Amino Acids and Proteins

Fibrous:

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

Globular:

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

• 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

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

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

Negative Charge, Hydrophilic

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

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+

• 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

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.

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

Twisted, pleated sheet, either parallel or antiparallel:

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

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

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

• 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

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

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

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

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)

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)

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

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)

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)