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Introduction to Bioinformatics

Lecture 14: Protein Folding

Centre for Integrative Bioinformatics VU (IBIVU)

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Introduction to Protein Structure

• Great book covering basics of Protein Structure:

– Short Introduction to Molecular Structures

– “Introduction to Protein Structure”

• Chapters 1 to 5• Carl Branden &

John ToozeISBN: 0-8153-2305-0

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Prelude: molecular structures

• John Dalton (1810)A new system of chemistry

• Elements, but no structures yet

• Mendeljev (1869)

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Johannes van ’t Hoff

• Chimie dans l’Espace“Proposal for the development of three-dimensional chemical structural formulae” (1875)

• Tetraedrical carbon atom

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Linus Pauling (1951)

• Atomic Coordinates and Structure Factors for Two Helical Configurations of

Polypeptide Chains

• Alpha-helix

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James Watson & Francis Crick (1953)

• Molecular structure of nucleic acids

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James Watson & Francis Crick (1953)

• Molecular structure of nucleic acids

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analysis and prediction

The building blocks:

•Chains of amino acids

•Three-dimensional Structures

•Four levels of protein architecture

•Amino acids: classes

•Disulphide bridges

•Histidine

•Proline

•Ramachandran plot: mainchain dihedral angles

•Rotamers: sidechain dihedral angles

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The Building Blocks (proteins)

• Proteins consist of chains of amino acids• Bound together through the peptide bond• Special folding of the chain yields structure• Structure determines the function

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Chains of aminoacids

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Three-dimensional Structures

• Four hierarchical levels of protein architecture

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Aminoacids: physicochemical classes

• Hydrophobic aminoacidsAlanine Ala A Valine Val V Phenylalanine Phe F Isoleucine Ile ILeucine Leu L Proline Pro PMethionine Met M

• Charged aminoacids

Aspartate (-) Asp D Glutamate (-) Glu E Lysine (+) Lys K Arginine (+) Arg R

• Polar aminoacids

Serine Ser S Threonine Thr TTyrosine Tyr Y Cysteine Cys CAsparagine Asn N Glutamine Gln Q Histidine His H Tryptophane TrpW

• Glycine (sidechain is only a hydrogen)Glycine Gly G

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Disulphide bridges

• Two cysteines can form disulphide bridges• Anchoring of secondary structure elements

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Ramachandran plot

• Only certain combinations of values of phi (and psi (angles are observed

phi

phipsi

psi

omega

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Rotamers: highly populated combinations of side-chain dihedral angles

Rotamers •are amino acid sidechain dihedral angles, numbered 1, 2, 3,... going outward from C atom •different numbers of -angles depending on amino acid type•are usually defined as low energy side-chain conformations. •the use of a library of rotamers allows the modeling of a structure while trying the most likely side-chain conformations, saving time and producing a structure that is more likely to be correct.

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analysis and prediction

Motifs of protein structure

• Secundary structure elements

• Renderings of proteins

• Alpha helix

• Beta-strands & sheets

• Turns and motifs

• Domains formed by motifs

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Motifs of protein structure

• Global structural characteristics:– Outside hydrophylic, inside hydrophobic (unless…)– Often globular form (unless…)

Artymiuk et al, Structure of Hen Egg White Lysozyme (1981)

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Secundary structure elements

Alpha-helix Beta-strand

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Renderings of proteins

• Irving Geis:

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Renderings of proteins

• Jane Richardson:

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Alpha helix

• Hydrogen bond: from N-H at position n, to C=O at position n-4 (‘n-n+4’)

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Other helices

• Alternative helices are also possible

– 310-helix: hydrogen bond from N-H at position n, to C=O at position n-3

• Bigger chance of bad contacts– -helix: hydrogen bond from N-H at position n, to

C=O at position n-4– -helix: hydrogen bond from N-H at position n, to

C=O at position n-5• structure more open: no contacts• Hollow in the middle too small for e.g. water• At the edge of the Ramachandran plot

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Helices

• Backbone hydrogen bridges form the structure– Often covers hydrophobic centre of protein

• Sidechains point outwards (‘Xmas tree’)– Possibly: one side hydrophobic, one side

hydrophylic (amphipathic helices)

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Beta-strands: beta-sheets

• Beta-strands next to each other form hydrogen bridges

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Parallel or Antiparallel sheets

Anti-parallel

Parallel

• Usually only parallel or anti-parallel

• Occasionally mixed• Sidechains alternating

(up-down)

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Turns and motifs

• Between the secundary structure elements are loops• Very short loops between two -strands: turn

• Different secondary structure elements often appear together: motifs– Helix-turn-helix– Calcium binding motif– Hairpin– Greek key motif– -motif

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Helix-turn-helix motif

• Helix-turn-helix important for DNA recognition by proteins

• EF-hand: calcium binding motif

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Hairpin / Greek key motif

• Different possible hairpins : type I/II

• Greek key:anti-parallel beta-sheets

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motif

• Most common way to obtain parallel -sheets

• Usually the motif is ‘right-handed’

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Domains formed by motifs

• Within protein different domains can be identified– For example:

• ligand binding domain• DNA binding domain• Catalytic domain

• Domains are built from motifs of secondary structure elements

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Alpha/beta barrels

• TIM barrel after triosephosphate isomerase• Usually 8 -strands, at least 200 aminoacids• Often hydrophobic interior

– alternating amino acids in the strands

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Alpha/beta barrels

• Active site formed by (variable) loop regions at top of the barrel• Exception:

active site in the core of methylmalonyl-coenzyme A mutase

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Summary

• Aminoacids form polypeptide chains• Chains fold into three-dimensional structure• Specific backbone angles are permitted or not:

Ramachandran plot• Secundary structure elements:

-helix, -sheet• Common structural motifs:

Helix-turn-helix, Calcium binding motif, Hairpin, Greek key motif, -motif

• Combination of elements and motifs: tertiary structure

• Many protein structures available: PDB

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Sequence

Structure

Function

Inverse folding,

Threading

Ab initio

BLAST

Folding: impossible but for the smallest structures

Function prediction from structure – very difficult

Knowledge based

Sequence-Structure-FunctionWhat can we do with bioinformatics?

•Ab initio prediction (based on first principles) is still not generally succesful (red)

•Many Bioinformatics methods are therefore knowledge-based (green)

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Active protein conformation

• Active conformation of protein is the native state• unfolded, denatured state

– high temperature– high pressure– high concentrations urea (8 M)

• Equilibrium between two forms

Denatured state Native state

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Anfinsen’s Theorem (1950’s)

• Primary structure determines tertiary structure.In the mid 1950’s Anfinsen began to concentrate on the problem of the relationship between structure and function in enzymes. […] He proposed that the information determining the tertiary structure of a protein resides in the chemistry of its amino acid sequence. […] It was demonstrated that, after cleavage of disulfide bonds and disruption of tertiary structure, many proteins could spontaneously refold to their native forms. This work resulted in general acceptance of the ‘thermodynamic hypothesis’ (Nobel Prize Chemistry 1972)."

www.nobel.se/chemistry/laureates/1972/anfinsen-bio.html

• Anfinsen performed un-folding/re-folding experiments

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Dimensions: Sequence Space• How many sequences of length n are possible?

N(seq) = 20 • 20 • 20 • … = 20n

e.g. for n = 100, N = 20100 10130, is nearly infinite– Only a subset of these will fold in a stable

conformation

• The probability p of finding twice the same sequence is p = 1/N, e.g. 1/10130

is nearly zero.

• Evolution: divergent or convergent– sequences are dissimilar,

in divergent and particularly in convergent evolution

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Dimensions: Fold Space• How many folds exist?

– Sequences cluster into sequence families and fold families

– some have many members, some few or only one:

• Using Zipf’s law:

n(r) = a / rb

• For sequence families:

b 0.64 ntotal 60000

• For fold families:

b 0.8 ntotal 14000

r is the rank of family, n(r) is the number of proteins in the r-th family, a is a scaling constant, depending on the number of proteins in the dataset. Constant b does not depend on the size of the dataset.

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Levinthal’s paradox (1969)

• Denatured protein re-folds in ~ 0.1 – 1000 seconds

• Protein with e.g. 100 amino acids each with 2 torsions ( en )

Each can assume 3 conformations (1 trans, 2 gauche)

3100x2 1095 possible conformations!

• Or:100 amino acids with 3 possibilities in Ramachandran plot (, , L): 3100 1047 conformations

• If the protein can visit one conformation in one ps (10-12 s) exhaustive search costs 1047 x 10-12 s = 1035 s 1027 years!(the lifetime of the universe 1010 years…)

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Levinthal’s paradox

Protein folding problem:– Predict the 3D structure from sequence– Understand the folding process

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From 1D to 3D…

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What to fold?…fastest folders

1

10

100

1000

10000

100000

Nanose

con

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CPU

-days

10

60

1

CPU

years

PPA alphahelix

betahairpinBBA5 villin

Pande et al. “Atomistic Protein Folding Simulations on the Submillisecond Time Scale Using Worldwide Distributed Computing” Biopolymers (2003) 68 91–109

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Rates: predicted vs experiment

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000experimental measurement

(nanoseconds)

Pre

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tim

e

(n

an

osecon

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PPA

alpha helix

betahairpin

villin

BBAW

Experiments:

villin: Raleigh, et al, SUNY, Stony Brook

BBAW:Gruebele, et al, UIUC

beta hairpin: Eaton, et al, NIH

alpha helix: Eaton, et al, NIH

PPA: Gruebele, et al, UIUC

Predictions:Pande, et al, Stanford

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Molten globule

• First step: hydrophobic collapse• Molten globule: globular structure, not yet correct folded• Local minimum on the free energy surface

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Folded state

• Native state = lowest point on the free energy landscape

• Many possible routes • Many possible local minima (misfolded structures)

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Folding energy

• Each protein conformation has a certain energy and a certain flexibility (entropy)

• Corresponds to a point on a multidimensional free energy surface

Three coordinates per atom3N-6 dimensions possible G = H – TS

In very rough generalities:

H relates to bond formation/breaking

S relates to configurational freedom and water ordering

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Hydrophobic Effect

Fundamental:The Hydrophobic Effect is a Solvent Effect

+Oil Water Oil

How is interfacial waterlayer ordered?

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Hydrophobic Effect in Protein Folding

Unfolded

More Hydrocarbon-Water Interfacial Area,

More Water Ordered

Less Hydrocarbon-WaterInterfacial Area,

Less Water Ordered

Folded

S = +HOH HOH

+

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Helper proteins

• Forming and breaking disulfide bridges– Disulfide bridge forming enzymes: Dsb– protein disulfide isomerase: PDI

• “Isomerization” of proline residues– Peptidyl prolyl isomerases

• Chaperones– Heat shock proteins– GroEL/GroES complex– Preventing or breaking

‘undesirable interactions’…

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Disulfide bridges

• Equilibriums during the folding process

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Proline: two conformations• Peptide bond nearly always trans (1000:1)

• For proline cis conformation also possible (trans:cis equilibrium = 4:1)

• For folding, all prolines need to be in trans conformation --Isomerization is bottleneck, cyclophilin catalyses

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Chaperones

• During folding process hydrophobic parts outside?– Risk for aggregation of proteins

• Chaperones offer protection– Are mainly formed at high temperatures (when needed)– Heat-shock proteins: Hsp70, Hsp60 (GroEL), Hsp10 (GroES)

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GroEL/GroES complex

• GroEL:– 2 x seven subunits in a ring– Each subunit has equatorial, intermediate and apical domain– ATP hydrolyse, ATP/ADP diffuse through intermediate domain

• GroES:– Also seven subunits– Closes cavity of GroEL

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GroEL/GroES mechanism

• GroES binding changes both sides of GroEL– closed cavity– open cavity

• cycle– protein binds side 1– GroES covers, ATP binds– ATP ADP + Pi– ATP binds side 2– ATP -> ADP + Pi

• GroES opens• folded protein exits• ADP exits

– New protein binds

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Alternative folding: prions

• Prion proteins are found in the brains

• Function unknown • Two forms

– normal alpha-structure– harmful beta-structure

• beta-structure can aggregate and form ‘plaques’– Blocks certain tissues and

functions in the brains

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Protein flexibility

• Also a correctly folded protein is dynamic– Crystal structure

yields average position of the atoms

– ‘Breathing’ overall motion possible

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B-factors

• The average motion of an atom around the average position

alpha helicesbeta-sheet

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Protein Tertiary Structure Tied to Function

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Conformational changes

• Often conformational changes play an important role for the function of the protein

• Estrogen receptor – With activator (agonist) bound: active– With inactivator (antagonist) bound: not active

active inactive

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Main points

• Anfinsen: proteins fold reversibly!• Levinthal: too many conformations for fast folding?

– First hydrophobic collapse, then local rearrangement• Protein folding funnel

– Assistance with protein folding• Sulphur bridge formation• Proline isomerization• Chaperonins

• Intrinsic flexibility: Breating / Conformational change– Conformational changes for

• Activation / Deactivation