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Biochemistry > 4. Protein > Flashcards

4. Protein Flashcards

1
Q

Explain protein variability (2)

A
  1. Simple - non-conjugates
  2. Complex - require non-amino acid cofactors/prosthetic groups for full activity
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2
Q

Explain cofactors (3)

A
  1. Can be inorganic - metal or phosphate
  2. Can be organic (coenzymes) - sugar, heme, flavin, lipid
  3. Covalently or non-covalently attached to protein
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3
Q

What are the ways proteins can be purified? (4)

A
  1. size
  2. charge
  3. solubility
  4. affinity
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4
Q

Explain size exclusion/gel sieving chromatography (6)

A
  1. Small beads of polymerized glucose, agarose or acrylamide
  2. Beads are manufactured with different pore sizes by crosslinking the polymers differently
  3. The pore containing beads are packed into a cylinder
  4. Protein mixture is applied to the top of the column
  5. Big proteins do not enter the porous beads - run through quickly
  6. Small proteins enter and exit the beads - run through the column slowly
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5
Q

Explain affinity chromatography (5)

A
  1. Uses a ligand - a molecule that is specifically bound by a protein
    - EX) ATP to hexokinase
  2. Ligands are attached to polymer beads and packed into a column
  3. A protein mixture is applied to the column
  4. Hexokinase binds to the ligand, all other proteins are washed out of the column
  5. ATP is added to the column where it competes for binding sites causing pure protein to unbind and elute from the column
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6
Q

Explain SDS paging (8)

A
  1. Separates proteins by electrophoresis to estimate protein masses
  2. Gel is a cross-linked polyacrylamide gel moolecular sieve
  3. The detergent SDS binds to the proteins and makes them highly negatively charged
    - 1SDS per 2AA
  4. SDS-coated proteins move through the gel by electrophoresis when an electrical potential is applied
  5. Small proteins move quickly and easily through the pores
  6. Big proteins move slowly through the pores
  7. After electrophoresis, the proteins are visualized by staining with Coomassie blue or silver
  8. The migration distance is proportional to the log10. The mass of an unknown protein can be determined by interpolation, using migration distances of proteins of known molecular weight
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7
Q

Explain what is found in each of the protein structures
1. Primary
2. Secondary
3. Tertiary
4. Quaternary

A
  1. Amino acid sequence
  2. Backbone conformation
  3. 3D polypeptide conformation
  4. Association of polypeptides
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8
Q

What determines the primary structure?

A

Amino acid analysis

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9
Q

Describe amino acid analysis (6)

A
  1. It does not determine the order of amino acids
  2. Hydrolyze (bond cleavage by hydrolysis) all peptide bonds in a pure protein using 6M HCl @ 110 degrees for 24 hours
  3. Seperate amino acids by ion exchange chromatography
  4. Quantify amino acids by reacting with Ninhydrin to produce a purple colour
  5. Measure the absorption of light at 540nm
  6. Use Beer’s Law to determine the concentration of each amino acid
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10
Q

Describe protein sequencing (4)

A
  1. Cleave the polypeptide into shorter peptides using proteolytic enzymes or chemicals and separate them
    - Chymotrypsin - cleaves after aromatic amino acids at C-end
    - Trypsin - cleaves after lys or arg at C-end
    - Cyanogen bromide - cleaves after met at C-end
  2. Uses edman degradation:
    - PITC (reagent) attaches to the first amino acid (N-terminus amino acid)
    - High ph levels breaks the bond between the first and second amino acid
    - The new PITC-amino acid is called the PTH-amino acid, can be determined using light absorbance
    - Once its identified, the PITC repeats the process with the new first amino acid that was just cleaved
  3. This is repeated up to 50 times
  4. The protein sequence is determined by analyzing pieces of the protein that overlap with each other (from enzymes)
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11
Q

How else can amino acids be determined by?

A
  1. Mass spectrometry
  2. Analyzing the gene sequence
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12
Q

Notes on protein sequences (6)

A
  1. The linear polypeptide (inactive form) contains the information to direct protein folding into a 3d conformation (active form)
  2. Amino acids sometimes contain signal sequences used for export, location, modifications, half-life
  3. Incorrect amino acid incorporation can lead to a loss of alteration of protein activity or folding (disease)
  4. 30% of human proteins are polymorphic - multiple variations but doesn’t really affect function
  5. Proteins that have the same function in difference species usually have similar sequences
  6. The greater the evolution history difference between species, the greater the amino acid differences in their protein
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13
Q

Explain disulfide bonds in terms of amino acids (3)

A
  1. Oxidization of 2 cysteines give cystine
  2. Bonds may be intrapolypeptide (aa + same polypeptide) or interpolypeptide (aa + a diff polypeptide)
  3. Hold distant parts of a polypeptide close together
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14
Q

Explain the secondary protein structure (3)

A
  1. Biologically active proteins are folded into well-defined 3D conformation
  2. Unfolding denaturation of a protein eliminates activity
  3. Conformation can be described by torsion angles about single bonds
  4. Backbone torsion angles: phi Φ, psy Ψ and omega Ω
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15
Q

Explain the significant of phi Φ, psy Ψ and omega Ω in torsion angles. Which one is fixed and why?

A

phi (Φ) - between amino acid and alpha c
psi Ψ - alpha-c and carbonyl
omega Ω - carbonyl and another amino acid group

Omega is fixed at 180 because the bond has a partial double bond due the delocalization of the N-lone pair electrons - the bond is strong and stable because of shared electrons, making it act like a straight line instead of allowing movement.

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16
Q

Explain phy Φ and psi Ψ and their involvement with steric interactions (2)

A
  1. It limits the amount of conformations possible. if they are too close together it causes hindrance or straining which affects function
  2. Uses the ramachandran to show the allowed combinations between the 2
17
Q

Explain the alpha helix (6)

A
  1. Phy = psi = -60 (right, CW), 60 (left, CCW)
  2. Inner rail = backbone, steps = amino acids
  3. Held together by hydrorgen bonds from C=O and N-H
  4. Most are right handed
  5. Each turn has 3.6 amino acids
  6. A d-amino acid will not fit into a right hand helix because of steric interactions - the shape and arrangement of atoms in the D-amino acid make it too bulky or awkward to fit properly into the tight spiral of the helix.
18
Q

Explain the certain combination of amino acids that from helices (5)

A
  1. Ala, met, glu favour the helix
  2. Pro puts bends in helices
    - Side chains clashes with the preceding C=O
    - It does not have an NH for H-bonding
  3. Clusters of Lys/Arg and Glu/Asp are not helical due to charge repulsion in helix
  4. Clusters of bulky amino acids like Ile and Trp are not helical for steric reasons
  5. Gly adds flexibility to the helix because of its small side chain
19
Q

Explain the significance of beta-strands (5)

A
  1. phi (Φ) - -120
    psy (Ψ) - +120
  2. Zigzag conformation
  3. Held together by hydrogen bonds
  4. They form beta sheets (2 beta strands running either parallel or antiparallel to eachother)
    - Antiparallel is stronger, have more energy and decrease free energy
20
Q

Which amino acids favour beta-sheets?

A

Val, ile, phe, tyr

21
Q

Explain the significance of turns (3)

A
  1. 4 amino acids tightly connected to 2 antiparallel b strands
  2. sharp 180 degree turns found at protein surface
  3. can also be loops that are important for binding metals and substrates
22
Q

What kinds of bonds stabilize secondary structure? (5)

A
  1. H-bonding
  2. VW
  3. Ionic interactions
  4. Disulfide bonds
  5. Metal binding
23
Q

Explain the tertiary protein structure (3)

A
  1. The arrangement of secondary structure form the 3D structure
  2. Dominated by long range side chain interactions
  3. X-ray diffraction is used to determine the 3D structures of crystallin protein
24
Q

Explain this photo in terms of tertiary protein structure (3)

A
  1. Superoxide dismutase determined by x-ray diffraction
  2. positive = blue and negative = red charges on protein surface
  3. active site = green (copper) - positive
  4. Substrate O2 and carries a negative charge and is attracted to the positively charged binding site
25
Q

Explain myoglobin and its relation to tertiary structure (4)

A
  1. Found in muscle and stores oxygen but needs a certain shape to fully function
  2. The heme cofactor (non-polar) is tucked in the hydrophobic pocket that is created from 8 alpha helicies
  3. Because of the tertiary structure, it allows iron thats needed in heme cofactor to sit directly in between the histidine residues
  4. Tertiary structure makes hydrophobic Interior, hydrophilic exterior which is important for solubility and ensures that heme attaches to oxygen and not water
26
Q

Explain cytochrome c and its relation to tertiary structure (3)

A
  1. Binds to heme and functions in electron transfer
  2. Mostly alpha helices and no beta strands
  3. Sequences of cytochrome c are different in different things but proteins fold into the same conformation
27
Q

Explain immunoglobulin and its relation to tertiary structure (2)

A
  1. Involved in vertebrate defence against foreign invaders
  2. Mostly beta sheets and some alpha
28
Q

Explain ribonuclease and its relation to tertiary structure (2)

A
  1. Secreted by the pancreas into the small intestine
  2. Hydrolyzes RNA
  3. Alpha and beta is even
29
Q

How can proteins with different sequences belong to the same structural family, and what leads to their functional differences? (2)

A
  1. Many different protein sequences fold into a similar conformation giving rise to a small number of structural families
    – Example: myoglobin and hemoglobin belong to the same family
  2. Different functions arise from subtle differences in conformation and/or critical amino acid
30
Q

What are 6 interactions that keeps the folded state of proteins

A
  1. hydrophobic effect
  2. hydrogen bonding
  3. electrostatic interactions
  4. van der waals interactions
  5. disulfide bonds
  6. binding or metal and other ligands
31
Q

What does it mean that proteins are “marginally stable,” and how does this stability affect their function? (6)

A
  1. Proteins are marginally stable:
    - G unfolding is ~20-70kJ/mol
  2. This is the difference in free energy between the folded and the unfolded protein
  3. Breaking 4-20 hydrogen bonds is enough to unfold a protein
  4. Proteins function via changes in conformation
  5. They are dynamic rather than static
  6. Their atoms are constantly in motion
32
Q

How does the disruption of structure lead to loss of biological activity? (6)

A
  1. Heat: break the weak bonds that hold a protein’s shape together.
  2. Cold: water molecules to become more ordered, making it harder for the protein to stay in its proper shape.
  3. Extreme pH: Too much acid or base can mess with the charges on a protein’s surface, causing parts of it to repel each other and unfold. - curdling of milk
  4. Mechanical Force: Even physical force can denature proteins. Beating egg whites is a perfect example of mechanical denaturation.
  5. Chemicals: Certain chemicals, like urea, can disrupt the forces that hold a protein together
  6. Detergents: interacting with the oily parts of proteins, preventing them from folding properly.
33
Q

Explain protein denaturation (2)

A
  1. Some proteins will re-fold following denaturation
    - Others will precipitate from solution
  2. In vivo, some proteins require molecule chaperones to prevent aggregation and increase the efficiency of folding
34
Q

Explain the significance of quaternary structure (1)

A

For biological activity many proteins require two or more polypeptide chains

35
Q

Explain the significance of keratin (6)

A
  1. Found in hair, feathers and nails
  2. A tough, insoluble material
  3. Forms the outer layer of human skin
  4. Two right-handed alpha helical chains form a left hand supercoiled rope
  5. The coils organize into supramolecular structures called protofilaments to protofibrils to microfibrils
    - Strength by cross-linking and disulfide bonds
36
Q

Explain the significance of collagen (4)

A
  1. Found in tendons and bone matrix and has a high tensile
    strength
  2. (Gly-Xxx-Pro) is repeated over and over again
  3. phi (Φ) - -60
    psy (Ψ) - +160
  4. Collagen helix - Three staggered polypeptides form
    a supercoiled right-handed triple helix called tropocollage
37
Q

Explain silk fibroins (1)

A
  1. Stacked antiparallel b-sheets rich in glycine and alanine that permit close packing of the sheets

Biochemistry (7 decks)

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