Details of Protein Structure Function, evolution & experimental - - PowerPoint PPT Presentation

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Thomas Blicher, Center for Biological Sequence Analysis Anne Mølgaard, Kemisk Institut, Københavns Universitet

Details of Protein Structure

Function, evolution & experimental methods

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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.

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Outline

Protein structure evolution and function

Inferring function from structure. Modifying function

Experimental techniques

X-ray crystallography NMR spectroscopy

Structure validation

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Watson, Crick and DNA, 1952

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"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 specific 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.

DNA Conclusions

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“Could the search for ultimate truth really have revealed so hideous and visceral-looking an

• bject?” Max Perutz, 1964, on protein structure

John Kendrew, 1959, with myoglobin model

Once Upon a Time…

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They provide a detailed picture of interesting biological features, such as active site, substrate specificity, allosteric regulation etc. They aid in rational drug design and protein engineering. They can elucidate evolutionary relationships undetectable by sequence comparisons. Why are Protein Structures so Interesting?

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In evolution structure is conserved longer than both function and sequence. Structure > Function > Sequence

Structure & Evolution

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Rhamnogalacturonan acetylesterase (A. aculeatus) (1k7c) Platelet activating factor acetylhydrolase (B. Taurus) (1WAB) Serine esterase (S. scabies) (1ESC)

Structure & Evolution

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COOH NH2

Asp His Ser

Topological switchpoint

Inferring biological features from the structure

1DEO

Structure to Function

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Platelet activating factor acetylhydrolase Serine esterase Rhamnogalacturonan acetylesterase

Mølgaard, Kauppinen & Larsen (2000) Structure, 8, 373-383.

Structure & Evolution

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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

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Hydrophobic Effect and Folding

Oil and water Clathrate structures Entropy Indirect consequence

• f attraction between

water molecules

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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

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Hydrophobic vs. Hydrophilic

Globular protein (in solution) Membrane protein (in membrane)

Myoglobin Aquaporin

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Hydrophobic vs. Hydrophilic

Globular protein (in solution) Membrane protein (in membrane)

Myoglobin Aquaporin Cross-section Cross-section

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Characteristics of Helices

Aligned peptide units Dipolar moment Ion/ligand binding Secondary and quaternary structure packing Capping residues The helix (ii+4) Other helix types! (310, )

N C

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• Sheets

Multiple strands sheet

Parallel vs. antiparallel Twist

Flexibility

• Vs. helices

Folding Structure propagation (amyloids) Other…

Thioredoxin

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• Sheets

Multiple strands sheet

Parallel vs. antiparallel Twist

Flexibility

• Vs. helices

Folding Structure propagation (amyloids) Other…

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• Sheets

Multiple strands sheet

Parallel vs. antiparallel Twist

Flexibility

• Vs. helices

Folding Structure propagation (amyloids) Other…

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• Sheets

Multiple strands sheet

Parallel vs. antiparallel Twist

Flexibility

• Vs. helices

Folding Structure propagation (amyloids) Other…

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• Sheets

Multiple strands sheet

Parallel vs. antiparallel Twist

Strand interactions are non-local Flexibility

• Vs. helices

Folding

Antiparallel Parallel

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Turns, Loops & Bends Revisited

Between helices and sheets On protein surface Intrinsically “unstructured” proteins

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Structure Levels

• Primary structure = Sequence
• 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
• f Secondary structure elements

MSSVLLGHIKKLEMGHS…

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• Myoglobin
• Hemoglobin

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.

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Grouping Amino Acids

Livingstone & Barton, CABIOS, 9, 745-756, 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

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http://www.ch.cam.ac.uk/magnus/molecules/amino/

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Proteins Are Polypeptides

The peptide bond A polypeptide chain

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Ramachandran Plot

Allowed backbone torsion angles in proteins

N H

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Torsion Angles

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Ramachandran Plots

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Im, Ryu & Yu (2004) Engineering thermostability in serine protease inhibitors PEDS, 17, 325-331.

Engineering Thermostability

Example: Serpin (serine protease inhibitor) Overpacking Buried polar groups Cavities

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Experimental Methods

Crystallography & NMR spectroscopy

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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

Methods for Structure Determination

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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.

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X-rays Fourier transform

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The Importance of Resolution

high low

4 Å 2 Å 3 Å 1 Å

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Key Parameters

Resolution R values

Agreement between data and model. Usually between 0.15 and 0.25, should not exceed 0.30.

B factors

Contributions from static and dynamic disorder

Well determined ~10-20 Å2, intermediate ~20-30 Å2, flexible 30- 50 Å2, invisible >60 Å2.

• No. of observations vs. parameters

Ramachandran plot

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NMR Spectroscopy

Upper limit for structure determination currently ~50 kDa. Protein molecules are in solution. Dynamics, protein folding. Slow. Especially suited for studies of protein dynamics of small to medium size proteins.

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NMR Basics

NMR is nuclear magnetic resonance NMR spectroscopy is done on proteins IN SOLUTION Only atoms 1H, 13C, 15N (and 31P) can be detected in NMR experiments Proteins up to 30 kDa Proteins stable at high concentration (0.5-1mM), preferably at room temperature

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NMR Spectroscopy

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Well-defined structures

RMSDs < 0.6 Å

Evalutation of NMR Structures

Atomic backbone RMSD:

Less well-defined structures

RMSDs > 0.6 Å

3GF1, Cooke et al. Biochemistry, 1991 1T1H, Andersen et al. JBC, 2004

( )

n x x RMSD

n i i

• =

1 2 '

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Evaluation of NMR Structures

What regions in the structure are most well-defined? Look at the pdb ensembles to see which regions are well-defined

1RJH Nielbo et al, Biochemistry, 2003

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Which Structural Model?

Normally NMR structure models are listed according to the total energy and the number of violations. Model 1 in the PDB file is often the one with lowest energy and fewest violations. Use that model as template for modelling.

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NMR versus X-ray Crystallography

Hydrogen atoms are observed! Only 13C,15N and 1H are observed Study of proteins in solution Only proteins up to 30-40 kDa No total “map” of the structure Information used is incomplete and used as restraints An ensemble of structures is submitted to PDB The solved structure can be used for further dynamics characterization with NMR

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Holdings of the Protein Data Bank (PDB): The PDB also contains nucleotide and nucleotide analogue structures.

PDB

• Sep. 2001 May 2006 Oct. 2007

X-ray 13116 30860 39706 NMR 2451 5368 6862 Other 338 200 250 Total 15905 36428 46818

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Summary

In evolution structure is conserved longer than both function and sequence.

X-ray crystallography

Proteins in crystal lattice Many details – one model Resolution, R-values, Ramchandran plot

NMR spectroscopy

Proteins in solution Fewer details – many models Violations, RMSD, Ramachandran plot

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Links

PDB (protein structure database)

www.pdb.org/

PyMOL home:

http://pymol.sourceforge.net/

PyMOL manual:

http://pymol.sourceforge.net/newman/user/toc.html

PyMOL Wiki:

http://www.pymolwiki.org/index.php/Main_Page

PyMOL settings (documented):

http://cluster.earlham.edu/detail/bazaar/software/pymol /modules/pymol/setting.py

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