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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 bond • A 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… Quaternary Structure • Assembly of monomers/subunits into protein complex • Myoglobin a – 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. • Haemoglobin a b b a 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 Surface Interior Hydrophobic vs. Hydrophilic • Globular protein (in solution) Myoglobin • Membrane protein Aquaporin Hydrophobic vs. Hydrophilic • Globular protein (in solution) Cross-section • Membrane protein Cross-section Myoglobin Aquaporin From Unfolded to Native State DG = DH - T×DS ------------------------DG: Free (Gibbs) energy DH: Enthalpy (interactions) DS: Entropy (conformations/ states) Transition state one or more narrow ensembles E T U DG Unfolded state, ensemble F Native fold, one structure 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 Ce Sd Cg • Side chain at Ca • Chiral, only one enantiomer found in proteins (L-amino acids) • 20 natural amino acids Cb N Ca C O Methionine Amino Acid Properties • Many features – Charge +/• Acidic vs. basic (pKa) – Polarity (polar/non-polar) • Type, distribution – Size • Length, weight, volume, surface area – Type (Aromatic/aliphatic) Grouping Amino Acids A – Ala C – Cys D – Asp E – Glu F – Phe G – Gly H – His I – Ile K – Lys L – Leu Livingstone & Barton, CABIOS, 9, 745-756, 1993 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 planarity • 2 degrees of rotational freedom per residue Ramachandran Plot • Allowed backbone torsion angles in proteins Peptide bond N H Residue Torsion Angles Characteristics of Helices C • Backbone interactions are local • Aligned peptide units Dipolar moment N Helix Types b-Sheets • Multiple strands sheet – Parallel vs. antiparallel – Twist • Strand interactions are non-local • Flexibility – Vs. helices – Folding Antiparallel Parallel b-Sheets Thioredoxin b-Sheets Thioredoxin b-Sheets Thioredoxin b-Sheets Thioredoxin Not All b-Sheets Are Flat • Nitrophorin • Thioredoxin Residue Patterns • Helices C – Helix capping – Amphiphilic residue patterns N • Sheets – Amphiphilic residue patterns – Residue preferences at edges vs. middle • Special residues – Proline • Helix breaker – Glycine • In turns/loops/bends 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 nonlocal interactions. • Secondary structure elements are associated with specific residue patterns. a-sheet and b-helices a-sheet Theoretical b-helix Real 1M8N