ionization (MALDI)
  2. Electrospray ionization (ESI)
58
Q

How does MALDI work?

A
  1. Crystalize the protein with a low molecular weight organic compound (matrix) on a target.
  2. When the matrix is excited by a laser, it ejects the protein into the gas phase (+1 charge).
  3. The charged protein is subjected to an electric field.
  4. Its time of flight (TOF) to the detector is a function of its mass and charge.
59
Q

How does ESI work?

A
  1. Spray a solution of the protein in a volatile solvent through a small capillary tube maintained at a high voltage.
  2. This gives highly charged droplets of protein solution. The solvent quickly evaporates.
  3. This leads to highly charged protein ions in the gas phase (+0.5 to +2 charges per kDa)

(More accurate than MALDI. Mass average can be obtained from multiple m/z ionic species.)

60
Q

Explain how protein identity is confirmed by MS/MS.

A
  1. Peptides generated by proteolysis are injected into an ESI-MS instrument with a second MS instrument.
  2. One peptide can be isolated and sent into a collision cell filled with helium.
  3. Collisions with the gas cause fragmentation of the peptide.
  4. The fragments travel into the second MS spectrometer, and m/z is determined.
  5. The difference between the peaks represents the mass of an amino acid.
61
Q

What’s a ligand?

A

A molecule that binds reversibly to a protein at a specific binding site

62
Q

How do ligands bind to the binding site?

A

Through the formation of many non-covalent interactions

There’s an energy cost in removing bound water molecules from the binding site and the ligand (desolvation).

63
Q

Proteins have a high _____ for a _____ number of ligands.

A

specificity; small

64
Q

Binding is _____ dependent.

A

concentration

(High concentration = More likely to collide)

65
Q

What is induced fit?

A

When proteins undergo some conformational change upon binding to optimize interactions

66
Q

What is the equation for the equilibrium constant for the release of ligand?

(Dissociation constant, Kd)

A

Kd = ([P][L])/[PL]

67
Q

The smaller the Kd, the ____ the affinity of the protein for the ligand.

A

greater

68
Q

Define θ and give the two equations.

A

The fraction of binding sites occupied by ligand

θ = [PL]/([PL]+[P])

θ = [L]/([L]+Kd)

69
Q

A plot θ against [L] gives a ______ curve.

A

hyperbolic

70
Q

What happens to the fraction of binding sites occupied as [L] increases?

A

It approaches saturation asymptotically (curve flattens off).

71
Q

Kd is the [L] at which __% of the binding sites are occupied.

A

50

72
Q

Explain the evolution of hemoglobin.

A
  1. Two billion years ago, the atmosphere changed from reducing to oxidizing.
  2. The electron-deficient properties of O2 were harnessed to increase the efficiency and diversity of oxidative metabolism.
  3. Large multicellular animals needed an oxygen transport molecule to deliver oxygen to tissues deep within the organism.
  4. An iron-containing organometallic molecule known as heme evolved with the globin protein fold to achieve this.
73
Q

O2 binds _____ to the Fe2+.

A

reversibly

74
Q

Under what conditions does O2 bind to Fe2+?

A
  1. The 4 electron-donating N ligands from the porpyrin stablize the Fe2+ over the Fe3+.
  2. To prevent the reaction of 2 free hemes with O2 (would lead to the irreversible oxidation to Fe3+), the heme is hidden in a globin protein. One of the 2 remaining ligand sites is occupied by a His, stabilizing Fe2+ and leaving only one binding site for O2.
75
Q

The globin fold is composed of mostly ______.

A

a-helices

76
Q

In modern higher vertebrates, hemoglobin is a ______.

A

α2β2 heterotetramer

77
Q

What causes sickle-cell disease?

A

A single point mutation on the beta chain of Glu to Val

Both copies of the gene must be mutated (homozygous).

78
Q

How does the sickle cell mutation alter the beta chain?

A

It puts a hydrophobic residue on the surface of the Hb.

79
Q

How does a red blood cell (with sickle cell disease) get the sickle shape?

A
  1. The HbS Val fits into a pocket of another deoxygenated Hb.
  2. The deoxyHbS polymerizes into fibers.
  3. These fibers stiffen and deforms them into the sickle shape.
  4. Deformation of red blood cells occurs at tissues following release of O2.
80
Q

The hemoglobin content in people with sickle-cell disease is about __% of that of normal individuals. Why?

A

50

Sickle cells are very fragile and rupture easily.

81
Q

In heterozygotes with one sickle gene, they only display phenotypic symptoms under what condition?

A

Severe hypoxia (Lack of O2)

82
Q

In heterozygotes with one sickle gene, only about __% of their cells sickle.

A

1

83
Q

A single copy of the sickle gene offers what benefit?

A

Resistance to lethal forms of malaria

84
Q

What’s myoglobin? Where is it mainly located, and what’s its function?

A

A single chain globin

It’s most abundant in muscle and acts to aid O2 transport through tissue. It also serves as an O2 storage in diving mammals.

85
Q

What’s the equation for the fractional binding to myoglobin?

A

θ = pO2/(pO2 + P50)

pO2 = Partial presure of O2
P50 = Partial pressure of O2 at which half the available ligand sites are occupied

86
Q

Hb is composed of __ subunits.

A

4 (α2β2)

87
Q

Hb can bind up to __ O2 molecules.

A

4

88
Q

Hb can undergo a conformational change between an __ state and a __ state. What changes between these states?

A

R; T

Extensive changes occur at the αβ interfaces. Some of the ion pairs/salt bridges that stabilize the T state are broken, and a few new ones are formed in the R state.

From the T state, Hisβ – Aspβ and Lysα–β are lost. Hisβ rotates into the center of the molecule, leading to the loss of both ion pairs.

89
Q

Which Hb state has a higher affinity for O2?

A

The R state

90
Q

O2 binding in the heme ____ ____ stablizes the R state.

A

distal pocket

91
Q

When O2 is absent, the __ state is more stable.

A

T

92
Q

Describe the proximal and distal positions in heme.

A

Proximal: Porphyrin ring is slightly puckered. Iron is pulled towards the proximal His ligand.

Distal: Porphyrin becomes planar, pulling the iron and proximal His into the plane of the porphyrin.

93
Q

Hb can transition from a low O2 affinity state (T) to a high affinity state (R). Why is this important?

A

Hb must bind O2 efficiently at the lungs and release it at the tissues.

94
Q

The transition from the R to the T state is positively cooperative. What does this mean?

A

As each O2 binds, the affinity for O2 in the remaining unbound subunits increases.

95
Q

Proteins are allosteric if they have what?

A

Multiple binding sites

96
Q

Define homotrophic and heterotrophic.

A

Homotrophic: A protein has binding sites for the same ligand

Heterotrophic: A protein has binding sites for different ligands

97
Q

What happens when a ligand binds to the allosteric site?

A

It causes a conformational change that alters the affinity of binding at the second site.

98
Q

What’s negative cooperativity?

A

The binding affinity at the second site is decreased by the ligand binding at the first site.

99
Q

When θ is plotted against pO2, what are the curve shapes for cooperative binding and non-cooperative binding?

A

Cooperative: Sigmoidal curve
Non-cooperative: Hyperbolic curve

100
Q

What is the equation for Kd in the case of multiple protein binding sites that are cooperative?

A

Kd = ([P][L]^n) / [PLn]

101
Q

What is the equation for fractional binding in the case of multiple protein binding sites that are cooperative?

A

θ = [L]^n / ([L]^n + Kd)

102
Q

What is the Hill equation used for?

A

The relative saturation; fraction of receptor bound to ligand and not bound to ligand

[L]^n / Kd

103
Q

What do you get when you take the log of both sides of the Hill equation?

A

nHlog[L] - logKd

Slope = nH (Hill coefficient)
X-intercept = (log Kd) / nH
Y-intercept = -log Kd

104
Q

nH is a measure of what?

A

The degree of cooperativity

105
Q
  1. nH = 1, ___ cooperativity
  2. nH > 1, ___ cooperativity
  3. nH < 1, ___ cooperativity
A
  1. no
  2. positive
  3. negative

BIOC4331: Biochemistry I (9 decks)

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