Levels of Protein Structure Primary to Quaternary Structure

Learning Objectives After today you should be able to: Define the structural levels of proteins. Identify the structural units of the protein backbone. Explain why some backbone conformations are “forbidden”, i.e. not found in natural proteins. Name properties on which the amino acids can be grouped. Name more amino acids than you could before One and three letter codes

Amino Acids Proteins are built from amino acids Amino group and acid group Side chain at Ca Chiral, only one enantiomer found in proteins (L-amino acids) 20 natural amino acids Ce Sd Cg Cb C Ca N O Methionine

L-amino acids in Living organisms

How to Group Them? Many features Charge +/- Polarity (polar/non-polar) Acidic vs. basic (pKa) Polarity (polar/non-polar) Type, distribution Size Length, weight, volume, surface area Type (Aromatic/aliphatic) 5

The Amino Acids Thr (T) Phe (F) Val (V) Ala (A) His (H) Arg (R) Ser (S) Leu (L) Cys (C) Met (M) Asp (D) Lys (K) Asn (N) Ile (I) Trp (W) Gln (Q) Glu (E) Tyr (Y) Pro (P) Gly (G)

Amino Acids A – Ala M – Met C – Cys N – Asn D – Asp P – Pro E – Glu F – Phe G – Gly H – His I – Ile K – Lys L – Leu M – Met N – Asn P – Pro Q – Gln R – Arg S – Ser T – Thr V – Val W – Trp Y - Tyr Livingstone & Barton, CABIOS, 9, 745-756, 1993

Blosum62 substitution matrix

The Evolution Way Based on Blosum62 matrix Measure of evolutionary substitution probability

The Simple Aliphatic Val (V) Ala (A) Leu (L) Met (M) Ile (I)

The Small Polar Ser (S) Cys (C) Thr (T) Cystin

The Unusual P, G Also aliphatic Structural impact Strictly speaking, proline is an imino acid Gly (G) Pro (P)

The Acidic and Their Derivatives Asp (D) Asn (N) Glu (E) Gln (Q)

The Basic Arg (R) Lys (K) His (H)

The Aromatic Phe (F) His (H) Trp (W) Tyr (Y)

Proteins Are Polypeptides The peptide bond A polypeptide chain

Ramachandran Plot Allowed backbone torsion angles in proteins N H

Small Exercise (5 minutes) For the polypeptide on the left discuss the following with your neighbour: Why is the lower right quadrant a ”forbidden” region in the Ramachandran plot? What makes Gly a special amino acid when it comes to Ramachandran plots? What about Pro?

Structure Levels Primary structure = Sequence (of amino acids) Secondary Structure = Helix, sheets/strands, bends, loops & turns (all defined by H-bond pattern in backbone) Structural Motif = Small, recurrent arrangement of secondary structure, e.g. Helix-loop-helix Beta hairpins EF hand (calcium binding motif) Many others… Tertiary structure = Arrangement of Secondary structure elements within one protein chain MSSVLLGHIKKLEMGHS…

Quaternary Structure Assembly of monomers/subunits into protein complex Backbone-backbone, backbone-side-chain & side-chain-side-chain interactions: Intramolecular vs. intermolecular contacts. For ligand binding side chains may or may not contribute. For the latter, mutations have little effect. Myoglobin Haemoglobin a a b

Hydrophobic Core Hydrophobic side chains go into the core of the molecule – but the main chain is highly polar. The polar groups (C=O and NH) are neutralized through formation of H-bonds. Myoglobin Surface Interior

Hydrophobic vs. Hydrophilic Globular protein (in solution) Membrane protein (in membrane) Myoglobin Aquaporin

Hydrophobic vs. Hydrophilic Globular protein (in solution) Membrane protein (in membrane) Cross-section Cross-section Myoglobin Aquaporin

Characteristics of Helices Aligned peptide units  Dipolar moment Ion/ligand binding Secondary and quaternary structure packing Capping residues The a helix (i→i+4) Other helix types! (310, p) C N

Helix Types

b-Sheets Multiple strands  sheet Parallel vs. antiparallel Twist Coil regions between the secondary structure elements Thioredoxin

b-Sheets Multiple strands  sheet Parallel vs. antiparallel Twist

b-Sheets Multiple strands  sheet Parallel vs. antiparallel Twist

b-Sheets Multiple strands  sheet Parallel vs. antiparallel Twist

b-Sheets Multiple strands  sheet Strand interactions are non-local Parallel vs. antiparallel Twist Strand interactions are non-local Flexibility Vs. helices Folding Antiparallel Parallel

Summary Amino acids in Living organisms have L-configuration One & three letter codes Groups of amino acids: hydrophobic ... The backbone of polypeptides form regular secondary structures. Helices, sheets & loops.

Building Blocks Ca Peptide unit (amide) COOH OH C/CH/CH2/CH3 Amide Indole N S/SH H-bond Phenyl Imidazole Guanidine NH2/NH3+

Building Blocks Ce Sd c3 Cg c2 Cb c1 Ca y N f C O O On-1 Nn+1 Ca Ca O Cn-1 c2 O Cb O c1 Ca y Single amino acid N Residue f C O O Methionine

Procedure Build backbone in extended conformation (strand). Twist to align all peptide units (look at the C=O groups). Add H-bonds (i+4) to construct an ideal a-helix. Add side chains along the way (pointing “down”). C N