Protein Structure Elements Primary to Quaternary Structure

Learning Objectives After this lesson you should be able to: –Define the structural levels of proteins. –Identify regular secondary structure elements. –Identify the structural units of the protein backbone. –Explain why some backbone conformations are favoured and some are “forbidden” (not found in natural proteins). –Name properties on which the amino acids can be grouped. –Explain the driving forces behind protein folding related to the properties of the backbone and the side chains.

Proteins Are Polypeptides The peptide bondA polypeptide chain

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…

Myoglobin Haemoglobin 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.     

A Bit About Protein Folding How and why proteins fold

Why Fold? Hydrophobic collapse –Hydrophobic residues cluster to “escape” interactions with water. –Polar backbone groups form secondary structure to satisfy hydrogen bonding donors and acceptors. –Initially formed structure is in molten globule state (ensemble). –Molten globule condenses to native fold via transition state

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 SurfaceInterior

Hydrophobic vs. Hydrophilic Globular protein (in solution) Membrane protein MyoglobinAquaporin

Hydrophobic vs. Hydrophilic Globular protein (in solution) Membrane protein MyoglobinAquaporin Cross-section

From Unfolded to Native State  G =  H - T×  S  G: Free (Gibbs) energy  H: Enthalpy (interactions)  S: Entropy (conformations/ states) E U F T GG Unfolded state, ensemble Native fold, one structure Transition state, one or more narrow ensembles

Protein Stability & Dynamics Folded proteins are: –Only marginally stable (enthalpy and entropy almost balance at physiological temperatures) Allows for easy degradation and reuse. Amyloid exception. –Dynamic “Breathing” motions on pico- to nanosecond scale. Allows substrates/products to enter/leave enzymes. Allows allosteric regulation of activity.

Amino Acids Proteins are built from amino acids Amino group and acid group Side chain at C  Chiral, only one enantiomer found in proteins (L-amino acids) 20 natural amino acids N O C CC CC CC CC SS Methionine

Amino Acid Properties Many features –Charge +/- Acidic vs. basic (pK a ) –Polarity (polar/non-polar) Type, distribution –Size Length, weight, volume, surface area –Type (Aromatic/aliphatic)

Grouping Amino Acids Livingstone & Barton, CABIOS, 9, , 1993 A – Ala C – Cys D – Asp 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

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

Backbone Properties Amide bond planarity2 degrees of rotational freedom per residue

Ramachandran Plot Allowed backbone torsion angles in proteins N H Residue Peptide bond

Torsion Angles

Characteristics of Helices Backbone interactions are local Aligned peptide units  Dipolar moment N C

Helix Types

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

 -Sheets Thioredoxin

 -Sheets Thioredoxin

 -Sheets Thioredoxin

 -Sheets Thioredoxin

Not All  -Sheets Are Flat NitrophorinThioredoxin

Residue Patterns Helices –Helix capping –Amphiphilic residue patterns Sheets –Amphiphilic residue patterns –Residue preferences at edges vs. middle Special residues –Proline Helix breaker –Glycine In turns/loops/bends N C

Turns, Loops & Bends Revisited Between helices and sheets On protein surface Intrinsically “unstructured” proteins

Summary The backbone of polypeptides form regular secondary structures. –Helices, sheets, turns, bends & loops. These are the result of local as well as non- local interactions. Secondary structure elements are associated with specific residue patterns.

 -sheet and  -helices  -sheet  -helix 1M8N TheoreticalReal