Amino Acids and Proteins

  • Created by: rosieevie
  • Created on: 08-01-17 19:41

Protein Classes

Fibrous:

  • Insoluble
  • Structures, strength and protection
  • e.g. coatings, webs, layers

Globular:

  • Soluble
  • Dynamic functions
  • e.g. enzymes, hormones, transport
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Protein Properties

  • Linear amino acid polymers that can fold
  • Wide range of funtional groups = chemically reactive
  • Interact with each other and other molecules
  • Can be flexible or rigid
  • 20 amino acid subunits
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Amino Acid Structure

(http://www.nutrientsreview.com/wp-content/uploads/2014/10/Amino-Acid-Structure.jpg)

(http://glossary.periodni.com/images/zwitterion.jpg)(http://www.mun.ca/biology/scarr/Karp_2_25_alanine_stereoisomers.gif)

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Non-Polar R Groups

Hydrophobic

  • Glycine, Gly, G - H
    • Cannot form L isomer
  • Alanine, Ala, A - CH3
  • Phenylalanine, Phe, F - CH2-Cyclic
  • Cytesine, Cys, C - CH2-SH
  • Methionine, Met, M - CH2-S-CH3
  • Valine, Val, V - C-,-CH3,CH3
  • Tryptophan, Trp, W - CH2, Weird Molecule w/NH
  • Lecuine, Leu, L - CH-C-,-CH3,CH3
  • Isoleucine, Ile, I - C-,-CH3,CH2-CH3
  • Proline, Pro, P - Complete cyclic compound w/ CH2s
    • Imino acid
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Polar Uncharged R Groups

Hydrophilic

  • Serine, Ser, S - CH2-OH
  • Threorine, Thr, T - CH-,-OH,CH3
  • Tyrosine, Tyr, Y CH2-Clyclic-OH
    • Phosphorylated in post-transcriptional modification
  • Asparagine, Asn, N - CH2-,-C=O,NH2
  • Glutamine, Gln, Q - CH2-CH2-C=O-NH2
    • Gluten
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Polar Acidic R Groups

Negative Charge, Hydrophilic

  • Aspartic Acid (Aspartate), Asp, D - CH2-COO-
  • Glutamaic Acid (Glutamate), Glu, E - CH2-CH2-COO-
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Polar Basic R Groups

Positive, Hydrophilic

  • Lysine, Lys, K - CH2-CH2-CH2-CH2-NH3+
  • Arginine, Arg, R -CH2-CH2-CH2-C=NH3+,-NH2
  • Histidine, His, H -CH2-Cyclic w/NH+
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Joining Amino Acids Together

  • Condensation reaction
  • Peptide bond 
    • Rigid as it provides a partial double bond character
  • Ribsosomes provide site of reaction
  • Polypeptides have residue seqence
  • Sequence always described from N to C
  • Molecular mass of a protein = Number of residues X 10
  • Amino acid sequence is primary structure

(http://oregonstate.edu/instruct/bb331/lecture01/peptidebond.gif)

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Pauling and Corey (1951)

Worked out stucture of fibrous protein a-keratin using x-ray diffraction. Created 3 rules:

  • No rotation around planar peptide bond
    • Resonance gives partial double bond
  • Other parts of the chain are rhythmically flexible
    • Amino group and a-carbon bond = Phi bond
    • Carboxyl and a-charbon bond = Psi bond
    • Can be positive bonds and rotate clockwise or negative and rotate anticlockwise
    • Different R-groups define rotation (repel/attract)
  • Structure must have maximum number of stabilising forces between residues
    • NOT R-groups
    • Hydrogen bondng between C=O and N-H

Suggested 2 possible structure types:

  • Alpha helix
  • Beta sheet

Very stable and have maximum number of hydrogen bonds.

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Secondary Structure - a-helix

Side view:

  • NH of one residue bonds to CO x+4 amino acids along
  • 5.4Å per complete turn
  • 3.6 amino acids per tern
  • 1.5Å length per residue

Top view:

  • Clockwise from N-C termini
  • Right-handed
  • Side chains project outwards 100° relative to neighbours

Disrupting folding:

  • Glycine - no chiral centre so more flexible
  • Proline - cyclic sidechain, restricts phi angle rotation to -50°. No hydrogen bonding = KINK

Amphipathic helicases - residues arrange so helix has 2 different sides to make either hydrophobic or hydrophillic

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Secondary Structure - B-sheets

Twisted, pleated sheet, either parallel or antiparallel:

  • Parallel - 2 H bonds
  • Antiparallel - 4 H bonds (more stable)
    • B-turns involve 2 residues 

(http://www.bio.miami.edu/tom/courses/protected/ECB/CH05/5_10.jpg)

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Secondary Structures - Prediction

Chou-Fasman

  • a-helix - 4/6 contiguous residues have a-helix values of >100
  • b-sheet - 3/6 contiguous residues have b-sheet values of >100

Ramachandran Plot

(http://www.cryst.bbk.ac.uk/PPS95/course/3_geometry/rama.gif)

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Supersecondary Structures

a-keratin:

  • Amphipathic helicases
  • Hydrophobic interactions make keratin strong

Helix-turn-helix:

  • DNA binding - binds to specific DNA sequence

Helix-loop-helix-EF hand:

  • a-helix then B-sheet then a-helix
  • Center binds to Ca2+ ions 
  • Specific signalling for molecules
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Disrupting Protein Structure

  • Heat - 20°C-40°C
  • pH - (normal pH7.2ish) - boil in acid or alkali (6M)
  • Ionic strength
  • Denaturing agents
    • Organic solvents
    • Chaotropic agents e.g. urea, guanidinum
  • Proteolytic enzymes - proteases (hydrolysis) - indiscriminate or sequence specific
    • Trypsin cuts C-terminal to Arginine (R) or Lysine (K)
    • Renin for cheese production cuts F-M bond in casein
    • In programmed cell death (apoptosis) caspases cleave after DxxD
  • UV/oxidative/radiation damage
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Tertiary Structures - Hydrophobic Interactions

Main driving force called hydrophobic collapse

Hydrophobic clusters:

  • Hydrophobic side chains make core
  • Occurs in water
  • Hydrophobic residues may be on outside if interacting with other proteins
  • Broken by organic solvents/denaturating agents = create a hydrophobic environment

pi-Bond Interactions (pi-stack/pi-overlap)

  • Aromatic residues only
  • Mixing of pi electron clouds
  • Weak - disrupted by heat
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Tertiary Structures - Hydrogen Bonds

  • Involve polar non-charged R-groups w/ oxygen or nitrogen
  • Broken down by heat or denaturing agents
  • Disrupted by water - stronger hydrogen bonding
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van der Waals Interactions

  • Not major cause of protein structure
  • Short range effects
  • Weak
  • Broken by heat and denaturing agents
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Electrostatic/Ionic Bonding

Salt bridges betweeb charged residues - ineffective unless surounded by hydrophobic interact.

  • Acidic (COO-)
  • Basic (NH3+)
  • Cysteine or tyrosin

Protonation state of R groups changes according to pH. Also why titration curves for certain amino acids have multple curve things.

Isoelectric point - point where the side chain has no net charge

Henderson Hasselbatch equation - relationships between weak amino acids:

  • HA ⇌ A- + H+
  • pH = pKa + log([A-]/[HA)
  • Each amino acid has a pKa (where 50% of ionisation has occured)
  • Charge of protein determined by pH and number/type of amino acid residues
  • pH changes or increasing ionic strength cause salt bridges/ionic bonds to break
  • Phosphorylation can make R-group charges negative (permenant with Aspartate or switchable with Alanine)
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Disulphide Bonds (Covalent)

Very strong - stops unfolding

Forms between two cystenine with O2 and B-,mercaptoethanol

Important for extracellular proteins = robust

Anifinsen Experiment on Ribonuclease

  • Denatured and renatured native ribonuclease
  • Shows folded, active protein form has lowest free energy
  • Shows all necessary info needed in primary structure

Not all proteins fold easily - some require protein disulphide isomerases which make and break disulphide bonds until correct conformation (chaperones)

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Definitions

Monomer - molecule that can bond to identical molecules to make a polymer

Dimer - molecule/complex consisting of 2 identical molecules

Trimer - polymer consisting of 3 monomer units

Oligomer - polymer with few repeating units

Polymer - substance with molecular structure forming a large number of similar units

Homomeric - substance made up of identical products/molecules

Heteromeric - substance made up of more than one subunit

Heterotetramer - protein with 4 non-covalently bound non-identical subunits e.g. haemoglobin

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Protein Interactions

Transient - interactions for a short time period

Stable - interactions for a long time period (can be permanent)

Interaction via:

  • Motifs - short, amino acid sequences (widespread)
  • Domains - larger, structural

Conformational changes allow new interaction sites e.g. PKR kinase activated when dsRNA present in the cell and binds to allow bimerisation and then kinase activation. Activated PKR phosphorylates a substrate whiich switches off general translation, inhibiting viral replication

Enzymes act as scaffold for others to bind onto - keep them together in one place, speeding up enzyme reaction rate. Good in intracellular signalling scaffolds.

Molecular mimicry - mechanism for autoimmune diseases where sequence similaries between foreign and own peptides to result in cross-activation of immune system

Motif/domain recognition is blind to the rest of the protein meaning that e.g. antibodies can cause autoimmune diseases

Proteins can be post-translationally modified at single residues or conjugates (reversible - combined with multiple contacts)

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Protein Interactions

Transient - interactions for a short time period

Stable - interactions for a long time period (can be permanent)

Interaction via:

  • Motifs - short, amino acid sequences (widespread)
  • Domains - larger, structural

Conformational changes allow new interaction sites e.g. PKR kinase activated when dsRNA present in the cell and binds to allow bimerisation and then kinase activation. Activated PKR phosphorylates a substrate whiich switches off general translation, inhibiting viral replication

Enzymes act as scaffold for others to bind onto - keep them together in one place, speeding up enzyme reaction rate. Good in intracellular signalling scaffolds.

Molecular mimicry - mechanism for autoimmune diseases where sequence similaries between foreign and own peptides to result in cross-activation of immune system

Motif/domain recognition is blind to the rest of the protein meaning that e.g. antibodies can cause autoimmune diseases

Proteins can be post-translationally modified at single residues or conjugates (reversible - combined with multiple contacts)

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