Lecture 13 February 16, 2016 Biotech 3

Sickle Cell Anemia Gln  Val Normally a benign mutation. No effects on the 2°, 3°, or 4° structures  of hemoglobin under normal oxygen concentration. Under low oxygen concentration, hemoglobin exposes a hydrophobic patch. The hydrophobic side chain of valine associates with the hydrophobic patch, causing hemoglobin to aggregate and form fibrous precipitates.

Amino Acid Residues Aliphatic Residues: G, A, V, L, I, P amino acids with a open chain hydrocarbon side groups, not aromatic rings. No reactive groups on side chain. Do not interact favorably with water. Interact favorably with each other (the “bricks” of a functional protein molecule) Imposes rigidity constraints Have effect on conformation of protein “backbone” Promotes turns Least hydrophobic Most hydrophobic Hydrophobicity

Amino Acid Residues Cont. Charged Residues: D, E, H, K, R (sometimes charged) Negative Charge Chain length confers differences in interactions Interact with amines Positive Charge

Amino Acid Residues Cont.2 Aromatic Residues: F, T, W

Aromatic Amino Acid Residue Absorption Spectrum Aromatic Residues: F, T, W Aromatic side chains are responsible for most of the UV absorbance and fluorescence. Spectral properties are sensitive to immediate environment of side chain. Useful to use as probes of protein structure. Phe least reactive. Hydroxyl group of Tyr is reactive. Trp is less frequent in proteins. Absorbance Fluorescence λmax λmax Phenylalanine Tyrosine Tryptophan 257.4 282 274.6 303 279.8 348

Amino Acid Residues - Histidine Nitrogen with the hydrogen is an electrophile and donor for hydrogen bonding Nitrogen without a hydrogen is a nucleophile and acceptor for hydrogen bonding Very versatile Can undergo numerous reactions Ex) Phosphorylation

Amino Acid Residues – Sulfur Sulfur Containing Residues: M and C Most reactive of any amino acid side chain Thiol group can complex with copper, iron, zinc, cobalt, molybdenum, manganese, cadmium, mercury, and silver. Form disulfide bonds! Codeed by the start codon; ATG Relatively unreactive Can be labeled ex) methyl iodide

-S – CH2 – -S – CH2 – Oxidation of Cysteine – CH2 – S – S – CH2 – Cysteine disulfide bond Reduced -S – CH2 – Oxidized “Cystine” residue – CH2 – S – S – CH2 – -S – CH2 –

-S – CH2 – -S – CH2 – Reduction of Cystine – CH2 – S – S – CH2 – Cysteine disulfide bond Reduced -S – CH2 – Oxidized “Cystine” residue – CH2 – S – S – CH2 – Can be effectively reduced by thiol-disulfide exchange with thiol reagents β-mercaptoethanol (BME) Dithiothreitol (DTT) Tris(2-carboxymethyl)phosphine (TCEP) -S – CH2 –

Protein Electrophoresis – Reducing agent a. Reducing agent Reduces disulfide bonds Separates multi-subunit proteins linked by disulfide bridges β-mercaptoethanol (BME), dithiothreitol (DTT), TCEP

Protein Structure Proteins perform many functions Structural support – cellular cytoskeleton Metabolism – enzymes and hormones Protection – antibodies Communication – signal transduction Regulation – transcription factors Two main groups: Fibrous – form cellular structures: keratin, myosin, collagen, etc Globular – signaling and chemical changes within cell: enzymes

Protein Structure Cont. The main driving force for folding water-soluble globular proteins is to pack hydrophobic side chains into interior of the molecule; form a hydrophobic core. Problem: the main chain (aka backbone) is highly polar and therefore hydrophilic! Solution: formation of secondary structures within the interior of the protein molecule. Two types of secondary structures: alpha helices and beta sheets Secondary structures are characterize by the hydrogen bonds between the main chain NH and C=O groups. Hydrogen bond acceptor Hydrogen bond donor

Alpha (α ) Helix in Protein Structures Amino terminus Spiral structure May be more loosely or tightly coiled 3.6 residues per turn May vary considerably in length All hydrogen bonds in an α helix point in the same direction Significant net dipole moment in α helix: partial positive charge at amino end partial negative charge at carboxy end Preferred amino acids in α helix Proline fits very well into the first turn of an α helix (may also form a bend) Ala (A), Glu (E), Leu (L), Met (M)  Good Pro (P), Gly (G), Tyr (Y), and Ser (S)  Poor Typically on the outside of protein May cross membranes (α helix will have more hydrophobic amino acids) 3.6 residues/turn Dipole moment Carboxyl terminus

Pi (π) Helix in Protein Structures Spiral structure May be more loosely or tightly coiled 3.6 residues per turn May vary considerably in length All hydrogen bonds in an α helix point in the same direction Significant net dipole moment in α helix: partial positive charge at amino end partial negative charge at carboxy end Preferred amino acids in α helix Proline fits very well into the first turn of an α helix (may also form a bend) Ala (A), Glu (E), Leu (L), Met (M)  Good Pro (P), Gly (G), Tyr (Y), and Ser (S)  Poor Typically on the outside of protein May cross membranes (α helix will have more hydrophobic amino acids)

Helical Wheel Representation of Alpha Helices 1. Leu Ser Phe Ala Ala Ala Met Asn Gly Leu Ala 2. Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly hydrophobic Charged Polar 3. Lys Glu Asp Ala Lys Gly Lys Ser Glu Glu Glu Helical wheel representation of α helices:

Beta (β ) in Protein Structures Built from several regions of the polypeptide chain into β-strands 5 – 10 residues per strand Strands are aligned adjacent to each other into β-sheets: Parallel: amino to carboxy terminal run in the same direction Antiparallel: amino to carboxy terminal alternate direction Mixed: combination of parallel and antiparallel (bias against mixed β-sheets)

Beta (β ) in Protein Structures - Parallel Hydrogen bonds: main chain NH (amide from peptide bond) and O atoms within a β sheet

Beta (β ) in Protein Structures – Anti parallel Hydrogen bonds: main chain NH (amide from peptide bond) and O atoms within a β sheet

Beta (β ) in Protein Structures – Hairpin Loop

β Sheet Topology Topology diagrams representation of β sheets:

Protein Structure Motifs Motif: simple combination of a few secondary structure elements with a specific geometric arrangement that are frequently found to occur in protein structures. (tertiary structures) Examples: Helix-turn-helix Hairpin Beta Greek Key Which amino acid would you expect to find in the ‘turn”

Protein Structure Continued Structural hierarchy; primary, secondary, tertiary, quaternary. Simple motifs combine to form complex motifs. Large polypeptide chains fold into several domains (domains typically are also units of function). Single domain or multiple domains. Domains are built from structural motifs. α domains Β domains α/β domains