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Bioc 450 Midterm > Dr. Nagar (80%) > Flashcards

Dr. Nagar (80%) Flashcards

1
Q

Why Study Proteins?

A

1) The most abundant macromolecules
- 50% of the cell’s dry mass
- No. of proteins around 4 times the number of their coding genes
- up to 15,000 different proteins in one cell

2) Functionally diverse
- Catalysis of metabolic processes
- Energy transfer
- Gene expression
- Transport of solutes across membranes
- Cellular communication
- Molecular recognition
- Defense
- Forming intracellular & extracellular structures

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2
Q

Protein Structure Hierarchy

A

1) Primary protein structure
-> Linear amino acid sequence of the polypeptide chain including PTM’s and disulfide bonds.

2) Secondary protein structure.
-> Local structure of linear segments of the polypeptide backbone atoms without regard to the conformation of the side chains - regular repeated structures; a-helix, b-strand, b-turns. Motifs: associations of secondary structural elements, e.g., B-a-B.

3) Tertiary protein structure.
-> The three-dimensional arrangement of all atoms in a single polypeptide chain. Overall folding involves interaction of distant parts of the chain. The domain is the fundamental unit of tertiary structure.

4) Quaternary protein structure.
-> The arrangement of separate polypeptide chains (subunits) into the functional protein.

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3
Q

Ionization state vs pH of amino acid

A
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4
Q

What is the amino acid with no side chain?

A

Glycine, Gly, G

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5
Q

What are the Nonpolar amino acids?

A

1) Alanine, Ala, A
2) Valine, Val, V
3) Leucine, Leu, L
4) Isoleucine, IIe, I
5) Proline, Pro, P
6) Methionine, Met, M

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6
Q

What are the Polar-uncharged amino acids?

A

1) Serine, Ser, S
2) Threonine, Thr, T
3) Cysteine, Cys, C
4) Asparagine, Asn, N
5) Glutamine, Gln, Q

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7
Q

What are the electrically charged amino acids?

A

1) Glutamate, Glu, E
2) Aspartate, Asp, D
3) Lysine, Lys, K
4) Arginine, Arg, R
5) Histidine, His, H

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8
Q

What are the aromatic amino acids?

A

1) Phenylalanine, Phe, F
2) Tyrosine, Tyr, Y
3) Tryptophan, Trp, W

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9
Q

Alanine (Ala, A) Structure

A
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10
Q

Valine (Val, V) Structure

A
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11
Q

Leucine (Leu, L) Structure

A
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12
Q

Isoleucine (IIe, I) Structure

A
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13
Q

Methionine (Met,M) Structure

A
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14
Q

Serine (Ser,S) Structure

A
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15
Q

Threonine (Thr,T) Structure

A
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16
Q

Cysteine (Cys, C) Structure

A
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17
Q

Asparagine (Asn, N)

A
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18
Q

Glutamine (Gln, Q) Structure

A
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19
Q

Lysine (Lys, K) Structure

A

Basic

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20
Q

Arginine (Arg, R) Structure

A

Basic

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21
Q

Histidine (His, H)

A

Basic

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22
Q

Aspartate (Asp, D) Structure

A

Acidic

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23
Q

Glutamate (Glu, E)

A

Acidic

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24
Q

Phenylalanine (Phe, F) Structure

A
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25
Q

Tyrosine (Tyr, Y) Structure

A
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26
Q

Tryptophan (Trp, W) Structure

A
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27
Q

Spectroscopic properties of aromatic amino acids

A

1) UV abs:
- Phe, λ max 256 nm
- Tyr, λ max 275 nm
- Trp, λ max 280 nm

2) Fluorescence
- Phe, λ max 282 nm
- Tyr, λ max 303 nm
- Trp, λ max 348 nm

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28
Q

Glycine (Gly, G) Structure

A
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29
Q

Proline (Pro, P) Structure

A
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30
Q

Draw out the side chain atom nomenclature for Lysine (Lys, K), Leucine (Leu, L) and Isoleucine (IIe, I) B-branched.

A
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31
Q

Peptide bond formation

A
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32
Q

Partial double bond character

A
  • arises because of resonance between the lone pair on the nitrogen and the carbonyl group in the peptide bond. This resonance leads to partial delocalization of electrons, given the C-N bond a character that is intermediate between a single and double bond.
  • around 20 kcal/mol barrier is the energy required for the peptide bond to switch between the cis and trans conformations. This barrier is relatively high, making the trans confirmation more stable and more commonly found in proteins.
  • C-N bond length: the bond length is about 1.47 Å. However, in a peptide bond, due to the resonance, the C-N bond is shorter, around 1.32 Å, reflecting its partial double bond character.
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33
Q

Cis vs trans peptide bond

A

-> peptide bonds are overwhelmingly trans,
- cis peptide bonds result in Ca-Ca repulsion.

THE EXCEPTION: cis Xaa-proline

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34
Q

What amino acid can form disulfide bonds?

A
  • Cysteines oxidized to cystine (a dimer of two cysteines)
  • Inter- and intra-chain S-S bonds possible.
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35
Q

What is the average MW of amino acid?

A

115 daltons

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36
Q

What is pKa of an acid?

A

It is the pH at which it is half-dissociated.

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37
Q

List the amino acids with ionizable side chains and their corresponding pkA’s?

A

1) Aspartic Acid = 3.9
2) Glutamic acid = 4.3
3) Histidine = 6.0
4) Cysteine = 8.3
5) Tyrosine = 10.1
6) Lysine = 10.5
7) Arginine = 12.5

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38
Q

Draw the resonance form of the Arg side chain and its corresponding pKa.

A
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39
Q

Draw the resonance form of Tyr side chain and its corresponding pKa.

A
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40
Q

Backbone pKa’s of free amino acids

A
  • In a polypeptide backbone
  • Amine pKa around 8
  • COOH pKa around 3
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41
Q

Are amides protonated?

A
  • No
  • Amide has delocalized electrons and partial double bond character
  • Same for internal amide bonds in a polypeptide
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42
Q

What are factors that affect pKa?

A

1) Inductive effect
- Through bond interaction
2) Charge effect
- Through space interaction
- NH3+ stabilizes COO-

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43
Q

Why does Aspartate have a low pkA?

A
  • partly buried Asp, accepts around 4 hydrogen bonds and near positively charged residues (charged form is ‘happy’, i.e. stabilized)
  • it prefers to remain de-protonated, so its pKa decreases from 4 to around 2 (i.e. harder to add a proton to it)
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44
Q

Why does Glutamate have a high pKa?

A
  • Partly buried Glu forms no favourable interactions with other residues (charged from ‘unhappy’, i.e. unstable
  • it prefers to be in its neutral protonated form, so its pKa shift up from 4.4. to around 6.
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45
Q

What is the Isoelectric point?

A
  • pH at which the molecule has no net charge
  • pI = (Pk1 + pK2)/2
  • Basis for purification techniques such as: isoelectric focusing, ion-exchange chromatography.
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46
Q

Draw a Titration Curve graph

A
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47
Q

Post-translational modifications (PTMs)

A
  • proteins undergo chemical reactions: hydrolysis, ester formation, amide formation, oxidation-reduction, nucleophilic attack, etc.
  • change of physical chemical properties = glycosylation increases stability and water solubility.
  • regulation of activity = hormone-induced phosphorylation turns activity of many enzymes on and off.
  • cellular trafficking = acylation serves as membrane anchor
  • regulation of half-life = ubiquitinylation tags proteins for proteolysis.
  • proteolytic cleavage, glycosylation, disulfide bond formation, hydroxylation, phosphorylation, acetylation, methylation, ubiquitination + many more.
48
Q

Proteolytic cleavage

A
  • some proteins/polypeptides are activated by snipping off part of the chain (proproteins)
    -> Proenzymes to enzymes, e.g. Cathepsin A
49
Q

Glycosylation

A
  • Most common form of PTM (but rare in bacteria) -> Takes place in Golgi, ER and extracellular surfaces.
  • O-glycosylation (Ser, Thr, hydroxy-lysine)
  • N-glycosylation (Asn, Asn-X-Ser, Asn-X-Thr- motif) X cannot be Pro, Asp
  • Common monomer units: galacyose, mannose, glucose, gucose, N-acetylglucosamine, N-acetylgalactosamine, sialic acids, xylose.
  • cell surface full of glycoproteins and proteoglycans, long thought to be only on extracellular side, now known to be also present in the nucleus and cytosol, intercellular recognition.
50
Q

Hydroxylation

A

1) Hydroxyproline, Hydroxylysine

2) Collagen:
- structure protein: bone, tendons, skin, ligaments, blood vessels
- (Gly-X-Y)n, one-third of X and Y are Pro
- Many of the Pro and Lys in Y position are hydroxylated, hydroxyPro stabilizes triple helical structure of collagen.
- Hydroxylysine may be glycosylated.
- Vitamin C deficiency inactivates hydroxylase (results in skurvy)

51
Q

Disulfide bond formation

A
  • Cys-Cys bond, intra-chain or interchain (cystine)
  • S-S can be converted back to S-H with DTT (dithiothreitol).
  • S-S bond formation rigidifies the overall structure, e.g., a-keratin cross-links in hair, fingernails
  • S-S bonds determine how curly a peron’s hair is (perms)
52
Q

Acylation

A
  • Fatty acid acylation alters physical and functional properties significantly
  • Effects include increasing affinity for membranes, stabilization of protein-protein interactions, inhibition of enzymatic activity
  • Acetylation of Lys in Histone proteins regulates gene transcription
  • Acylation is usually reversible
53
Q

Phosphorylation

A
  • Most abundant PTM in eukaryotes (1/3 of all protein are phosphorylated at a given time)
  • Occurs on Ser, Thr, Tyr (in prokaryotes - also His) but also on Arg, Lys, Asp/Glu and Cys
  • Mechanisms: conformational change, ligand binding, catalytic residues
  • Kinases phosphorylated (using ATP), phosphatases dephosphorylate
  • phosphorylation/dephosphorylation regulates protein activity (enzyme activation (including the kinases themselves), membrane transport, signalling networks, protein-protein interactions etc.
54
Q

Methylation

A
  • occurs at a-amino or Lys, Arg, His side chains
  • Example: Histone methyl transferases put a methyl group on Lysine residues in epigenetic control of gene expression (SAM donates the methyl group).
55
Q

Ubiquitination

A
  • Ubiquitin is a small protein tag
  • Lys48 (proteosomal degradation)
  • Lys63 (DNA repair, endocytosis, signalling)
56
Q

What forces determine a protein’s structure/shape?

A

I. Bonding forces (covalent)
1) Bond length
2) Bond angles
3) Dihedral (torsion) angles (determines protein fold)

II. Non-bonded forces (between non-covalent atoms)
1) Van der Waal forces
2) Ionic
3) Hydrogen bonds
4) Hydrophobic

57
Q

Non-covalent interactions

A
  • much weaker than covalent bonds, make biomolecules flexible, and allow them to interact reversibly with each other.
  • Drive the folding of the protein chain into a specific 3D structure.
  • Drive the binding of ligands (substrate, hormone, messenger, allosteric modulator) to a binding/catalytic site in the proteins.

1) Electrostatic
- charge-charge (ionic)
- charge-dipole
- dipole-dipole:
-> permanent dipoles (Keesom interactions)
—–> Hydrogen bond
-> Induced dipoles (vdW interactions)
——> Repulsive
——> Attractive (dispersions)

2) Nonpolar (hydrophobic effect)

58
Q

Electrostatic interactions (ionic)

A
  • Attractive interaction between oppositely charged groups (repulsive between like charges)
  • e.g. Arg (+) — Asp (-) at pH 7.0
  • e.g Arg (+) — Lys (+) leads to repulsion.
  • energy of interaction depends on Coulomb’s law.
  • Typical bond lengths between electronegative atoms range from 2.4 and 4.0 Å.
  • typical energy is 3kcal/mol (range from 1 to 5 kcal/mol)
59
Q

Why are some side chains of amino acids polar and some non-polar?

A

1) Polar-side chains:
- due to difference in electronegativity (O, N)
- due to difference in electron density (S)

2) Non-polar side-chains
- contain only C-H, C-C and C-S-C bonds

60
Q

Electrostatic interactions (types) & their relative strength

A

Charge-charge> dipole-dipole > nonpolar-nonpolar
Ionic interactions are long range
Dipolar and vander waals interactions are short range

61
Q

Electrostatic interactions (aromatic)

A
62
Q

Van der Waals Attraction (Disperson)

A
  • Interaction between induced dipoles (all atom types)
  • Strength generally increases with MW
  • highly distance-dependent (optimal only at short distances)
  • both attractive and repulsive
  • The van der Waals distance signifies the atomic boundary
63
Q

Hydrogen bonding

A
  • higher than expected boiling points of NH3, H20 and HF are due to intermolecular hydrogen bonding
  • hydrogen bonds are between a proton covalently bound to an electronegative atom with another electronegative atom
  • can be between the protein and water (inter) or within the protein (intra-critical for higher order protein structure)
  • typical distance: 2.6 to 3.2 Å
  • typical energy: 1-3 kcal/mol
  • like ionic interactions hydrogen bonds depend on q,r and ε
  • unlike ionic interactions hydrogen bonds are geometry-dependent
  • hydrogen bonds are weaker and more distance-dependent than ionic interactions (r3 instead of r)
64
Q

Side chain hydrogen bonding - which one is the hydrogen bond acceptor and which is the donor?

A
65
Q

Solvated Interaction Energy (Electrostatic Component) - Sodium Chloride Hydration

A
  • The energy involved when a solute (like sodium chloride) dissolves in a solvent (like water)
  • In sodium chloride (NaCl), Na+ and Cl- ions interact with the water molecules through electrostatic forces.
  • When NaCl dissolves in water, Na+ is attracted to the oxygen atoms of water (which are slightly negative) and Cl- is attracted to the hydrogen atoms of water (which are slightly positive)
  • this ion-dipole interaction stabilizes the ions in solution, contributing to the electrostatic part of the hydration energy.
66
Q

Hydrophobic effect

A
  • Nonpolar groups tend to associate in water: in aqueous environments, nonpolar (hydrophobic) molecules aggregate to minimize their contact with water.
  • Entropically driven: this aggregation is driven by an increase in entropy, as water molecules become more ordered around nonpolar solutes, leading to a decrease in the system’s free energy.
  • cost of water ordering: the presence of nonpolar molecules disrupts the hydrogen bonding network of water, causing water molecules to form a structured “cage” around the solute, which reduces their mobility and the overall entropy of the system.
67
Q

Dihedral angles (Torsion angles)

A
  • The dihedral angle between four atoms A-B-C-D is defined as the angle between the planes ABC (marked by red lines) and BDC (marked in purple)
68
Q

Dihedral angle projections

A
69
Q

Peptide mainchain torsion (dihedral) angles

A
  • three dihedral angles exist in the peptide backbone
  • dihedral angles determine protein structure and conformation
  • omega is restricted, the peptide bond possesses partial double bond character and is planar, however, interconversion can occur leading to a cis/trans isomerization.
70
Q

Phi/Psi angle (what is the difference)?

A

1) Phi - look along N-Ca bond
angles between carbonyls
positive is clockwise C’N to Calpha-C’

2) Psi - look along Calpha-C’ bond
angles between nitrogens
positive is clckwise NCalpha-C’N

Positive when read from N to C using backbone atoms with “cis” being a zero angle

  • Values of phi and psi angles determine the conformation of the protein
  • All values of phi/psi are not possible due to steric clash
  • Restriction of phi/psi it one reason why proteins can fold into defined structures
  • Ramachandran plot visualizes phi/psi distribution
71
Q

Ramachandran plot for alanine

A
  • Typical amino acid
  • Phi essentially limited to -45 to -170
  • Shown are the conformations of alanine in 30 high-resolution protein X-ray structures.
72
Q

Ramachandran plot for glycine

A
  • least sterically hindered amino acid
  • largest range of possible phi and psi angles
  • shown are the conformations of glycine residues in 30 high-resolution protein X-ray structures.
73
Q

What are the characteristics of right-handed alpha-helix?

A

—->
1) Around 30% of residues in globular proteins
2) 5-40 resides (10-25 average)
3) 3.6 residues per turn (5.4 Å pitch)
4) 1.5 Å rise per residue (compact)
5) Around 5 Å diameter

—–> Stabilization:
- nonpolar & vdW interactions
- hydrogen bonds - but weaker (exists also in the unfolded state)

—-> Electric properties:
- peptide unit has an intrinsic dipole moment
- all the peptide units point in the same direction
- results in significant net dipole for the helix as a whole and a +0.5 charge at N-term and -0.5 at C-term.

—-> Amphipathic helices:
- Polar/nonpolar residues face oppoite directions.
- Same-type residues appear every 3 or 4 positions.
- Generally found on protein surfaces.

74
Q

Helix capping

A
  • refers to stabilizing the ends of an alpha helix by providing hydrogen bond (H-bond) partners.
  • (Unsatisfied H-bonds) the first and last 4 H-bonds at the ends of the helix are not satisfied because there not enough partners to form bonds.
  • N-cap (N-terminus) and C-cap (C-terminus): polar residues often fill in to satisfy the missing H-bonds at the ends in 35-50% of cases.
  • The rest of the unsatisfied H-bonds are often satisfied by amino acids brought into place during protein folding or by water molecules.
  • there are strong preferences for specific amino acids at the ends of helices to stabilize them.
75
Q

Side chains preferences of helices

A
  • good helix formers: Ala, Glu, Leu, Met
  • poor helix formers: pro, gly, tyr
  • Pro is a good helix started but causes bends in the middle.
  • Ser, Asp, Asn compete with main-chain H bonds
  • B-branched sidechains (IIe, Thr, Val) destabilize helices
76
Q

What are some other types of helices and their characteristics?

A
77
Q

β-sheets

A
  • Less compact than a-helices
  • β-sheets are composed of several regions of polypeptide chains, usually in strands of 5-12 residues
  • Length is 3.3 Å per residue
  • 2.0 residues per ‘turn’
  • Peptide bonds of adjacent residues point in opposite directions
  • Alternating side chains point in opposite directions
  • way more common to be anti-parallel than parallel
  • β-sheets have a twist:
    -> most sheets observed in globular proteins are twisted (0 to 30 deg. per residue)
  • anti-parallel sheets are generally more twisted than parallel
  • twists is left-handed when viewed from the side
78
Q

Secondary structure conformations in the Ramachandran plot

A
79
Q

What side chains are good Beta sheet makers?

A
  • Tyr, Trp, Val, IIe, Phe, Thr
80
Q

What are ‘Loops’?

A
  • connect secondary structure elements
  • usually hydrophilic (polar residues, unsatisfied backbone H-bonds)
  • on the surface
  • often create binding/active sites of receptors and enzymes
  • loops are not periodic
  • insertions and deletions during evolution often occur in loops
81
Q

What are ‘Turns’?

A
  • Turns also connect helices and strands but are much shorter (around 4 residues)
  • Unlike loops, turns are structured and classified by their dihedral angles
  • 2nd position - often cis-Pro (creates a kink)
  • 4th position - Gly (accommodates the kink)
82
Q

What side chains have preference of turns?

A

Gly and Pro

83
Q

Side chain torsion angles. What is most common and what is rare?

A
84
Q

Motifs

A
  • Combination of a few secondary structure elements (helices, sheet and turns)
  • Sometimes motifs have specialized functions
  • Generally, a motif is not stable when isolated.
85
Q

What are some common motifs?

A
86
Q

Tertiary Structure

A
  • the folding of the polypeptide chain (or subunit) into its native state
  • most basic unit of protein tertiary structure is the ‘domain’
  • No distinction between domain and subunit (a subunit can consist of one or many domains)
  • a secondary structure is the a-helix or b-sheet
  • a tertiary structure is the b sheet alpha helix and domain which makes a protein subunit (monomer)
87
Q

Tertiary structure stability

A
  • proteins have stabilization energies ranging from -5 kcal/mol to -15 kcal/mol
  • a typical protein may have hundreds of h-bonds, salt-bridges and van der waals interactions each contributing between 0.2 - 3kcal/mol of stability (assume on average 1 kcal/mol x 100 interactions = 100kcal/mol)
87
Q

What forces give rise to tertiary structure?

A
  • Hydrogen bond
  • Ionic bond
  • Van der Waals interaction
  • Disulphide bond
88
Q

Domains

A
  • usually range 40-350 residues
  • are separate “parts” of a protein (but still in the same chain - part of tertiary structure)
  • they can be considered independent functional units of the protein
  • useful for predictions
  • modular domains make up protein kinases (domain shuffling to create new proteins)
89
Q

Tertiary structure core packing

A
  • residues in the protein core are tightly packed with complementary surfaces
  • hydrophobic residues are generally found in the interior of a protein (consequence of the hydrophobic effect)
  • hydrophilic residues are usually found on the exterior of a protein
  • the conformation of the secondary structure in the packed state is close to that in isolation
  • helices pack ridge into groove, eg. sidechains of adjacent helices interdigitate
  • helices pack onto sheets with axes nearly aligned, due to complementary twist
  • b-sheets pack aligned or orthogonal
90
Q

What are the different classifications of Proteins based on Structure?

A

1) Alpha
2) Beta
3) Alternating alpha/beta
4) Segregated alpha and beta
5) membrane proteins

91
Q

All alpha proteins:

A
  • helices produces many classes of proteins including coiled-coils, globins (transport), four-helix bundles, membrane proteins (signal transduction) and fibrous proteins (structural)
  • includes Myoglobin - the first protein structure every determined. Oxygen storage protein and it is found in muscles.

Coiled-coils:
- The Gcn4
-Leucine zipper
- helices intertwine around each other
- forms a left-handed supercoil
- required a heptad repeat where 4th residue is a leucine -> leucine zipper forms the hydrophobic core for the dimer

Four-helix bundle:
- helices pack according to the ridges in grooves model

92
Q

All beta proteins:

A
  • beta-barrels (Porin trimer in the membrane - extracellular view), beta-propeller (Neuraminidase - influenza virus)
  • includes enzymes, antibodies, receptors and virus coat proteins
  • predominately anti-parallel strands
  • antibodies:
    greek-key motifs B-Sandwich
    Ig-fold has variable loops
    Antigen phosphorylcholine
93
Q

Alpha/beta proteins

A
  • most frequent domain structure found
  • consists of a central parallel (or mixed) B-sheet surrounded by helices
  • includes all enzymes of glycolysis, metabolite transport proteins
  • 3 main classes: 1) TIM barrel (triose phosphate isomerase), 2) Rossman fold (phosphofructokinase) 3) horshoe fold (ribonuclease inhibitor)
94
Q

alpha + beta proteins

A
  • protein kinase domain
  • insulin receptor kinase
95
Q

Membrane proteins

A
  • membrane proteins do not flip flop - contributes to the asymmetry of the membrane
  • include channels
  • are often glycosylated on the extracellular side
  • involved in light-conversion reactions and ATP production
  • involved in signal transduction for neurotransmitters, growth factors and hormone receptors
  • cell cell adhesion and cell recognition

1) Bacteriorhodopsin
-> photosensitive pigment (also found in human eyes to capture light). Light energy used to pump protons across membrane (light converted to chemical energy), when oxygen is low or light is intense, BR synthesis is increased.

2) Protassium channel.

3) Single spanning TM receptors
-> No 3D structures yet available for complete receptors because they are large, flexible and difficult to crystallize.

96
Q

What are the two major classes of proteins?

A

1) Fibrous proteins - structural, simple sequences, simple in shape (cables), insoluble in water, e.g. coiled coils: keratins (hair, nails, wool), triple helix: collagen (connective tissue), Elastin (heart, lung), B-sheets: silk (spider webs)

2) Globular proteins - folded up into - spherical shapes, most are water soluble, compact, functional - eg. enzymes, transport proteins (hemoglobin), protective (antibodies), contractile (ATP -> mechanical energy), receptors, DNA/RNA binding, hormones

97
Q

Coiled-coil fibrous proteins

A
  • include myosin, fibrinogen, spectrin, keratin (intermediate filaments)
  • keratins are structural proteins of skin, hair, wool, horn, feathers
  • exhibit heptad repeats in the central helical regions (around 300 residues), eg. collagen
98
Q

What are the characteristics of collagen?

A
  • forms a triple helix
  • forms supramolecular structures in the extracellular space
  • collagen fibrils found in tendons, cartilage, basement membranes (most abundant protein in the body 33%)
  • each chain forms a poly-pro type II helix
  • less compact left-handed helix (3.3 residues/turn, 9.6 A rise/turn -> around 1000 residues and around 3000 A length)
  • 3 left-handed helices trimerize to form a right-handed superhelix
  • collagen is a trimer with repeat sequence: Gly-X-Y where X is usually Pro and Y is usually hydroxyPro
  • HydroxyPro formed by PTM of Pro
  • Gly must repeat at 3rd position which ends up at centre of superhelix (all other sidechains are on the outside)
  • Hydrogen bonding stabilizes the collagen triple helix
99
Q

Quaternary Structure

A
  • Quaternary structure is the spatial arrangements of subunits (ie. each polypeptide chain) and the nature of their interactions - mainly driven by hydrophobic effect
  • monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, decamer, dodecamer, viral - often arranged symmetrically
  • molecular machines are often multimers, eg. proteosome = 4 heptameric rings (28-mer), ribosome (protein + RNA)
  • also have multiprotein complexes (more than one protein comes together to form a stable complex via protein-protein interactions)
  • roles of quaternary structure:
    Control or regulation - allostery or cooperativity
    Stability
    Partial active sites - eg. 2 subunits are required for full active site
100
Q

Physiological role of quaternary structure in hemoglobin

A
  • Hemoglobin is made up of 4 subunits that work together to transport oxygen efficiently (quaternary structure)
  • Lower pH (Bohr effect), when CO2 is dissolved in blood (increased acidity)

-> hemoglobin’s affinity for oxygen decreases, this helps release oxygen to tissues where its needed most.

-> CO binds strongly to hemoglobin (CO poisoning), prevents oxygen from binding. Disrupts the quaternary structure’s normal function, reducing oxygen delivery to tissues.

101
Q

What is the different symmetry in protein quaternary structure?

A

1) Cyclic symmetry
- designated Cn has N identical units related by a single N-fold rotational axis. That is, successive rotations of a single unit through (360/N) result in positions of the other units. Therefore, cyclic symmetries of N=2 to infinity are permitted.

2) Dihedral symmetry
- designated Dn has 2xN identical units related by a single N-fold rotational axis and N 2-fold rotational axes.

3) Helical symmetry
- the units in a helical symmetry are related by a screw axis (a translation and rotation operation)

102
Q

Image of cyclic symmetry

A
103
Q

Image of dihedral symmetry

A
104
Q

How would you study protein structure?

A
  • Determine the structure and how it contributes to function
  • Understand how proteins fold
  • how do proteins bind their substrates
  • what is their mechanism of action
  • what is their function within a cellular context
105
Q

What construct (version of a protein engineered for research) to make?

A
  • first approach: work with full-length protein
    -good idea to also consider working on domains (these are often more ‘crystallizable’) - trial and error
  • vast majority of protein structure in the PDB are not full-length, but rather single domains or combinations
106
Q

What is limited proteolysis?

A
  • It is a technique where enzymes (proteases) are used to cut a protein at specific sites.
  • It helps identify stable regions or domains of a protein by cleaving only at accessible, flexible parts.

1) Estimate MW based on migration.
2) Cut out band and subject to tryptic digestion to obtain peptide finger-print and determine sequence coverage.

1) Cut out band (or send proteolysis reaction) for mass spec to accurately measure the MW

107
Q

Sub-cloning Desired Construct

A
  • Sub-cloning: the process of transferring a specific DNA sequence (the construct) into a new vector for further study.
  • Transformation: introducing the sub-cloned DNA into bacteria to amplify and express the desired construct.
108
Q

Protein purification

A

Step 1:
–> cells or tissue
rupture cells
(sonication)
–> crude homogenate (protein, cell debris, other stuff)
-clarify by centrifugation
—> crude lysate (concentrated protein solution)

Step 2:
- affinity
- ion-exchange
- gel-filtration (size exclusion)

109
Q

Affinity Chromatography

A
  • separation based on the ability of the protein to carry out specific binding.
  • affinity tags are very cmmon
    1) Hexahistidine (His6) - short polypeptide binds to Ni2+ resin, elute protein with imidazole gradient.
    2) Glutathione-S-transferase (GST) - small 25 kDa highly soluble protein binds to glutathione resin, elute with reduced gluthathione.
  • tags are easily engineered into protein of interest with recombinant techniques.
  • tags are cleaved from protein with engineered enzyme cleavage site such as thrombin.

Example: glucose-binding protein attaches to glucose residues (G) on beads, addition of glucose (G), glucose-binding proteins are released on addition of glucose.

110
Q

Ion-exchange chromatography

A
  • separates proteins based on their net charge at a particular pH (isoelectric point, pI)
    1) Cation exchange (pKa = 4)
    2) Anion exchange (pKa = 9)
  • increase [NaCl] gradient elutes protein
  • proteins with a low density of charge will tend to emerge first
  • positively charged proteins binds to negatively charged bead
  • negatively charged protein flows through
111
Q

Gel-filtration chromatography

A
  • separation based on the size (mass) and shape (diameter) of the protein.
  • assumptions: a) protein is spherical (elongated proteins give larger than expected masses) b) no interaction with the sephadex gel
  • gel filtration chromatography used for 1) purification, 2) desalting (faster than dialysis), 3) to estimate the MW of the protein in its native state (ie. quaternary structure)
112
Q

Gel electrophoresis (SDS-PAGE)

A
  • protein travels from negative to positive end
  • Native PAGE: SDS-PAGE without the SDS (non-denaturing)
    -> proteins travel according to charge/mass ratio and conformation
    -> assessing conformational changes
    -> relative changes in quaternary structure
    -> binding events (protein: protein or protein:ligand)

A technique used to separate proteins based on their size, coats proteins with a negative charge and denatures them, so they all have uniform shape and charge-to-mass ratio. Proteins move through this gel when an electric current is applied, smaller proteins move faster, while larger ones move slower. After separation, proteins are stained to see as bands on the gel.

113
Q

What is supporting evidence that the structure obtained of a protein from a Crystal is similar to that in the cell?

A

1) Solvent content: protein crystals are 40-60% solvent, making the environment similar to the inside of the cell.
2) Multiple crystal structures: proteins crystallized in different ways show the same structure, meaning crystal packing does not affect the protein’s structure.
3) NMR comparison: structures obtains from NMR ( in solution) agree with crystal structures.
4) Some enzymes remain active in crystals, proving the protein retains its functional structure.

114
Q

7 stages of X-ray crystallogrpahy

A

1) Crystallization of protein of interest
2) Characterize the crystals (i.e. get space group and unit cell dimensions)
3) Diffraction data collection from crystals
4) Phase determination (alpha of X-ray)
5) Electron density map interpretation
6) Refinement of model so it fits the data
7) Interpretation of molecular model with other biochemical data

-> protein crystal is placed in X-ray beam
- diffraction pattern consisting of sharp spots
- diffraction pattern can be transformed into the protein electron density once phase problem is solved
- model built into map

115
Q
A

Bioc 450 Midterm (2 decks)

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