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BMB 401 > Exam 2 > Flashcards

Exam 2 Flashcards

1
Q

What are the three steps of protein sequencing?

A
  1. Break disulfide bonds
    2.fragment protein chains
  2. Use ESI or mass spec to determine sequence
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1
Q

What is the purpose of protein sequencing?

A

To determine the primary structure of a protein from N-C

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2
Q

How is a full fragment reconstructed?

A

By looking for overlap between the sequences of each fragment

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3
Q

What is an endopeptidase?

A

It is an enzyme that recognizes and cleaves certain amino acids

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4
Q

Do all endopeptidases have the same specificity?

A

NO!

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5
Q

What bond does the endopeptidase break?

A

The peptide bond in the backbone of the amino acid sequence

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6
Q

What does ESI do?

A

It allows the mass of the protein to be determined. Keeps the protein in tact, and the charges for each peak are different

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7
Q

How does charge change on an ESI chart?

A

Decreases to the right

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8
Q

What does tandem MS/MS do?

A

It breaks up the protein to identify the mass of each individual amino acid, charge stays the same for all peaks

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9
Q

What does MS1 do?

A

Isolates individual peptides

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10
Q

What does MS2 do?

A

Separates the fragments and measure m/z

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11
Q

What is the b-series?

A

N- terminus

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12
Q

What is the y-series?

A

C-terminus

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13
Q

How are the b and y peaks distinguished from each other?

A

By their charge!

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14
Q

Are all amino acids able to be determined using MS/MS?

A

No! some AA’s have identical or similar MW

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15
Q

What is primary structure?

A

Sequence of amino acids

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16
Q

What is secondary structure?

A

The local folding/ arrangement of the backbone atoms
ex. B sheets and a helices

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17
Q

What is tertiary structure?

A

3D structure of the chain, and is a combination of secondary structures

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18
Q

Why are proteins folded?

A

To minimize steric repulsions and maximize favorable non-covalent interactions

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19
Q

What are the properties of the peptide bond?

A

Has partial double bond character which restricts rotation of the bond. It is quite rigid/ planar and has a dipole moment

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20
Q

Which configuration of the peptide bond is more favorable?

A

The trans configuration, minimizes steric clash between R groups

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21
Q

Which amino acid favors the cis conformation?

A

Proline because the R group is covalently connected to the backbone

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22
Q

What is the psi angle?

A

The angle around the alpha carbon and the carbonyl carbon bond

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23
Q

What is the phi angle?

A

The angle around the alpha carbon and the amide nitrogen

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24
Q

What do favorable phi angles do?

A

minimize repulsion between the R group and the C=O (n-1 AA)

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25
Q

What does the psi angle do?

A

Minimize repulsion between the R group and the C=O

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26
Q

Why are some combinations of psi and phi angles unfavorable?

A

They cause steric crowding between atoms

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27
Q

Why are some combinations of psi and phi atoms more favorable?

A

They allow H-bonding interactions along the backbone

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28
Q

What is a Ramachandron plot?

A

It shows the POSSIble distribution of allowed psi and phi angle combinations

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29
Q

What characteristics do all regular secondary structures have?

A

Phi and psi angles, hydrogen bond patterns

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30
Q

What is an alpha helix?

A

A secondary structure that is right-handed and proceeds up and to the right. One turn is 3.6 residues.

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31
Q

What hydrogen bonds in an alpha helix?

A

The nth residue carbonyl oxygen to the N-H from the n+4 residue

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32
Q

How do side chains project in an alpha helix?

A

The project outwards and down

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33
Q

Is there a net dipole in an alpha helix?

A

Yes, goes from the N-terminus to the C-terminus

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34
Q

What is a helical wheel?

A

A diagram that shows the 3D arrangement of sidechains in an alpha helix. They often have complementary properties like ability to h-bond, or positive and negative charge

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35
Q

Is the core of an alpha helix tightly packed?

A

Yes! The backbone atoms are in contact via van der waals contact

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36
Q

What is a beta strand?

A

A stretch of polypeptide in an extended conformation

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37
Q

What is a beta sheet?

A

Formed from H-bonding between B strands

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38
Q

How are B sheets and A helices similar?

A

Both secondary structures and have H-bonding between backbone atoms

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39
Q

How are B sheets and A helices different?

A

B sheets are more extended and also have H-bonding between neighboring B strands

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40
Q

Which B sheet conformation is more stable and why?

A

Antiparallel is more stable because of the orientation of the atoms in the H-bond. They are in a straight line rather than angled.

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41
Q

What are strong helix formers?

A

Small hydrophobic residues like alanine

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42
Q

What amino acid is not favored in A helix or B sheet?

A

Proline because it cannot form H-bonds with other backbone atoms

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43
Q

Which amino acid is also not favored in a helix?

A

Glycine because it has large conformational flexibility and the incorporation limits disorder and is entropically unfavorable

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44
Q

What is tertiary structure in regards to secondary structure?

A

The combination of different secondary structures

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45
Q

What is a B turn?

A

It is the simplest turn in a polypeptide, and is comprised of 4 amino acids. Different types exist via the arrangement of atoms, and certain positions are enriched by certain amino acids

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46
Q

What is the difference between the Type 1 and Type 2 B turn?

A

The second amino acid in the turn is often a proline, which causes the peptide to kink. This kink is stabilized by H-bonding between N and N+4. The difference is the orientation of the carbonyl on the second amino acid. For Type 2, the oxygen crowds the beta carbon on the third amino acid. So to reduce this strain, the third amino acid is often glycine.

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47
Q

What are the two types of proteins?

A

Globular and Fibrous

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48
Q

What are the characteristics of a globular protein?

A

Typically round in shape and somewhat soluble in water

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49
Q

What are the characteristics of a fibrous proteins?

A

Extended secondary structure and tend to be insoluble in water

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50
Q

Are a majority of proteins globular or fibrous?

A

Globular

51
Q

What is an example of a fibrous protein?

A

collagen

52
Q

What are the characteristics of collagen?

A

It is made of three individual peptides that assume a lefthand helical conformation with 3 amino acids per turn

53
Q

What is the distinct amino acid composition of collagen?

A

repeating units of Gly-X-Y where:
-X is often proline
-Y is often a modified AA

54
Q

What is the collegan triple helix stabilized by?

A

H-bonds between the backbone atoms, H-bonds between different polypeptides, Pro C=O h-bond with N-H of Gly

55
Q

What stabilizes certain tertiary structures in globular proteins?

A

interactions between non-backbone atoms

56
Q

How does location of amino acids in globular proteins vary?

A

Polarity!

57
Q

What are motifs?

A

Specific combinations of secondary structure, also known as super-secondary structure

58
Q

What are domains?

A

A portion of a protein with a defined fold. They can fold independently and indicate clues about function. In domains, structure is more conserved than sequence

59
Q

Where do nonpolar residues lie?

A

mostly in the interior of the protein, and contributes to hydrophobic packing which is primary determinant of tertiary structure

60
Q

Where do polar residues lie?

A

usually located on the outside of the protein

61
Q

What are some examples of motifs?

A

B-A-B, B motif, A-loop-A, greek key

62
Q

What does it mean for a protein to be dynamic?

A

They have conformational stability, they exhibit breathing motions and local unfolding

63
Q

Which structure dictates tertiary structure?

A

primary

64
Q

What is the native state?

A

The correctly folded state of a protein and ensemble of low energy conformations

65
Q

What forces stabilize proteins?

A

Greatest influence- Hydrophobic effect
H-bonds, ionic interactions and dipoles, less role in stabilizing, greater role in determining native state structure

66
Q

How do we measure stabibility?

A

Delta G

67
Q

What are the three denaturing components?

A

Heat- can break non-covalent interactions
pH- alters ionization states, affects ionic bonds
Chemical- interfere with hydrophobic interactions and increase nonpolar solubility in water

68
Q

Whats a Chaotrope?

A

A salt that decreases the hydrophobic effect. It disorders the waters around the protein and denatures it. disrupts the secondary structure

69
Q

What is protein Tm?

A

Temp. at which 50% of the protein has become unfolded/

70
Q

What is a molten globule state?

A

with the secondary structure, hydrophobic amino acids form interior part of the protein, leads to loosely defined tertiary structures and well formed secondary structures

71
Q

What is the width of the free energy funnel?

A

Entropy

72
Q

What is the height of the free energy funnel?

A

Free energy difference, free energy decreases downwards

73
Q

What do the local minima represent in a free energy funnel?

A

immediate folded states

74
Q

What is a chaperone?

A

help proteins fold to their native structure. They bind to folding intermediates to prevent aggregation, provide a closed chamber, and catalyze disruption and reformation of peptide and disulfide bonds

75
Q

What is an amyloid?

A

extended oligomer beta sheet structure caused by aggregation of misfolded proteins

76
Q

What are the two requirements for amyloid formation?

A

native state with beta strands that unfolds into a molten globule state and aggregates

77
Q

What are the computational methods of modeling?

A

Homology modeling and de novo structure prediction

78
Q

What is homology modeling?

A

uses related protein, or protein w/ similar sequence to predict structure of new target

79
Q

What is de novo structure prediction?

A

purely based on amino acid characteristics ab initio prediction (works best for small proteins)

80
Q

What are the three ways of determining protein structure?

A

X-ray crystallography, NMR, and Cryo-EM

81
Q

What is X-ray crystallography?

A

provides atomic-level structural info, reconstruct image of electron density

82
Q

What is NMR?

A

NMR structures are often presented as a “sample of structures” that are consistent with the data, and is often used for proteins that fail to crystallize, or to confirm locations of residues that may be perturbed during crystallization

83
Q

What is Cryo-EM?

A

A beam of electrons is fired at a frozen protein solution, the emerging scattered electrons pass through a lens to create a magnified image on the detector, then a 2D imaged can be generated based on hundreds of these images

84
Q

What is myoglobin?

A

made of one polypeptide chain, has a high affinity for oxygen in muscle cells, no quaternary structure

85
Q

What is hemoglobin?

A

$ polypeptide chains, 2 subunits (2a, 2b), variable affinity for oxygen based on cooperativity, involved in oxygen transport

86
Q

What is the equation for the association constant?

A

Ka= [PL]/[P][L]. (m-1)

87
Q

what is the equation for the dissociation constant?

A

kd=[P][L]/[PL]. (M)

88
Q

How do the association and dissociation constants relate to affinity?

A

As affinity increases, Ka increases and Kd decreases

89
Q

What is Y?

A

The fractional saturation aka the amount of protein bound to ligand

Y= [PL]/ [P]+[L]

90
Q

What happens when Y=.5?

A

[L]= kd, aka kd is the concentration of ligand required to occupy 50% of binding sites

91
Q

How are the helices in in globins named?

A

Alphabetically, A is N-terminus, E an F sandwich heme, and C and D cap the heme

92
Q

What residues are conserved between myoglobin and hemoglobin?

A

His F8 which binds heme and His E7 which interacts with O2

93
Q

What are some challenges to O2 binding?

A

oxygen has low solubility in aqueous solutions, oxygen can bind to transition metals but generates free radicals, and does not bind to amino acid side chains reversibly

94
Q

What are the solutions to O2 binding?

A

Oxygen binding proteins use an Fe(II)-heme cofactor that is inside the protein and it can bind reversibly

95
Q

What about the heme group?

A

The Fe(II) is in its oxidated state and has one available covalent bond to oxygen, the heme cofactor is covalently bonded to His F8

96
Q

What is the Curve of Myoglobin binding?

A

It is a hyperbolic curve and is indicative of O2 binding independently with Mb

97
Q

How is hemoglobin stabilized?

A

noncovalent interactions between alpha and beta subunits

98
Q

What are the two forms hemoglobin can adopt?

A

oxy and deoxy, a conformational change occurs based on binding of oxygen and affects the affinity of ligands

99
Q

What is the requirement for cooperativity?

A

a protein must
have multiple binding sites for the same ligand

100
Q

what is positive cooperativity?

A

Binding the first ligand
increases the affinity at the other ligand binding
sites

101
Q

What is negative cooperativity?

A

Binding the first ligand
decreases the affinity at other ligand binding
sites.

102
Q

What is allostery?

A

ligand binding at one site affects binding at another site, can be any ligand site

103
Q

what is homotropic allostery?

A

allostery observed
between two of the same ligand ( cooperaivity)

104
Q

What shape is the hemoglobin binding curve?

A

sigmoidal, which indicates cooperativity

105
Q

What is the hill equation?

A

An equation that describes the sigmoidal binding curve

106
Q

What is n?

A

The hill coefficient which represents the degree of cooperatively, no cooperativity n=1, greater than 1 indicates cooperativity, is the slope of the binding curve

107
Q

How do we estimate affinity from a Hill plot?

A

Lower asymptote, low binding affinity (int. binding)
upper asymptote, higher affinity state (sub. binding)

108
Q

What is the hemoglobin conformational change as O2 binds?

A

Hb binds to O2, there is a conformational change, and this increases the affinity for additional binding

109
Q

What Hb form is associated with the oxy state?

A

flattened

110
Q

What Hb form is associated with the deoxy state?

A

domed

111
Q

What interactions does the C-terminus particpate in?

A

Backbone and sidechain interactions

112
Q

When the c-terminus interactions are lost, what state does the hemoglobin shift to?

A

Relaxed (oxy) state, the c-terminal his of the beta chain falls into the central cavity reducing space

113
Q

What is the bohr effect?

A

the impact of pH on
hemoglobin affinity for oxygen

114
Q

What does higher pH do?

A

promotes hemoglobin’s binding for
oxygen, R state (increases affinity)

115
Q

What does lower pH do?

A

promotes hemoglobin releasing
oxygen, T state (decreases affinity)

116
Q

What is heterotropic allostery?

A

Binding of other molecules can also affect Hb’s
affinity for oxygen

117
Q

What is the shape of an antibody?

A

Y shaped

118
Q

What is the quaternary structure?

A

E2upsidedown y2

119
Q

What are the chains in an antibody linked by?

A

disulfide bonds
heavy to heavy and heavy to light?

120
Q

What structures are different in antibodies?

A

primary and quaternary ( they have different functions)

121
Q

what structures are the same in antibodies?

A

secondary and tertiary

122
Q

What is the constant region?

A

AA sequence that is the same in all antibodies of the same organism

123
Q

What is the variable region?

A

AA region differs based on antigen

124
Q

How do antigens bind?

A

via noncovalent interactions in the hypervariable loop

125
Q

What is an epitope?

A

The antibody binding sites on an antigen. One antigen can have multiple epitopes

BMB 401 (3 decks)

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