Details of Protein Structure Function, evolution & experimental methods Thomas Blicher, Center for Biological Sequence Analysis Anne Mølgaard, Kemisk Institut, Københavns Universitet CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Learning Objectives � Outline the basic levels of protein structure. � Outline key differences between X-ray crystallography and NMR spectroscopy. � Identify relevant parameters for evaluating the quality of protein structures determined by X-ray crystallography and NMR spectroscopy. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Outline � Protein structure evolution and function � Inferring function from structure. � Modifying function � Experimental techniques � X-ray crystallography � NMR spectroscopy � Structure validation CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Watson, Crick and DNA, 1952 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
DNA Conclusions "We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest…. …It has not escaped our notice that the speci fi c pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." J.D. Watson & F.H.C. Crick (1953) Nature, 171, 737. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Once Upon a Time… “Could the search for ultimate truth really have revealed so hideous and visceral-looking an object?” Max Perutz, 1964, on protein structure John Kendrew, 1959, with myoglobin model CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Why are Protein Structures so Interesting? � They provide a detailed picture of interesting biological features, such as active site, substrate speci fi city, allosteric regulation etc. � They aid in rational drug design and protein engineering. � They can elucidate evolutionary relationships undetectable by sequence comparisons. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Structure & Evolution � In evolution structure is conserved longer than both function and sequence. Structure > Function > Sequence CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Structure & Evolution Rhamnogalacturonan acetylesterase (A. aculeatus) (1k7c) Serine esterase Platelet activating (S. scabies) (1ESC) factor acetylhydrolase (B. Taurus) (1WAB) CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Structure to Function Inferring biological features from the structure 1DEO NH 2 Asp His Topological switchpoint Ser COOH CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Structure & Evolution Rhamnogalacturonan acetylesterase Serine esterase Platelet activating factor acetylhydrolase Mølgaard, Kauppinen & Larsen (2000) Structure, 8, 373-383. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Why Fold? � Hydrophobic collapse � Hydrophobic residues cluster to “escape” interactions with water. � Indirect effect of attraction between water molecules. � Polar backbone groups form secondary structure to satisfy hydrogen bonding donors and acceptors. � Interactions with � Initially formed structure is in molten globule state (ensemble). � Molten globule condenses to native fold via transition state CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Hydrophobic Effect and Folding � Oil and water � Clathrate structures � Entropy � Indirect consequence of attraction between water molecules CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
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 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Hydrophobic vs. Hydrophilic � Globular protein (in � Membrane protein (in solution) membrane) Myoglobin Aquaporin CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Hydrophobic vs. Hydrophilic � Globular protein (in � Membrane protein (in solution) membrane) Cross-section Cross-section Myoglobin Aquaporin CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Characteristics of Helices � Aligned peptide C units � Dipolar moment � Ion/ligand binding � Secondary and quaternary structure packing � Capping residues � The � helix (i � i+4) � Other helix types! (3 10 , � ) N CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
� -Sheets � Multiple strands � sheet Thioredoxin � Parallel vs. antiparallel � Twist � Flexibility � Vs. helices � Folding � Structure propagation (amyloids) � Other… CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
� -Sheets � Multiple strands � sheet � Parallel vs. antiparallel � Twist � Flexibility � Vs. helices � Folding � Structure propagation (amyloids) � Other… CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
� -Sheets � Multiple strands � sheet � Parallel vs. antiparallel � Twist � Flexibility � Vs. helices � Folding � Structure propagation (amyloids) � Other… CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
� -Sheets � Multiple strands � sheet � Parallel vs. antiparallel � Twist � Flexibility � Vs. helices � Folding � Structure propagation (amyloids) � Other… CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
� -Sheets � Multiple strands � Antiparallel Parallel sheet � Parallel vs. antiparallel � Twist � Strand interactions are non-local � Flexibility � Vs. helices � Folding CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Turns, Loops & Bends Revisited � Between helices and sheets � On protein surface � Intrinsically “unstructured” proteins CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Structure Levels Primary structure = Sequence � MSSVLLGHIKKLEMGHS… Secondary Structure = Helix, � sheets/strands, loops & turns Structural Motif = Small, � recurrent arrangement of secondary structure, e.g. � Helix-loop-helix � Beta hairpins � EF hand (calcium binding motif) � Etc. Tertiary structure = Arrangement � of Secondary structure elements CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Quaternary Structure � Assembly of Myoglobin � monomers/subunits � into protein complex � Backbone-backbone, backbone-side-chain & side-chain-side-chain interactions: Hemoglobin � � Intramolecular vs. intermolecular contacts. � For ligand binding side � � chains may or may not contribute. For the latter, mutations have little effect. � � CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Grouping Amino Acids A – Ala M – Met C – Cys N – Asn D – Asp P – Pro E – Glu Q – Gln F – Phe R – Arg G – Gly S – Ser H – His T – Thr I – Ile V – Val K – Lys W – Trp L – Leu Y - Tyr Livingstone & Barton, CABIOS , 9 , 745-756, 1993 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
http://www.ch.cam.ac.uk/magnus/molecules/amino/ CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Proteins Are Polypeptides � The peptide bond � A polypeptide chain CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Ramachandran Plot � Allowed backbone torsion angles in proteins N H CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Torsion Angles CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Ramachandran Plots CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Engineering Thermostability Example: Serpin (serine protease inhibitor) � Overpacking � Buried polar groups � Cavities Im, Ryu & Yu (2004) Engineering thermostability in serine protease inhibitors PEDS, 17, 325-331. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Experimental Methods Crystallography & NMR spectroscopy CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
Methods for Structure Determination � X-ray crystallography � Nuclear Magnetic Resonance (NMR) � Modelling techniques � More exotic techniques � Cryo electron microscopy (Cryo EM) � Small angle X-ray scattering (SAXS) � Neutron scattering CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
X-ray Crystallography � No size limitation. � Protein molecules are ”stuck” in a crystal lattice. � Some proteins seem to be uncrystallizable. � Slow. � Especially suited for studying structural details. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU
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