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Biochem > Unit 2 > Flashcards

Unit 2 Flashcards

1
Q

What is a prosthetic group?

A

Permanently associated chemical component in a protein that is NOT an amino acid (could be lipids, metallic ions, sugars, etc)

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2
Q

Cofactors

A

Additional chemical component, needed by an enzyme for the enzyme’s activity, not necessarily permanently attached

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3
Q

Coenzyme

A

A specific type of cofactor which is a complex organic or metal organic molecule

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4
Q

Define conjugated protein

A

Protein that contains permanently associated chemical components in addition to amino acids (prosthetic groups).

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5
Q

List the several types of conjugated proteins and modifications or prosthetic groups found in these conjugated proteins

A

“lets go print his f* money”
- Lipoproteins
- Glycoproteins
- Phosphoproteins
- Hemoproteins
- Flavoproteins
- Metalloproteins

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6
Q

What is the prosthetic group for lipoproteins?

A

Lipids

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7
Q

What is the prosthetic group for Glycoproteins?

A

Carbohydrates

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8
Q

What is the prosthetic group for Phosphoproteins?

A

Phosphate groups

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9
Q

What is the prosthetic group for Hemoproteins?

A

Heme group

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10
Q

What is the prosthetic group for Flavoprotein

A

Flavin nucleotides

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11
Q

What is the prosthetic group for Metalloproteins?

A
  • Iron
  • ZInc
  • Calcium
  • Molybdenum
  • Copper
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12
Q

Primary structure

A

A description of all covalent bonds (mainly peptide bonds and disulfide bonds) linking amino acid residues in a polypeptide chain

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13
Q

Secondary Structure

A

Secondary structure of a protein consists of regularly repeating conformations of the polypeptide backbone such as alpha-helices and Beta pleated sheets. These always involve hydrogen bonds

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14
Q

Tertiary Structure

A

Completely folded 3D, biologically active (or native) conformation of a single polypeptide protein

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15
Q

Quaternary Structure

A

When a protein has multiple polypeptide subunits, their arrangements is quartenary

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16
Q

Point out and discuss the planar nature of the peptide bond. Identify the atoms found within the peptide plan formed by a peptide bond

A

6 atoms in a pane
Both the C-alpha (two of them) , C, O, N, and Hydrogen are included in one plane

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17
Q

Can the peptide bond rotate? Why or why not?

A

Only the C-alpha – C and the N – C-alpha bond can rotate. The C-N bond cannot rotate due to the partial double bond character on the nitrogen (this is seen in the resonance structure).

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18
Q

Within a peptide bond, is the hydrogen of the amino group usually trans or cis to the relative oxygen of the carbonyl.

A

It is usually trans

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19
Q

Is the alpha-helix left or right handed?

A

Right-handed

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20
Q

Explain the arrangement of the side chains on an alpha-helix relative to the main axis of the helix

A

The hydrophobic side chains are all on the inner (or opposite) side of the alpha helix. Since the alpha helix has hydrogen bonds every three to 4 amino acids, that positions the hydrophilic side chains on one side of the structure while the hydrophobic side chains are on the opposite side of the structure.

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21
Q

Is there a hole in the middle of the alpha helix?

A

No, it gives the impression because the balls to not represent the van Der Waals radii of individual atoms

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22
Q

Name the type of bonds that stabilize the alpha helix. Specifically, what residues are connected through these non-covalent bonds.

A

Hydrogen bonds stabilize the alpha helix. The bond is between the nitrogen atom’s hydrogen and the electronegative carbonyl oxygen atom of the 4th amino acid on the amino terminal side of that peptide bond

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23
Q

At the ends of an alpha-helican segment… there are always…

A

Three or four amine carboxyl or amino groups that cannot participate in this helical pattern of hydrogen bonding. These may be exposed to the other surrounding solvent, where they hydrogen-bond with water, or other parts of the protein may cap the helix to provide the needed hydrogen-bonding partners

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24
Q

How many amino acids are present in a turn of the alpha helix? How long is a single turn?

A

3.6 residues, a single turn is 5.4 Angstrom long

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25
Q

Is the alpha helical structure rigid and rodlike?

A

Yes

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26
Q

An alpha helix is built from one continuous region of a polypeptide. Is this also true for a Beta sheet?

A

No, Beta sheets must be arrangment of several Beta-strands side by side

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27
Q

Is the peptide bond in a Beta sheet planar?

A

Yes, it’s the same peptide bond

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28
Q

Name and discuss the bonds that stabilize the Beta sheets?

A

Hydrogen bonds form between the backbone atoms of adjacent segments of polypeptide chain within the sheet

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29
Q

Where is the position of hydrogen bonds in parallel vs antiparallel sheets ?

A

The hydrogen bonds for antiparallel sheets are more linear but parallel sheets are making bonds on an angle so they are a tiny bit weaker

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30
Q

Note the arrangement of the side chains relative to the plane of the sheet (for beta sheets)

A

The side chains alternate between pointing up at you and being behind the polypeptide. All the hydrophobic residues will be on the same side while the hydrophilic residues will be sticking out of the opposite side.

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31
Q

Compare parallel to anti-parallel sheets

A
  • The length of the repeat period is shorter in the parallel sheets. The parallel sheets are 6.5 Angstrom while the antiparallel sheets are 7 Angstrom
  • Additionally, the hydrogen bonds in anti-parallel sheets are more linear than they are in parallel sheets.
  • Parallel means all the sheets are pointing in the same direction while antiparallel is the opposite
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32
Q

How can you tell the direction a polypeptide chain is going in if you were given if on an exam?

A

Follow the N –> C-alpha –> C direction. This way you can determine which is parallel and antiparallel

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33
Q

Loops and Beta Turns

A
  • Beta-turns are the turns needed to connect antiparallel Beta strands. The structure is a 180º turn involving 4 amino acid residues with the carbonyl oxygen of the first residue forming a hydrogen bond with the 4th residue (the central two aren’t involved)
  • Loops and turns do not need to have the same hydrogen bonding pattern because they can form hydrogen bonds with water which is why they are found on the exterior
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34
Q

What does a loop connect?

A

A loop connects either two alpha-helices or an alpha helix and beta strand

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35
Q

What do B-turns connect?

A

Beta strands (antiparallel)

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36
Q

What is the Beta-alpha-Beta unit?

A

A Beta strand separated from a parallel Beta strand by an alpha helix

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37
Q

How do you answer super-secondary problems?

A
  • FIrst, determine the direction the polypeptides are moving in (draw arrows at the top)
  • If they are antiparallel, you can just connect them with turns
  • If they are parallel, you must use alpha helices to get too the N terminus
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38
Q

Distinguish between a domain and a subunit

A
  • A domain is a part of a polypeptide chain that is independently stable or could undergo movements as a single entity with respect to the entire protein (tertiary structure )
  • A subunit is a polypeptide chain. You can have multiple subunits make up a protein
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39
Q

Does a domain speak to the function of a protein?

A

Yes

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40
Q

How can you tell if you have a domain or if you have a subunit?

A

If you denatured a protein and you got separate pieces, you would know you have subunits that make up the quaternary structure. However, if you denatured the protein and still had a single polypeptide (tertiary structure) this is how you know you had a domain (because it appeared to look like different subunits since they are independently stable)

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41
Q

Are side chain hydrogen bonds important in the stabilization of tertiary structure?

A

True

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42
Q

Are the hydrogen-bonding modes of the ionizable residues pH-dependent?

A

Yes, because at certain pH’s, certain residues will lose their hydrogen, meaning they can no longer be hydrogen bond donors and can only be acceptors

43
Q

What other types of interactions are involved in the stabilization of the native tertiary structure besides hydrogen bonds?

A
  • Disulfide bonds
  • Hydrophobic Effect
  • Ionic Interactions
44
Q

Where are the hydrophobic and hydrophilic side chains located in a myoglobin molecule?

A

Hydrophobic residues are positioned towards the inner protein while hydrophilic residues face the outside (so they can interact with water)

45
Q

Where are the loops on myoglobin locate ? Why does this make sense?

A

The loops are located on the surface of the protein. The reason why is because the loops can make hydrogen bonds with the solvent (water) to stabilize themselves since they aren’t part of the alpha helix hydrogen bonding pattern.

46
Q

Discuss the evidence that the amino acid sequence of ribonuclease specifies its three dimensional structure.

A

When ribonuclease is placed in concentrated urea solution in the presence of reducing agents (Beta-mercaptoethanol), the protein denatures due to the reducing agent cleaving the 4 disulfide bonds to yield 8 cysteine residues. The urea disrupts the hydrophobic effect and hydrogen bonds. However, when the urea and Beta-mercaptoethanol were removed, the denatured ribonuclease spontaneously refolded correctly and restored its catalytic acitvity. This shows that all a protein needs to fold is its primary sequence.

47
Q

What does urea disrupt?

A

The hydrophobic interactions and hydrogen bonding

48
Q

What does Beta-mercaptoethanol disrupt?

A

Disulfide linkages. It broke 4 of them

49
Q

Can the disulfide bonds in ribonuclease be readily reduced by Beta-mercaptoethanol?

A

No, you need the protein to already be partly unfolded by agents like urea or guanidine hydrochloride.

50
Q

Describe the conformation of ribonuclease that has been treated with urea and Beta-mercaptoethanol. What is the meaning of teh phrase “to denature a protein”?

A
  • After treatment, the ribonuclease is in a denatured state
  • A loss of 3D structure sufficient to cause a loss of function is called denaturation. The denatured state doesn’t always equate with complete unfolding of the protein and randomization of conformation
51
Q

How can the denatured and reduced ribonuclease be converted to its native form?

A

If the urea and mercaptoethanol are removed

52
Q

Why did the Anfinsen experiment prove that the primary sequence specifies the native structure?

A

Because one the agents were removed from the ribonuclease, the polypeptide was able to refold again without the help of any other cell component, showing that it didn’t need them

53
Q

Discuss the role of molecular chaperones and chaperonins in protein folding

A

Not all proteins fold spontaneously as they are synthesized in the cell. Folding for many proteins requires chaperones, proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways, or providing microenvironments in which folding can occur.

54
Q

Most proteins that contain disulfide bonds are

A

Extracellular

55
Q

Discuss the role of PDI

A

PDI is an enzyme that catalyzes the interchange or shuffling of disulfide bonds until the bonds of teh native conformation are formed. Among its functions, PDI catalyzes the elimination of folding intermediates with inappropriate disulfide cross links

*The last sentence essentially means that PDI breaks disulfide bonds that are improperly placed on the polypeptide to give it another chance to make teh disulfide bond correctly

56
Q

Discuss the role of PPI

A

PPI catalyzes the interconversion of the cis and trans isomers of peptide bonds formed by Proline residues. For B-turns, the Proline has to be in the cis conformation, but for the Proline to react with other things (which is what it normally needs to do), it needs to be in the trans state.

57
Q

Crude extract

A

The first step in any protein purification procedure is to break open these cells, releasing their proteins in to a solution called a crude extract

58
Q

Differential centrifugation

A

The process of taking a homogenized tissue, centrifuging it at a speed so teh heaviest things can sink such as cells, nuclei, cytoskeleton etc. Then taking a supernatant of that from the top and centrifuging it again so you can get protein.

59
Q

Sucrose density centrifugation

A

Layering a sample on top of a gradient of sucrose solutions with varying concentrations. As the sample is centrifuged, the particles move through the gradient until they reach a sucrose concentration matching their density

60
Q

Fractionation

A

Once crude extract is ready, it is subject to treatments that separate the protein into different fractions based on a property such as size or charge. Early fractionation steps utilize difference in protein stability, which is a complex function of pH, temp, salt concentration and other factors

61
Q

Dialysis

A

A procedure that separates proteins from small solutes by taking advantage of the protein’s larger size. This is usually done when you have a solution containing the protein of interest.

62
Q

Ion Exchange Columns

A

You charge the resin so you can grab a specific protein.
- Anion exchangers: positively charged resin will bind to anions so you will be able to separate protein
- Cation exchangers: negatively charged resin will be cation

To get the positively charged anions off, you have to elute. Either:
1) Flush the column with salt –> the salt will interact with the protein and outcompete the protein for binding to the resin
2) Change the pH of the solution –> changing the pH could change the charge of the protein which would help it fall off

63
Q

Size Exclusion Column

A

Resin is made up of beads that have micropores.
- For larger proteins going through this resin, they cannot fit into the mini holes so they will leave the column extremely fast
- Smaller ones will be able to travel through the micropores meaning it would take them a while to leave

64
Q

Affinity Column

A

Resin is covalently bonded to the ligand that will interact with the protein of choice

To elute:
Flush the column with free ligand so it will outcompete the ligand that is bound to the resin, bind tighter to the protein, and leave

65
Q

On what basis are proteins separated on for Ion Column Chromatagrophy?

A
  • Charge
66
Q

What is the overall charge on a protein when pH<pI, pH=pI, pH>pI

A

pH<pI charge is +1
pH=pI charge is 0
pH>pI charge is -1

67
Q

Absorbance of UV light at 280nm is often used as a crude measure of protein concentration. Why do protein solutions absorb light at 280 nm?

A

Because most proteins have aromatic rings - tryptophan, tyrosine, (a bit of phenylalanine),. Tryptophan and tyrosine absorb UV light at 280 nm.. so the protein will if it has these aromatic groups

68
Q

For a size-exclusion chromatography, what are the axis and what do they mean?

A

Y - absorbance (280 nm) proves there’s a certain amount of protein
X- Fraction number. Fraction 1 is the first liquid to emerge and be put in a tube

69
Q

If Protein A has a lower fraction number than Protein B, which one is larger?

A

Protein A because it didn’t go through the microbeads since it was too large. As a result, it eluted first

70
Q

Describe the separation of proteins by SDS page

A

1) Loading buffer is added to protein containing SDS to break interactions and denature protein, glycerol to make solution more dense so it sinks to the bottom, and a charged dye so you can see the gel
2) Add the proteins to the gel with the ladder - proteins of known mass
3) The electric difference between the negative and positive sides of the gel will direct migration. Protein is negatively charged due to the SDS
4) Smaller polypeptides move quicker and travel farther than larger proteins
5) Proteins can be visualized after electrophoresis by treating the gel with a stain to bind to protein - not teh gel itself

71
Q

What are the polar and nonpolar parts of the SDS?

A
  • Has a polar sulfate group and a nonpolar chain
    *just remember it is an amphipathic molecule
    *draw
72
Q

Just remember this”

A

Mixtures of SDS and protein are heated to denature the protein. When cooled, the SDS preserves the denatured form by hydrophobic interactions forming an elongated micelle around the extended peptide backbone

73
Q

What is the approximate ratio of SDS to the number of amino acids? Describe how the charge on the complex compares with the original charge on the protein?

A
  • A protein will have 1 molecule of SDS for every amino acid residue
  • This means that the overall charge of the protein will be negative since SDS contributes so much charge. The original charge of the protein is essentially useless
74
Q

In the presence of SDS, what property of the protein determines the rate of migration?

A

Mass

75
Q

Will small protein molecules move faster or slower than larger ones on SDS?

A

Small protein molecules will move faster on SDS because they can navigate through the gel webs easier

76
Q

How does an SDS gel determine purity of a protein and its subunit composition

A
  • Each band on a gel represents a different protein or protein subunit
  • If there is one bad in a lane, that is how you know you purified your sample well and have a pure sample
  • If you know that you have a pure substance but still see bands, that signifies that there are multiple subunits –> this is because adding SDS can break down the bonds resulting in the separation of multipe polypeptides.subunits if they were originally in a quartenary structure
77
Q

Be able to determine the molecular weight of an unknown protein using the ladder. Why is panel B (the log graph) used to determine a more exact molecular weight?

A

Panel B is able to get linear data for the relative migration vs log of molecular weight. This allows for the molecular weight of the unknown sample to be read more easily. They plotted the ladder with the sample.

78
Q

Activity

A

Total units of enzymes in solution

one unit of enzyme activity is the AMOUNT of enzyme that converts a certain amount of substrate into product per unit of time

79
Q

Specific Activity

A

Units enzyme/ milligram total protein

*this should increase during purification because we are getting rid of other protein

80
Q

What are the ideal results of protein purification?

A

The most effective purification procedures will result in a large increase in specific activity and minimal loss of total activity

81
Q

Mass Spectrometry

A
  • Macromolecules in solution are forced from liquid phase to gas phase
  • A solution of analytes is passed through a charged needle that is kept at a high electrical potential, dispersing the solution into a fire mist of charged microdroplets
  • The solvent surrounding the macromolecules evaporates leaving positively charged molecules to hit the vacuum interface
  • The dispersion of molecules hitting the vacuum interface is analyzed to find the m/z ratio
82
Q

What is mass spec used to do?

A
  • Measure protein’s molecular weight
  • Determine primary structure
  • Protein identification
  • Identification of post-translational modifications to a protein
83
Q

What is an antibody?

A

A defense protein synthesized by the immune system of vertebrates and circulated in the blood

84
Q

Polyclonal antibody

A

Are produced by many different B lymphocytes responding to one antigen. This, polyclonal antibodies recognize different epitopes or parts of a protein

85
Q

Monoclonal antibodies

A

Synthesized by a population of identical B cels grown in cell culture

86
Q

Difference between activity and specific activity

A

In summary, enzyme activity quantifies the overall catalytic performance of an enzyme sample, while specific activity provides a normalized measure of enzyme activity relative to the protein content. Both parameters are important for characterizing and comparing enzyme preparations in biochemical research and industrial applications.

87
Q

What is the purpose of a Western, or immunoblot assay?

A

To reveal/detect protein in a solution or its location in a gel. Western blotting has the sensitivity to detect a specific protein

88
Q

How is immunoblot prepared? (by itself)

A
  • Proteins are first separated by size in a SDS gel. Detecting a specific protein with SDS is usually not possible bc it solely discriminates on size
  • Proteins are transferred from SDS gel to polymer (nitrocellulose) sheet through electrophoresis. The polymer is positively charged so the negatively charged proteins move from the SDS to the sheet
  • Proteins stick to the sheet in the same position they had on the gel
  • Once your sheet is coated with sample, add nonspecific protein to fill in gap to prevent the antibody binding to teh polymer sheet
  • Then add a primary antibody that is specifically made for the protein you are trying to find
  • Then add a secondary antibody that has a colored product/enzyme or fluorescence linked to it.
  • For add a substrate to bind to the secondary antibody so it can release colored product to show you exactly where the protein was on the SDS gel.
89
Q

Explain why only a single band is visible in each lane of the immunoblot while several are visible in the SDS gel

A

With SDS, you have the detection of all proteins so it might not exactly be the protein of your choice unless its been repeatedly purified until you have one bad

With Immunoblot, even though all the proteins are carried over to the nitrocellulose sheet, the only one that appears (have color) will be the ones that had antibody attached to it. Therefore, it should only have 1 bond

90
Q

What is determined using X-ray crystallography?

A

The spacing of atoms in a crystal lattice (the 3D arrangement of atoms, or molecules can be determined

91
Q

What are the steps of X-ray crystallography?

A

1) X-ray hits the crystal of the protein and the x-ray diffraction patterns are generated from
2) Data extracted from the diffraction patterns are used to calculate a 3D electron density map
3) Regions of greatest electron density reveal the location of the atomic nuclei, and this information is used to piece together the final structure
4) you have your final structure

92
Q

What does NMR stand for?

A

Nuclear magnetic resonance

93
Q

What is 3D NMR used to determine?

A

Protein structure and dynamic characteristics such as folding and interactions

94
Q

List two advantages of using NMR over X-ray crystallography

A
  • NMR is carried out on molecules in solution, whereas X-ray crystallography is limited to molecules that can be crystallized
  • NMR can also illuminate the dynamic side of protein structure including conformational changes, protein folding, and interaction with other molecules. X-ray crystallography can only identify the crystal structure of a protein (and identify
95
Q

What does crystallization of proteins refer to?

A

The process of inducing proteins to form highly ordered, 3D arrangements of molecules known as crystals. When proteins are studied in solution, they are in their natural state

96
Q

What can single-particle Cryo-EM be used to do?

A

Determine the 3D strucutres of large macromolecular complexes

97
Q

Describe how Cryo-EM works?

A
  • A sample containing many individual copies of the structure of interest is quick frozen in noncrystalline ice and it kept frozen while being observed in 2D with electron microscope, greatly reducing damage to teh specimen by electron beams
  • Using the different angles of the molecules (since you have multiple copies they are not oriented in the same way) , you can construct teh 3D structure of teh macromolecule
98
Q

Can mass spec determine protein strucutre?

A

No, it determines primary structure

99
Q

Which is more damaging to the sample, X-ray crystallography or Cryo-EM?

A

Both of their main purpose is to determine protein structure; however, X-ray crystallography’s beams can be more damaging to the sample

100
Q

List potential advantages of this approach over other structural determination techniques

A
  • Cryo-EM is less damaging to the sample than X-ray crystallography is
  • Cryo-EM allows you to actually visualize your proteins since you are seeing it
  • Cryo-EM doesn’t take much work to prepare, relatively easy compared to the others.
101
Q

What are the ways to purify sample?

A
  • Ion exchange chromatography
  • Size exclusion chromatography
  • Affinity chromatography
102
Q

What are ways to detect you’ve purified your sample?

A
  • SDS
  • Immunoblot
103
Q

What are ways to analyze your sample? identification?

A
  • Mass spec (tells you primary strucutre)
  • X-ray crystallography
  • NMR
  • Cryo-EM
104
Q

What are ways to determine protein structure?

A
  • NMR
  • X-ray crystallography
  • Cryo-EM

Biochem (15 decks)

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