Proteins Proteins -- Structure, Structure, folding and domainsfolding and domains

Tommi Tommi KajanderKajanderXX--ray crystallography labray crystallography labInstitute of BiotechnologyInstitute of BiotechnologyUniversity of HelsinkiUniversity of Helsinki

OutlineOutline

basics on proteinsbasics on proteinsstability and foldingstability and foldingstructures and domainsstructures and domainsXX--ray crystallographyray crystallography

protein structureprotein structure

primary structure, amino primary structure, amino acid sequence, the peptide acid sequence, the peptide bond, polypeptide runs from bond, polypeptide runs from amino(N) to amino(N) to carboxy(Ccarboxy(C))--terminus of the chain. terminus of the chain.

secondary structure, tertiary secondary structure, tertiary structure (the folded state) structure (the folded state) + + oligomericoligomeric state state (quaternary structure).(quaternary structure).

cellular functionscellular functions

produced by living cells, translated from produced by living cells, translated from the encoding gene. the encoding gene. may function inside the cell (in its various may function inside the cell (in its various compartments, e.g. nucleus compartments, e.g. nucleus --DNA binding DNA binding proteinsproteins-- or or cytosolcytosol or on the cell or on the cell membrane or be secreted out (e.g. membrane or be secreted out (e.g. microbial enzymes such as microbial enzymes such as cellulasescellulases etc, etc, animals growth factors, antibodies, animals growth factors, antibodies, lysozymelysozyme, proteases etc (food processing in , proteases etc (food processing in the stomach, gut). the stomach, gut).

Protein functional classesProtein functional classes

enzymesenzymesstructural proteins (collagen etc)structural proteins (collagen etc)ion channels ion channels transporters (hemoglobin, throughtransporters (hemoglobin, through--membrane transport)membrane transport)immune system proteins (binding)immune system proteins (binding)other binding proteins (growth factors, other binding proteins (growth factors, DNA binding proteins, chaperones)DNA binding proteins, chaperones)often functionalities are specific to specific often functionalities are specific to specific domains (domains (see belowsee below))

Building blocksBuilding blocks

the 20 natural Lthe 20 natural L--amino acids (no Damino acids (no D--amino acids) amino acids) the peptide bondthe peptide bondamino and amino and carboxycarboxy--termini, direction of termini, direction of the polypeptidethe polypeptidezwitterions, zwitterions, chiralchiralwith different “side with different “side chains”chains”

The 20 amino acidsThe 20 amino acids

20 natural amino acids encoded by DNA (one 20 natural amino acids encoded by DNA (one amino acid per base triplet, 64 triplets)amino acid per base triplet, 64 triplets)all Lall L--amino acids (stereochemistry), amino acids (stereochemistry), chiralchiralaround around CCα−α−atom.atom.hydrophobic and polar and charged.hydrophobic and polar and charged.what does this mean, implications? learn them!what does this mean, implications? learn them!some with special properties (some with special properties (GlyGly, Pro, , Pro, CysCys..)...).atom nomenclature: atom nomenclature: –– heavy atoms:heavy atoms: N,C,O, CN,C,O, Cα (α (peptide unit) + peptide unit) + side chain side chain

atoms Catoms Cββ, C, Cγγ, X, Xδδ etc with distance from etc with distance from CCα.α.–– some side chains are branched (numbering 1,2..)some side chains are branched (numbering 1,2..)–– three letter and one letter codes (three letter and one letter codes (GlysineGlysine, , GlyGly, G), G)

hydrophobic amino acidshydrophobic amino acids

inside proteinsinside proteinsthe hydrophobic core, the hydrophobic core, sticky binding pockets.sticky binding pockets.aromatic rings, aromatic rings, stacking. delocalization stacking. delocalization of the of the ππ--electronselectrons

(Tyr, (Tyr, TrpTrp, hydrogen , hydrogen bonds)bonds)volume and shapevolume and shapeProlineProline ring, ring, iminoimino acidacid

polar and chargedpolar and charged

SulphurSulphur containing amino acidscontaining amino acids

• Cysteine and methionine

• Cys the most reactive amino acid, thiol group can oxidize and deprotonate, pKa ca 8-9.

• disulhide brigdes, structurally important. also non-spesificdimerization/aggregation via free -SH groups

•Met potentially nucleophilic, hydrophobic.

Acidic groupsAcidic groups

ApsAps, , GluGlu. . pKapKa = 4 can vary with environment = 4 can vary with environment substantially (proteins tune the substantially (proteins tune the functional group functional group protonationsprotonations))active sites/enzymesactive sites/enzymesmetal ion binding (also other polar metal ion binding (also other polar residues (e.g His and transition residues (e.g His and transition metals)metals)acid/base catalysisacid/base catalysisthermostabilitythermostability/ionic networks/ionic networks

Basic groups: Basic groups: ArgArg, Lys, , Lys, HisHis

positively charged under positively charged under physiological pH (neutral)physiological pH (neutral)His, metal coordination and His, metal coordination and catalysiscatalysispKpK around 6around 6--7, 7, protonationprotonation state state variable (mostly not charged?)variable (mostly not charged?)Lys, catalytic base in active sitesLys, catalytic base in active sitesArgArg, ion, ion--pairs, electrostatic pairs, electrostatic interactions in active sites and interactions in active sites and ligandligand (e.g PO(e.g PO44) binding) binding

Other polarOther polar

AsnAsn, , GlnGln–– hyrdogenhyrdogen bonding, bonding, AsnAsn

can can deaminatedeaminate..–– AsnAsn, N, N--linked linked

glycosylationglycosylation of proteinsof proteins

Ser, Ser, ThrThr, Tyr, Tyr–– HH--bonds, Ser in bonds, Ser in catalysicatalysi, ,

Ser, Ser, ThrThr, Tyr are , Tyr are phosporylatedphosporylated and and dephosphorylateddephosphorylated in in cellular cellular signallingsignalling events events (most common (most common signallingsignalling method)method)

““turn” residuesturn” residuesGlysineGlysine, , ProlineProline–– common in turns, break secondary structure (not always)common in turns, break secondary structure (not always)–– Pro is an Pro is an iminoimino acid, generates kinks, if not turns.acid, generates kinks, if not turns.–– both both destablizedestablize secondary structure in any case (helices and secondary structure in any case (helices and

strands)strands)–– Pro and hydroxylPro and hydroxyl--Pro in collagen triple helix (also Pro in collagen triple helix (also GlyGly).).–– GlyGly has NO side chain.has NO side chain.–– Pro is really an Pro is really an iminoimino acid acid ciscis or trans form, or trans form, usuallyusually trans. Pro is rigid, trans. Pro is rigid, GlyGly can have variable peptide bondcan have variable peptide bondphi and phi and psipsi angles.angles.

pKapKa, , pIpI

pKapKa --loglog1010 of the acid dissociation constant:of the acid dissociation constant:–– KKaa = [H+][A= [H+][A--]/[HA] for HA <]/[HA] for HA <--> H+ + A> H+ + A--

pIpI, the , the isoelectricisoelectric point, net charge of protein is zero.point, net charge of protein is zero.–– can be calculated from sequence (can be calculated from sequence (googlegoogle protparamprotparam), ),

approximately, or experimentally ,by approximately, or experimentally ,by isoelectricisoelectric focussingfocussing. . –– so this tells whether protein has + or so this tells whether protein has + or -- charge at specific pH.charge at specific pH.–– implications on solubility?implications on solubility?

ppKKaass of amino acidsof amino acids

residue residue ppKKaa physiological pH (7.4)physiological pH (7.4)AspAsp 4.54.5 charged(charged(--))GluGlu 4.64.6His 6.2His 6.2 neutral/chargedneutral/chargedCysCys 9.19.1--9.59.5 --SH(neutrSH(neutr.).)TyrTyr 9.79.7Lys Lys 10.410.4 --NH3+ charged(+)NH3+ charged(+)ArgArg ca. 12ca. 12 charged(+)charged(+)

roles of residues in roles of residues in proteinsproteins

hydrophilic out, hydrophobic inhydrophilic out, hydrophobic in (or more (or more precisely the groups not necessarily whole side precisely the groups not necessarily whole side chains, chains, e.g.e.g. lysine has a long aliphatic lysine has a long aliphatic hydrocarbon chain)hydrocarbon chain)functional/active site specificity is in functional/active site specificity is in shapeshape(conformation and hydrophobic) (conformation and hydrophobic) hydrogen hydrogen bonding bonding (polar/charged)(polar/charged)catalytic residues (acids, bases, catalytic residues (acids, bases, nucleophilesnucleophiles, , metal ion binding)metal ion binding)

about enzymesabout enzymestypes of catalysis:types of catalysis:–– general acid/base catalysis (Lys, Asp, general acid/base catalysis (Lys, Asp, GluGlu, His), His)–– electrostatic catalysis (exclusion of solvent)electrostatic catalysis (exclusion of solvent)–– nucleophilicnucleophilic and and electrophilicelectrophilic catalysis catalysis

Ser, Ser, CysCys (proteases) most common (proteases) most common nucleophilesnucleophiles, , metals as metals as electrophileselectrophiles

(+ Reduction of entropy on binding, high effective (+ Reduction of entropy on binding, high effective concentration, catalysis in preordered active concentration, catalysis in preordered active site)site)

The Serine protease catalytic triad (electron transfer chain)

amino acids in the amino acids in the polypeptide contextpolypeptide context

polypeptide backbone + side chains of polypeptide backbone + side chains of the amino acid residues = protein the amino acid residues = protein

protein synthesisprotein synthesis

peptide bonds are formed (in cells) on the peptide bonds are formed (in cells) on the ribosome on translation of the of ribosome on translation of the of messengerRNA(mRNAmessengerRNA(mRNA), amino acid gets ), amino acid gets transferedtransfered from the from the transferRNA(tRNAtransferRNA(tRNA) and ) and added to the polypeptide Cadded to the polypeptide C--terminus. terminus.

Structure formationStructure formationproperties of the properties of the side chainsside chains determine the higher order determine the higher order structure of proteins, and functionality, mostly.structure of proteins, and functionality, mostly.hydrogen bonds between and from the peptide “backbone” hydrogen bonds between and from the peptide “backbone” amide and carbonyl groups are important for secondary amide and carbonyl groups are important for secondary structure (still defined by side chains)structure (still defined by side chains)peptide bond is planarpeptide bond is planar (important!)(important!)proteins can be a) proteins can be a) fibriousfibrious/filamentous /filamentous (collagen, silk, (collagen, silk, muscles myosin and muscles myosin and actinactin), b) ), b) solublesoluble (e.g. enzymes, growth (e.g. enzymes, growth factors, insulin etc etc), or in the cell lipid membrane c) factors, insulin etc etc), or in the cell lipid membrane c) “membrane proteins”“membrane proteins” (hydrophobic/lipid soluble) (ion (hydrophobic/lipid soluble) (ion channels and pumps, control of cell channels and pumps, control of cell homeostatishomeostatis, transporters , transporters and receptors (Gand receptors (G--protein coupled receptors, protein coupled receptors, signallingsignalling), ), typically cell surface receptors have a typically cell surface receptors have a intramembraneintramembrane domain domain + + extracellularextracellular ligandligand binding domain + intracellular region binding domain + intracellular region for for signallingsignalling inside the cell.inside the cell.

Secondary structureSecondary structure

fold/structure of the protein stabilized in fold/structure of the protein stabilized in secondary structure, secondary structure, αα--helices or helices or ββ--strandsstrandssequencialsequencial arrangement (topology) and arrangement (topology) and spacialspacial organization of these elements organization of these elements defines the FOLD of the proteindefines the FOLD of the proteinamino acids have different propensities for amino acids have different propensities for forming particular secondary structure (e.g. forming particular secondary structure (e.g. Ala and nonAla and non--β β brancedbranced residues in residues in αα--heliceshelices

stericsteric restraintsrestraints

RamachandranRamachandran plot of polypeptide plot of polypeptide main chain variable angles (phi, main chain variable angles (phi, psipsi))only a restricted set of conformations only a restricted set of conformations available for different secondary available for different secondary structures (and amino acids)structures (and amino acids)stericsteric effects, due to side chains effects, due to side chains ----> > GlyGly has most freedom, hence has most freedom, hence important in turns.important in turns.

polypeptide “backbone”polypeptide “backbone”anglesangles

3 main chain 3 main chain dihedralsdihedralsOnly two really varyOnly two really vary–– Create all of the Create all of the

backbone structures backbone structures seen in proteins.seen in proteins.

RamachandranRamachandran plotplot

Beta strands Beta strands -- spread outspread out

Alpha (Alpha (lhlh) helices ) helices --mostly mostly glycineglycineAlpha (Alpha (rhrh) helices ) helices --bunched upbunched up>90% of residues should >90% of residues should be in most be in most favouredfavouredareasareasGlycinesGlycines in many areasin many areasfrom MLE, code 1MUC

αα--helixhelix3.6 residues/turn3.6 residues/turnmain chain hmain chain h--bondingbondingfrom i to i+4. from i to i+4. 1.5 Å rise/residue1.5 Å rise/residue5.4 Å rise/turn5.4 Å rise/turnIndependent Independent stabilization by Hstabilization by H--bonding.bonding.SidechainsSidechains point back point back like a Christmas tree.like a Christmas tree.helices often have helices often have different facesdifferent faceshelix dipole/capping helix dipole/capping (N/C)(N/C)

helical wheel and packinghelical wheel and packingamphilicityamphilicity, binary pattern (polar/aliphatic, , binary pattern (polar/aliphatic, helical packing), “helical packing), “heptadheptad repeat”repeat”packing: “grooves to ridges” (in real life more packing: “grooves to ridges” (in real life more variable, variable, Bowie Nat. Bowie Nat. StructStruct. Biol. 1997. Biol. 1997))

ββ--strandstrand

FlatFlat--ishishTwo varietiesTwo varieties–– parallelparallel–– antiparallelantiparallel

Often distortedOften distorted–– Twisted, BulgedTwisted, Bulged

Certain proteins can also Certain proteins can also form form amyloidamyloid fibrils fibrils =“infinite Beta=“infinite Beta--sheets” sheets” ((Altzheimers’sAltzheimers’s, , prionsprions, , other other amyloidamyloid diseases, diseases, e.g.e.g. lysozymelysozyme and betaand beta--micromicro--globulin and globulin and transerythrintranserythrin variants)variants)

antiparallelantiparallel ββ--sheetsheet

ββ--turnsturns

• nearly 1/3 of residues involved in turns (various types)

•hairpin loops, connect beta-strands

• classification (here, type I and II), torsion angles

SupersecondarySupersecondary structurestructure

Regular secondary Regular secondary structure patternsstructure patterns–– Sometimes Sometimes

functionalfunctionalHTH motif (DNA HTH motif (DNA binding element)binding element)

–– Sometimes Sometimes structuralstructural

ββ−−αα−−ββ β− β−hairpinhairpin

sequence analysissequence analysis

conserved domains or motifs (web tools).conserved domains or motifs (web tools).membrane vs. soluble proteinsmembrane vs. soluble proteins–– hydrophobicityhydrophobicity plots(scales), again the www.plots(scales), again the www.–– types of membrane proteins: (alphatypes of membrane proteins: (alpha--helical e.g. helical e.g.

77--TM proteins, ionTM proteins, ion--channels etc), betachannels etc), beta--barrel, barrel, singlesingle--span alphaspan alpha--helical (many receptors and helical (many receptors and adhesion molecules).adhesion molecules).

GABA-transporter

domains..domains..

DomainsDomains

Defined as independentlyDefined as independently--foldable units.foldable units.Often mark different functional units.Often mark different functional units.Separated by linker regions.Separated by linker regions.

MLE, code 1MUCGoldman, Helin et al.

DomainsDomains

Mammalian proteins Mammalian proteins often are “beads on often are “beads on a string”a string”–– Repeated domains Repeated domains

often occuroften occur–– Different functions, Different functions,

structural variationsstructural variations–– See e.g. SMART or See e.g. SMART or

PFAM databases PFAM databases (Google) for domain (Google) for domain compositions of compositions of proteinsproteins

Domains 19-20 of Factor HJokiranta et al & Goldman, EMBO J, 2006

Proteins FoldsProteins Folds

Some protein folds are unique, but the “foldSome protein folds are unique, but the “fold--space” space” must be limited (must be limited (structural genomicsstructural genomics attempsattemps to to map this).map this).many proteins have several domainsmany proteins have several domainsMost proteins fold into alreadyMost proteins fold into already--known structuresknown structures–– Even Even when there is no sequence homology (<10when there is no sequence homology (<10--20%)20%)–– Structure preserved Structure preserved much much longer than sequence.longer than sequence.

Some common foldsSome common folds–– β/αβ/α barrelbarrel–– 77--TM receptorTM receptor–– 44--helix bundlehelix bundle−− ββ--sanwichsanwich domains (immunoglobulindomains (immunoglobulin--like) etc.like) etc.

β/αβ/α barrelbarrel

β/αβ/α barrelbarrel–– 40% of unique 40% of unique

proteinsproteins–– Usually enzymesUsually enzymes

–– 8 repeated 8 repeated β/αβ/α unitsunits

77--TM foldTM fold

77--TM foldTM fold–– Example is Bovine Example is Bovine

rhodopsinrhodopsin–– >500 such folds in >500 such folds in

humanshumans–– 7 7 transmembranetransmembrane

heliceshelices–– Signal transductionSignal transduction

signals very variedsignals very varied

PDB code: 1F88

Repeat proteinsRepeat proteins

Ankyrin repeat

TetratricopeptideRepeat (TPR)

HEAT repeat

-protein-protein interactions in various cellular contexts

-structural scaffolds

Leucine rich repeat

TheThe ββ--propeller propeller (a repeat (a repeat protein of sorts)protein of sorts)

Neurospora crassa CMLE, Kajander et al. (2002)

-All β-sheet-4-7 ”blades” with 4 strands-Neuraminidase, WD-40 repeat proteins(Gproteins).-binding site oftenin the center

β-structures:”Sandwich”, barrels, propellers

The The ββ−−sandwich foldsandwich fold

-Antibodies (Immunoglobulins), adhesion molecular and receptors NCAM, ICAM etc, RAGE, FGFR etc.

Antibody structureAntibody structure

multidomainmultidomain structure..structure..

integrin αvβ3 Hsp90 complex with p23

protein complexesprotein complexesStableStable–– RibosomesRibosomes ((smallsmall and and largelarge subunitsubunit) () (proteinprotein--

RNARNA))–– FoF1 FoF1 ATPaseATPase (ATP (ATP synthasesynthase, , protonproton pumppump))–– ProteosomeProteosome, , etcetc etc.etc.

TransientTransient–– NucleicNucleic acidacid polymerasepolymerase complexescomplexes ((proteinprotein--

DNADNA))–– chaperoneschaperones and and theirtheir ((unfoldedunfolded) ) substratesubstrate

proteisproteis–– signallingsignalling complexescomplexes (e.g (e.g GG--proteinsproteins and and theirtheir

receptorsreceptors))

viral viral capsidscapsids

Protein foldingProtein folding

““the protein folding problem” the protein folding problem” –– complex, complex, vsvs e.g. DNA/RNA structuree.g. DNA/RNA structure–– Can’t predict the structure Can’t predict the structure abab initio initio –– but there are cases of success for small but there are cases of success for small

proteinsproteins

what what stabilisesstabilises the folded (native) state the folded (native) state driving forces driving forces mechanismsmechanisms

protein foldingprotein foldingLevinthal’sLevinthal’s paradoxparadox–– folding via sampling all conformations (contact order) would takfolding via sampling all conformations (contact order) would take e

more more than age of the universe than age of the universe ----> folding landscape/not all conformations > folding landscape/not all conformations are explored. are explored.

–– Original example: 3Original example: 3100 = 100 = 5 X 105 X 1047, 47, 10101313 per second, or 3 x 10per second, or 3 x 102020 per year, it per year, it will take 10will take 102727 years….years….

hydrophobic hydrophobic vsvs hydrophilic:hydrophilic:–– the most basic idea of protein folding is binary patterning the most basic idea of protein folding is binary patterning

(used in (used in protein designprotein design of helical bundles, most simple case). of helical bundles, most simple case). KamtekarKamtekar et al & , Hecht MH. Protein design by binary et al & , Hecht MH. Protein design by binary patterning of polar and patterning of polar and nonpolarnonpolar amino acids. amino acids. ScienceScience (1993)(1993)

folded/native statefolded/native state

free energy minimum, typical free energy minimum, typical functional proteins have a single functional proteins have a single defined native state defined native state hydrophobic and aromatic residues hydrophobic and aromatic residues inside inside charged/polar residues on the outside charged/polar residues on the outside OR hydrogen bonded (OR hydrogen bonded (solvationsolvation by by the protein)the protein)

hydrophobic and aromatic residues inside hydrophobic and aromatic residues inside charged/polar residues on the outside OR hydrogen charged/polar residues on the outside OR hydrogen bonded (bonded (solvationsolvation by the protein)by the protein)–– one observed feature of one observed feature of thermophilicthermophilic proteins is increased proteins is increased

number and extent of ionic networks number and extent of ionic networks minimal “alphabet” designs (polar, hydrophobic, turn, e.g minimal “alphabet” designs (polar, hydrophobic, turn, e.g funcitonalfuncitonal SH3 domain by Riddle et al & Baker D. amino SH3 domain by Riddle et al & Baker D. amino acids: Ile, Lys, acids: Ile, Lys, GluGlu, Ala, , Ala, GlyGly))Both cases used combinatorial methods (selection or Both cases used combinatorial methods (selection or screening) from mutant libraries (screening) from mutant libraries (KamtekarKamtekar et al & Riddle et al & Riddle et al ...and others)et al ...and others)

Thermodynamics of Thermodynamics of foldingfolding

large opposing and favoring termslarge opposing and favoring terms–– the the hyrdophobichyrdophobic effect (release of water from hydrophobic effect (release of water from hydrophobic

surface, entropy driven)surface, entropy driven)–– hydrogen bonding of the backbone, favorable enthalpyhydrogen bonding of the backbone, favorable enthalpy

CooperativityCooperativity, e.g. helix and beta, e.g. helix and beta--sheet formation, main chain hydrogen bonds sheet formation, main chain hydrogen bonds define folded topology.define folded topology.all buried side chains solvated by Hall buried side chains solvated by H--bondingbonding

–– loss of conformational freedom of the protein chain loss of conformational freedom of the protein chain (unfavorable entropy)(unfavorable entropy)

–– other effects? charge burial? small proteins <100 residues, other effects? charge burial? small proteins <100 residues, sometimes stabilized by bound metals or sometimes stabilized by bound metals or disulphidesdisulphides..

SubstratctingSubstratcting two large values: two large values: proteins are marginally proteins are marginally stable (stable (ca.ca. 55--20 kcal/mol) ...and one hydrogen bond is (220 kcal/mol) ...and one hydrogen bond is (2--10 10 kcal/mol)kcal/mol)

Thermodynamic studies Thermodynamic studies of protein stabilityof protein stability

denaturationdenaturation by heat or chemical by heat or chemical denaturationdenaturation 00--6 M 6 M GuGu--HClHClCD spectroscopyCD spectroscopy (secondary structure)(secondary structure)TrpTrp--fluorescencefluorescence quenching (tertiary quenching (tertiary structure) structure)

ProteinProtein foldingfolding

CI2: collapse & formation of secondary structure simultaneously:nucleation-condensation (a basic foldon)

typically single domain proteins exhibit twotypically single domain proteins exhibit two--state unfolding with sharp state unfolding with sharp transitiontransitionmultidomainmultidomain or or oligomericoligomeric proteins not.proteins not.SPECIAL CASESPECIAL CASE (?): repeat proteins: The 1(?): repeat proteins: The 1--D D IsingIsing model and model and mechanical unfolding (stepmechanical unfolding (step--wise + wise + elasticitityelasticitity, hearing (hair cells in ear), hearing (hair cells in ear)

AFMAFM

• a two-state transition• Increase in stability with increasing number of repeats

• Increase in cooperativity (slope of transition) with number of repeats

(in addition to dominant local interactions and regular structure retained as repeats are added)

The 1D-Ising model captures this behavior (as shown for helix-coil transition by the Zimm-Bragg model):

=(spin up, +1) folded helix =(spin down, -1) unfolded helix

= description of folded/unfolded helices (in TPRs)

1D-Ising model: Stability of TPRs vs. number of TPR repeats

each “spin” up-down interaction will cost energy

Kajander et al. & Regan (2005) J. Am. Chem. Soc.

FoldingFolding statesstates

EquilbrEquilbr. U. U-->N, U>N, U-->I>I-->N (>N (endend statesstatesobservableobservable))SeenSeen byby severalseveral transitionstransitions, , orordifferencedifference in in fluorescencefluorescence and and farfar--UVUVCDCD--spectroscopyspectroscopyKineticsKinetics telltell aboutabout the the foldingfolding pathwaypathway

FoldingFolding kineticskinetics

TwoTwo--statestate kineticskinetics vsvs intermediatesintermediatesFirstFirst--orderorder kineticskinetics ifif 22--statestate

TranstionTranstion statestate theorytheory::–– kkff==υκυκ expexp ––∆∆GG‡‡/RT/RT

∆∆∆∆GG‡‡=RTln=RTln kkff’’//kkff

ChevronChevron plotsplots

• measure folding & unfolding (dilution to or from e.g. GuHCl)• deviations from: effects of intermediates, mutations / what they affect.

Folding, Folding, φφ--value analysisvalue analysisin order to study the folding pathway one needs to look at kinetin order to study the folding pathway one needs to look at kinetics (e.g. ics (e.g. trptrp--fluorescence by stoppedfluorescence by stopped--flow rapid mixing)flow rapid mixing)e.g. phie.g. phi--value analysis of mutants (value analysis of mutants (FerhstFerhst & co& co--workers)workers)Range from 0 to 1 (effect of mutation on denatured or folded staRange from 0 to 1 (effect of mutation on denatured or folded state).te).

–– 0=(already) unfolded in transition state (0=(already) unfolded in transition state (mutmut: no effect), 1=structured in : no effect), 1=structured in transition state.transition state.

Mutate away

CTPR2 & CTPR3 –NH hydrogen exchange (Main et al. & Regan PNAS 2005)

Variability in local stability

protection factor (pf) = ku/kex

H/D exchange with NMR.H/D exchange with NMR.

3D3D--Structural Techniques Structural Techniques of of biomoleculesbiomolecules

XX--ray (and neutron) scattering & crystallography.ray (and neutron) scattering & crystallography.–– Elucidates structureElucidates structure--function relationships at the atomic level. function relationships at the atomic level. –– Very large size range (40 Very large size range (40 DaDa to > 1 to > 1 MDaMDa) (ribosome, viruses)) (ribosome, viruses)

NMR spectroscopy also provides detailed structural data.NMR spectroscopy also provides detailed structural data.–– Complementary to crystallographic efforts and results.Complementary to crystallographic efforts and results.–– NMR has been restricted to molecules below 40 NMR has been restricted to molecules below 40 kDakDa. .

Electron microscopy, single particle and diffraction.Electron microscopy, single particle and diffraction.–– Low resolution, but highest molecular mass (Low resolution, but highest molecular mass (egeg ClathrinClathrin pits, pits,

whole ribosome, enveloped viruses)whole ribosome, enveloped viruses)

Strategy in protein structure Strategy in protein structure determination via Xdetermination via X--ray ray crystallographycrystallography

Obtain pure preparations in milligram quantities Obtain pure preparations in milligram quantities of the macromolecule(s) of interest.of the macromolecule(s) of interest.Screen for preliminary crystallization conditions Screen for preliminary crystallization conditions on an incomplete factorial basis. Consider the on an incomplete factorial basis. Consider the biochemistry.biochemistry.Refine crystallization conditions / Mutants / Refine crystallization conditions / Mutants / protein chemical modifications.protein chemical modifications.Collect diffraction data from suitable crystals.Collect diffraction data from suitable crystals.Solve the “Phase” problem.Solve the “Phase” problem.Analyze the electron density maps and build the Analyze the electron density maps and build the atomic model.atomic model.

Cloning Protein Expressionand Purification

Crystallization Trials

Model Buildingand Refinement

Data Collection Optimization

Landscape of Structural Biology

crystallizationcrystallization

typically by typically by vapourvapourdiffusiondiffusionalso also microdialysismicrodialysis or or “batch” (no diffusion).“batch” (no diffusion).or diffusion in a or diffusion in a capillarycapillaryuse 24/48/96 welluse 24/48/96 well--platesplatesdifferent temperatures different temperatures (4/RT/other)(4/RT/other)start with various start with various random screensrandom screens

CrystallisationCrystallisation automatinautomatin withwithroboticsrobotics

http://www.biocenter.helsinki.fi/bi/xray/automation/

CrystalsCrystals

Why do we need a Why do we need a crystal?crystal?–– Scattering from a Scattering from a

single molecule is single molecule is undetectable.undetectable.

need a latticeneed a lattice

–– Crystals have Crystals have translational translational symmetrysymmetry

–– Can resolve features Can resolve features at atomic resolution.at atomic resolution.

Crystals and SymmetryCrystals and Symmetry

Unit CellUnit Cell

Smallest object from Smallest object from which you can make which you can make the entire crystal by the entire crystal by translationtranslation along the along the edges.edges.Bounded by Bounded by lattice lattice points.points.Edges: a, b, cEdges: a, b, cAngles: Angles: αα, , ββ, , γγ

WhatWhat is in a is in a unitunit cellcell??

A A unitunit cellcell is is builtbuilt fromfrom asymmetricasymmetric unitsunits..AsymmetricAsymmetric unitunit–– the the smallestsmallest unitunit fromfrom whichwhich a a unitunit cellcell cancan bebe

builtbuilt byby applicationapplication of of crystallographiccrystallographicsymmetrysymmetry operatorsoperators..

ExamplesExamplesL

L

L

L

L

L L

LL

L

LL

L

LL

LLL

Translational Symmetry Translational Symmetry

Cover spaceCover space–– Screw axesScrew axes

sliding rotationssliding rotations2211, 3, 311, 3, 322, 4, 411, 4, 422, 4, 433

repeats that extend repeats that extend across unit cellsacross unit cells

–– (Glide planes)(Glide planes)sliding mirrorssliding mirrors

21 41 42

Asymmetric unitAsymmetric unit

The The smallestsmallest objectobject fromfrom whichwhich the the unitunit cellcell cancan bebebuiltbuilt upup, , byby applicationapplication of of crystallographiccrystallographicsymmetrysymmetry..ContentsContents of an of an asymmetricasymmetric unitunit–– At At leastleast oneone macromoleculemacromolecule per per asymmetricasymmetric unitunit. . –– CanCan bebe moremore -- ifif therethere is is nonnon--crystallographiccrystallographic symmetrysymmetry: :

a a dimericdimeric proteinprotein maymay bebe in the in the asymmetricasymmetric unitunit ((oror notnot).).the 5the 5--fold fold symmetricsymmetric virus virus coatcoat is is alwaysalways in the in the asymmetricasymmetricunitunit..

SymmetrySymmetry

Crystallographic (Space group) symmetryCrystallographic (Space group) symmetry–– The symmetry elements apply The symmetry elements apply throughoutthroughout the the

crystal. crystal. A 2A 2--fold axis in unit cell A will be coincident with other fold axis in unit cell A will be coincident with other 22--fold axes in different unit cells.fold axes in different unit cells.

NonNon--crystallographic (local) symmetrycrystallographic (local) symmetry–– The symmetry elements apply The symmetry elements apply locallylocally only to the only to the

atoms around a particular lattice point.atoms around a particular lattice point.Axes are Axes are parallelparallel but not (except by chance) coincident but not (except by chance) coincident with each other.with each other.

CrystalCrystalCrystal latticeCrystal lattice–– Asymmetric unit + symmetry = unit cellAsymmetric unit + symmetry = unit cell

2

What is a protein What is a protein crystal?crystal?Crystal lattice is a periodic, symmetric system:Crystal lattice is a periodic, symmetric system:–– Proteins are asymmetric and fit periodicity poorlyProteins are asymmetric and fit periodicity poorly

Inorganic crystals are hard and dryInorganic crystals are hard and dry–– protein crystals are moist and include up to 70% protein crystals are moist and include up to 70%

solvent .solvent .–– Solvent channels between molecules: Solvent channels between molecules: –– Possible to soak in chemicals; enzymes can be active Possible to soak in chemicals; enzymes can be active

in the crystalin the crystal

BravaisBravais latticeslattices

BravaisBravais, 1850:, 1850:–– 14 different crystal lattices 14 different crystal lattices

possible:possible:–– Due to symmetry of unit cell.Due to symmetry of unit cell.–– Constraints on cell lengths & Constraints on cell lengths &

angles.angles.230 space groups; 65 230 space groups; 65 biological onesbiological onesPoint groupPoint group–– Symmetry around a point.Symmetry around a point.

Space groupSpace group–– Symmetry arrangement that Symmetry arrangement that

covers space.covers space.The word “group” here has its The word “group” here has its full mathematical meaning.full mathematical meaning.

Space GroupsSpace Groups

Space Group Percentage (1997)P212121 23 %P21 11 %P3221 8 %P21212 8 %C2 8 %

CertainCertain spacespace groupsgroups areare moremore common in common in proteinprotein (and (and otherother) ) crystalscrystals..60% of the PDB 60% of the PDB structuresstructures belongbelong to 5 to 5 spacespace groupsgroups::

Space groups Space groups

230 possible space 230 possible space groupsgroups–– All available symmetry All available symmetry

operations on all 14 operations on all 14 BravaisBravais lattices.lattices.

Biological ones are Biological ones are chiralchiral–– 65 of those.65 of those.

Protein crystalsProtein crystalsOnly limited amount of symmetries are allowed Only limited amount of symmetries are allowed for proteins: no mirror!for proteins: no mirror!Symmetry has to fill the space: 2Symmetry has to fill the space: 2--fold, 3fold, 3--fold, fold, 44--fold, 6fold, 6--fold (no 5 or 7 fold etc!! Can’t build a fold (no 5 or 7 fold etc!! Can’t build a crystal lattice!!)crystal lattice!!)Screw axisScrew axis–– Ex. P2Ex. P211

CrystalsCrystals areare notnot likelikesolutionsolution??ProteinProtein crystalscrystalscontainingcontaining 3535--75% 75% (vol.) (vol.) waterwaterCrystalsCrystals cancan bebe activeactiveEnzymeEnzyme activityactivityTimeTime--resolvedresolvedcrystallographycrystallographyBacteriorhodopsinBacteriorhodopsinphotobleachingphotobleaching–– PumpingPumping in the D96N in the D96N

variantvariant crystalcrystal ((colourcolourchangeschanges))

Myoglobin CO photolysis

DiffractionDiffractionAre these good ?Are these good ?For our purposes For our purposes they are good they are good when they diffract when they diffract XX--rays well.rays well.

Data Data collectioncollection

Data Data fromfrom a a synchrotronsynchrotron

MicroscopyMicroscopy

HowHow itit worksworks–– SpecimenSpecimen

diffractsdiffracts parallelparallel lightlight

–– objectiveobjective lenslensfocussesfocusses diffracteddiffractedimageimage

DiffractionDiffraction

different ordersof diffraction (waves)1,2,3,4,5....

Crystal LatticeCrystal LatticeA theoretical A theoretical conceptconceptLattice planes (Lattice planes (HilaHilatasottasot) ) –– Can be defined by Can be defined by

their intersection their intersection with the axeswith the axes

–– Spacing, Spacing, dd,, between between the planes used in the planes used in Bragg’s law.Bragg’s law.

in phase from identical lattice points

Braggs law:nλ=2d sinθ

Fourier transformFourier transform

• adding up the components of nth (of increasing frequency) order will give the sum = repeating unit.• the more terms the finer the detail

∑∑∑ ++−=h k l

lzkyhxiihklxyz eeF

V)(2||1 παρ

Measured Phase Fourier Transform

The “phase problem”The “phase problem”

?

Obtaining the phase(s)Obtaining the phase(s)

to reconstruct the diffracted waves need to know to reconstruct the diffracted waves need to know the phase, intensities collected from diffraction the phase, intensities collected from diffraction give amplitude (of the wave) I = |F|give amplitude (of the wave) I = |F|22

Only the Intensities can be directly obtained by Only the Intensities can be directly obtained by the Xthe X--ray detector.ray detector.

pattersonpatterson functionfunction

Phkl =1V

Ihkle−2πi(hx+ky+ lz )

l∑

k∑

h∑

•methods (in practise):•heavy atom derivatization, inclusion of (other) anomalous scattering elements (e.g. SelenoMethionine-labeling, Br-nucleotides), or using a model (homologous (ca. >30-40 % id.) structure) and find the position and orientation of this in the crystal unit cell (asymmetric unit more exactly).

•Find the heavy atom structure with patterson functioncalculation

(a fourier transform without the phases)

DifferenceDifference pattersonpatterson functionfunction(I(IPHPH--IIPP=I=IHH))CrossCross vectorvector peakspeaks betweenbetweenatomsatoms relatedrelated byby symmetrysymmetryoperatorsoperators of the of the spacespace groupgroupfoundfound on on specialspecial sectionssections calledcalledthe the harkerharker planesplanes..FromFrom thisthis cancan bebe obtainedobtainedH (H (x,y,zx,y,z))

StructureStructure solutionsolution

ObtainObtain initialinitial electronelectron densitydensity withwith the heavy the heavy atomatom coordinatescoordinates ((initialinitial phasephase refinementrefinement))BuildBuild a a modelmodel (and/(and/oror useuse nonnon--crystallographiccrystallographicsymmetrysymmetry averagingaveraging and and solventsolvent flatteningflattening to to improveimprove mapsmaps, , possiblypossibly phasephase extensionextension to to higherhigher resolutionresolution ifif data data availableavailable))AutomatedAutomated chainchain tracingtracing programsprograms + + handhandbuildingbuilding on on graphicsgraphics

A A mapmap phasedphased withwith 4 4 PtPt--atomsatoms

finishing the structure finishing the structure --refinementrefinement

fit model back to X-ray data via a minimizationtarget function(minimize an energy, including knowngeometric/chemical terms)

The meaning of resolution (1.9The meaning of resolution (1.9--2.52.5--3.0Å)3.0Å)

lattice plane separation d, smaller the separation the wider the diffraction angle = higher resolution of “image”

HowHow dodo youyou knowknow whenwhen the the crystalcrystal structurestructure is is rightright??

R =Fo − Fc∑

Fo∑

–– Data Data completenesscompleteness, , –– signalsignal//noisenoise (I/sigma)(I/sigma)

–– RR--factorfactor ((RRworkwork, , RRfreefree))

–– GeometryGeometry ((RamachandranRamachandran plotplot, ,

r.m.s.dr.m.s.d. of . of bondsbonds and and anglesangles))

–– look at the look at the electronelectron densitydensity mapsmaps

TrueTrue measuresmeasures of of resolutionresolution

Links and literatureLinks and literatureGoogle: PDB, Google: PDB, www.expasy.comwww.expasy.com (various seq. analysis tools + (various seq. analysis tools + swissmodelswissmodel), SMART, PFAM + web courses on crystallography.), SMART, PFAM + web courses on crystallography.

EDS: EDS: http://eds.bmc.uu.se/eds/http://eds.bmc.uu.se/eds/ (view maps for PDB structures)(view maps for PDB structures)

(protein interaction domains: (protein interaction domains: http://pawsonlab.mshri.on.ca/index.php,http://pawsonlab.mshri.on.ca/index.php, doenstdoenst include all...)include all...)Free PC/Mac Free PC/Mac sofwaresofware for structure viewing and analysis: for structure viewing and analysis: PymolPymol(best by far), Swiss(best by far), Swiss--PDBviewerPDBviewer, , RasmolRasmol (see also PDB site).(see also PDB site).ReadingReading–– Introduction to protein structure, Introduction to protein structure, BrändenBränden C and C and ToozeTooze J.J.–– Crystallography made crystal clearCrystallography made crystal clear, Rhodes G. , Rhodes G. –– OutlineOutline of Crystallography for Biologistsof Crystallography for Biologists, Blow D., Blow D.–– Structure and mechanism in Protein ScienceStructure and mechanism in Protein Science, , FershtFersht A.A.