function, evolution & experimental methods - cbs · center for biological sequence...

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU

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

Details of Protein Structure

Function, evolution &experimental methods


Learning Objectives

Outline the basic levels of protein structure.

Outline key differences between X-raycrystallography and NMR spectroscopy.

Identify relevant parameters for evaluatingthe quality of protein structures determinedby X-ray crystallography and NMRspectroscopy.


Outline

Protein structure evolution and functionInferring function from structure.Modifying function

Experimental techniquesX-ray crystallographyNMR spectroscopy

Structure validation


Watson, Crick and DNA, 1952


"We wish to suggest a structure for the salt ofdeoxyribose nucleic acid (D.N.A.). This structurehas novel features which are of considerablebiological interest….…It has not escaped our notice that the specificpairing we have postulated immediatelysuggests a possible copying mechanism for thegenetic material."

J.D. Watson & F.H.C. Crick (1953) Nature, 171, 737.

DNA Conclusions


“Could the search for ultimate truth really haverevealed so hideous and visceral-looking anobject?” Max Perutz, 1964, on protein structure

John Kendrew, 1959, with myoglobin model

Once Upon a Time…


They provide a detailed picture ofinteresting biological features, such asactive site, substrate specificity, allostericregulation etc.

They aid in rational drug design and proteinengineering.

They can elucidate evolutionaryrelationships undetectable by sequencecomparisons.

Why are Protein Structures soInteresting?


In evolution structure is conserved longer thanboth function and sequence.

Structure > Function > Sequence

Structure & Evolution


Rhamnogalacturonanacetylesterase

(A. aculeatus) (1k7c)

Platelet activatingfactor acetylhydrolase

(B. Taurus) (1WAB)

Serine esterase(S. scabies) (1ESC)



COOH

NH2

Asp His Ser Topological switchpoint

Inferring biologicalfeatures from the structure

1DEO

Structure to Function


Platelet activatingfactor acetylhydrolase

Serine esterase

Rhamnogalacturonanacetylesterase

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



Why Fold?

Hydrophobic collapseHydrophobic residues cluster to “escape” interactionswith water.

Indirect effect of attraction between water molecules.

Polar backbone groups form secondary structure tosatisfy hydrogen bonding donors and acceptors.Interactions withInitially formed structure is in molten globule state(ensemble).Molten globule condenses to native fold via transitionstate


Hydrophobic Effect and Folding

Oil and water

Clathrate structures

Entropy

Indirect consequenceof attraction betweenwater molecules


Hydrophobic Core

Hydrophobic side chains go into the core ofthe molecule – but the main chain is highlypolar.The polar groups (C=O and NH) areneutralized through formation of H-bonds.

Myoglobin

Surface Interior


Hydrophobic vs. Hydrophilic

Globular protein (insolution)

Membrane protein (inmembrane)

Myoglobin Aquaporin


Hydrophobic vs. Hydrophilic

Globular protein (insolution)

Membrane protein (inmembrane)

Myoglobin Aquaporin

Cross-section Cross-section


Characteristics of Helices

Aligned peptideunits DipolarmomentIon/ligand bindingSecondary andquaternarystructure packingCapping residuesThe helix(i i+4)Other helix types!(310, )

N

C


-Sheets

Multiple strands sheet

Parallel vs. antiparallelTwist

FlexibilityVs. helicesFoldingStructure propagation(amyloids)Other…

Thioredoxin


-Sheets



FlexibilityVs. helicesFoldingStructure propagation(amyloids)Other…


-Sheets



Strand interactionsare non-local

FlexibilityVs. helicesFolding

Antiparallel Parallel


Turns, Loops & Bends Revisited

Between helicesand sheets

On protein surface

Intrinsically“unstructured”proteins


Structure LevelsPrimary structure = Sequence

Secondary Structure = Helix,sheets/strands, loops & turns

Structural Motif = Small,recurrent arrangement ofsecondary structure, e.g.

Helix-loop-helixBeta hairpinsEF hand (calcium binding motif)Etc.

Tertiary structure = Arrangementof Secondary structure elements

MSSVLLGHIKKLEMGHS…


Myoglobin

Hemoglobin

Quaternary Structure

Assembly ofmonomers/subunitsinto protein complex

Backbone-backbone,backbone-side-chain &side-chain-side-chaininteractions:

Intramolecular vs.intermolecular contacts.For ligand binding sidechains may or may notcontribute. For the latter,mutations have littleeffect.


Grouping Amino Acids

Livingstone & Barton, CABIOS, 9, 745-756, 1993

A – AlaC – CysD – AspE – GluF – PheG – GlyH – HisI – IleK – LysL – Leu

M – MetN – AsnP – ProQ – GlnR – ArgS – SerT – ThrV – ValW – TrpY - Tyr


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


Proteins Are PolypeptidesThe peptide bond A polypeptide chain


Ramachandran Plot

Allowed backbone torsion angles in proteins

N

H


Torsion Angles


Ramachandran Plots


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

Engineering Thermostability

Example: Serpin (serineprotease inhibitor)OverpackingBuried polar groupsCavities


Experimental Methods

Crystallography&

NMR spectroscopy


X-ray crystallographyNuclear Magnetic Resonance (NMR)Modelling techniques

More exotic techniquesCryo electron microscopy (Cryo EM)Small angle X-ray scattering (SAXS)Neutron scattering

Methods for StructureDetermination


X-ray Crystallography

No size limitation.Protein molecules are ”stuck” in a crystallattice.Some proteins seem to be uncrystallizable.Slow.

Especially suited for studying structuraldetails.


X-rays

Fourier transform


The Importance of Resolution

high

low4 Å

2 Å

3 Å

1 Å


Key Parameters

ResolutionR values

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

B factorsContributions 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. parametersRamachandran plot


NMR Spectroscopy

Upper limit for structure determinationcurrently ~50 kDa.Protein molecules are in solution.Dynamics, protein folding.Slow.

Especially suited for studies of proteindynamics of small to medium size proteins.


NMR Basics

NMR is nuclear magnetic resonance

NMR spectroscopy is done on proteins INSOLUTION

Only atoms 1H, 13C, 15N (and 31P) can be detectedin NMR experiments

Proteins up to 30 kDa

Proteins stable at high concentration (0.5-1mM),preferably at room temperature


NMR Spectroscopy


Well-defined structuresRMSDs < 0.6 Å

Evalutation of NMR Structures

Atomic backbone RMSD:

Less well-defined structuresRMSDs > 0.6 Å

3GF1, Cooke et al. Biochemistry, 19911T1H, Andersen et al. JBC, 2004

( )n

xxRMSD

n

ii

= 1

2'


Evaluation of NMR Structures

What regions in the structure are most well-defined?

Look at the pdbensembles to seewhich regions arewell-defined

1RJH

Nielbo et al, Biochemistry, 2003


Which Structural Model?

Normally NMR structure models are listedaccording to the total energy and thenumber of violations.Model 1 in the PDB file is often the one withlowest energy and fewest violations.Use that model as template for modelling.


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 dynamicscharacterization with NMR


Holdings of the Protein DataBank (PDB):

The PDB also containsnucleotide and nucleotideanalogue structures.

PDB

Sep. 2001 May 2006 Oct. 2007X-ray 13116 30860 39706NMR 2451 5368 6862Other 338 200 250Total 15905 36428 46818


Summary

In evolution structure is conserved longerthan both function and sequence.

X-ray crystallographyProteins in crystallatticeMany details – onemodelResolution, R-values,Ramchandran plot

NMR spectroscopyProteins in solutionFewer details – manymodelsViolations, RMSD,Ramachandran plot


LinksPDB (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


Other Courses

27617Protein Structure and Computational BiologyMaster’s level course13 weeks, spring semester5 ECTSMax. 40 students

function, evolution & experimental methods - cbs · center for biological sequence...

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