Gruber Kap 3

7/29/2019 Gruber Kap 3

1/28

01/2011 Protein Folds 1

Chapter 3: Fold families, protein

folding


A taxonomy of protein structures yieldsa classification of domain structures

into three main groups: domains, domains, and / domains. In

structures the core is built upexclusively from helices; in structures the core comprises

antiparallel sheets and there areusually two sheets packed againsteach other. The / structures aremade from combinations of --motifs that form a predominantly

parallel sheet surrounded by ahelices


2/28


unique topologies (folds)according to CATH

http://www.cathdb.info/

Limited number

of folds


Alpha-Domain Structures

The first globular protein structure that was determined, myoglobin,belongs to the class of alpha-domain structures. Alpha helices are

sufficiently versatile to produce many very different classes ofstructures. Inmembrane-bound proteins, the regions inside the

membranes are frequently helices whose surfaces are covered byhydrophobic side chains suitable for the hydrophobic environmentinside the membranes.

Alpha helices are also frequently used to produce structural andmotile proteins with various different properties and functions. Thesecan be typical fibrous proteinssuch askeratin, which is present in

skin, hair, and feathers, or parts of the cellular machinery such asfibrinogenor the muscle proteinsmyosin anddystrophin.


3/28


Coiled-coil helicesDespite its frequent occurrence in proteins an isolated helix is onlymarginally stable in solution. Alpha helices are stabilized in proteins by

being packed together through hydrophobic side chains. The simplestway to achieve such stabilization is to pack two helices together. Theside-chain interactions are maximized if the two helices are not straightrods but are wound around each other in a supercoil, a so-called coiled-coil arrangement. Coiled-coils in fibers can extend over many hundreds ofamino acid residues to produce long flexible dimers.


Coiled-coil helices contain arepetitive heptad amino acid

sequence patternA left-handed supercoil of two right-handed helicesreduces the number of residues per turn in eachhelix from 3.6 to 3.5. Such sequences are repetitivewith a period of seven residues, the heptadrepeat. Theresidues within one such heptadare labeled a-g, andthe d-residue is hydrophobic (Leuor Ile). Within acoiled-coil structure, the d-side chains pack againsteach other every second turn of the helices. Thisarrangement is termed aleucine zipper.


4/28


The leucine zipper

Every seventh residue (d) in both helicesis a leucine. Due to the heptadrepeat, the

d-residues pack against each other.Residues labeled a are also usuallyhydrophobic and participate in formingthe hydrophobic core.

-helical coiled-coil structures occur indifferent proteins with diverse functions,e.g. Fibrinogen(blood coagulation);RNA- and DNA-binding proteins;

collectins(cell-surface recognitionproteins); spectrin, dystrophinand myosin(muscle contraction)

Leu-Zipper


Coiled-coil structures

Salt bridges can stabilizecoiled-coil structures andare sometimes important forthe formation ofheterodimericcoiled-coilstructures. The residueslabeled e and g in theheptadsequence are closeto the hydrophobic core andcan form salt bridges

between the two helices,the e-residue in one helixwith the g-residue in the

second and vice versa.


5/28


"Knobs-in-holes" model

Schematic diagram of packing side chains in the hydrophobic coreof coiled-coil structures according to the knobs in holesmodel. The positions of theside chains along the surface of the cylindrical a helix is projected onto a plane

parallel with the helical axis for both helices of the coiled-coil. The side-chain positions of the first helix, the knobs, superimpose between the side-chain positions in the second helix, the holes.

Knobs in Holes


The four-helix bundle

The simplest and most

frequent -helical domainconsists of four helicesarranged in a bundle with

the helical axes almostparallel to each other. Thisarrangement ischaracterized by ahydrophobic core in themiddle of the bundle alongits length, where the sidechains are so closelypacked that water is

excluded.


6/28


The four-helix bundle can have

different topologies

Antiparallel -helices: e.g.myohemerythrin(non-hemeoxygen-transport), cytochromec and cytochrome b562(heme-containing electron carriers),ferritin(iron storage), tobaccomosaic virus coat protein.

Parallel -helices: e.g. humangrowth hormone

parallel antiparallel


The Globin Fold

The globinfold has been found in a largegroup of related proteins, includingmyoglobin, hemoglobinsand thephycocyanins.Thepairwise arrangementsof the

sequential helices in the globinfoldare different from the antiparallelorganization found in the four-

helix-bundle structures. The globinstructure is a bundle of eight helices,usually labeled AH arranged so thatthe helices form a pocket for the activesite, which binds a heme group. With the

exception of the last two helices (Gand H), all packing interactions areformed between pairs of a helices thatare not sequentially adjacent.

Myoglobin


7/28


Geometric considerations determine

-helix packingThe geometry of-helix packingin the globin fold is different fromcoiled coils or four-helix bundles,

where there is an angle of about20 between the helical axes. Inthe globin fold the angles betweenthe helical axes are often around50.

The side chains of an helix formridgesseparated bygroovesonthe surface. Alpha helices packwith the ridges on one helix into

the grooves of the other and viceversa. The ridges and grooves areformed by amino acids that arethree or four residues apart. Helices


Geometric considerations determine-helix packing(a) Two helices, I and II, withridges from side chains separatedby four residues. In panel 3 the blue helix is turned over through 180in order to form an interface with

the red helix. In panel 4 theorientation of the helices has beenrotated 50to pack the ridges of onehelix into the grooves of the other.

(b) In the red helix the ridges areformed by side chains separated by

four residues and in the blue helixby three residues. The helices arerotated 20in order to pack ridgesinto grooves, in a direction oppositethat in (a).


8/28


Case Study: Potassium channel

Most channel proteins in theplasmamembranes of plant and animal

cells havenarrow and highly selective poresthat are concernedspecifically with thetransport of inorganic ions, and so are referred toas ion channels. The function of such channels is to allow specificinorganic ions, mainly K+, Na+, Ca2+and Cl- to diffuse rapidly across thelipid bilayer and therefore balance differences in electric charge betweenthe two sides of the membrane.

The K+leak channels arehighly selective for K+ions by a factor of10,000 over Na+. In spite of their high selectivity, they exhibit a

throughput of108 ions per second, which approaches the diffusion-limited rate.



9/28


Angew. Chem. Int. Ed.43(33), 4265-4277.


The K+ channel is a tetrameric molecule withone ion pore in the interface between the

four subunits

The potassium channelviewed perpendicular to theplane of the membrane. The

molecule is tetrameric with ahole in the middle that formsthe ion pore (purple). Eachsubunit forms twotransmembrane helices, theinner and the outer helix. Thepore helix and loop regionsbuild up the ion pore in

combination with the innerhelix.


10/28


The ion pore has a narrow ion

selectivity filter

Diagram showing twosubunits of the K+channel,illustrating the way theselectivity filter is formed.Main-chain atoms line thewalls of this narrowpassage withcarbonyloxygen atoms pointing into

the pore, forming bindingsites for K+ions.


The selectivity filter is highlyconserved

In thestructure, theglycine residues

arelocatedin theleft-handed--helical

regionof theRamachandran-plot.


11/28


The ion pore of the K+ channel

From the cytosolic side the porebegins as a water-filled channelthat opens up into a water-filled

cavity near the middle of themembrane. A narrow passage, theselectivity filter, links this cavity tothe external solution. Threepotassium ions (purple spheres)bind in the pore. Thepore helices(red) are oriented such that theircarboxyl end (with a negativedipole moment) isoriented

towards the center of the cavityto provide a compensating dipolecharge to the K+ions.


Cation vs. anion channel

Theoverall architectureof K+channelsand ClC Cl--transportproteinsisvery different butcertaingeneral

featuresaresimilar. One similarity isthedirectionof-helicestoward theionpathway. Thenegative C-

terminal end charge(red) pointsto theK+ion, whereasthepositive N-terminal end charge(blue) pointsto theCl-

ion.


12/28


High resolution

structure of the

K+-channel

Thepotassiumionsites(greenspheres) areonly 50% occupied.

hydratedK+-ion

dehydratedK+-ionsin theselectivityfilter region


Carbonyloxygen atoms

mimic the

water

coordination

Potassium channel


13/28


Cation vs. anion channel

K+and Cl- selectivity filtersmakeuseof mainchainatomsto coordinateions:carbonyl oxygenatomsforK+ions(green) and amidenitrogenatomsfor Cl-

ions(red). Bothfilterscontainmultiple close-spacedionbindingsites.


Mechanism of the K+ channel

First, the main-chain atoms of the selectivity filter create a stackofsequential oxygen ringsalong the passage, providing severalclosely spaced binding sitesof the required dimensions forcoordinating naked, dehydrated K+ions. Side chain packing

firmly fixes the positionsof the carbonyl oxygen atoms withthe correct dimensions to providestrong binding sites for K+.

When an ion enters the selectivity filter itdehydrates. Bindingto the carbonyl oxygen atoms in the filter compensates theenergetic cost of dehydration. The dimensions of the bindingsites are such that a K+ion fits in the filter precisely, but thefirm packing of the side chains prevents the carbonyl oxygenatoms from approaching close enough to compensate for thecost of dehydration of a Na+ion.


14/28


Histogram of K+O=C distances


Histogram of Na+O=C distances


15/28


Ion transport

TwoK+ionsin theselectivity filter arehypothesizedto existpredominantly in thetwospecificconfigurations1,3 and 2,4.


TheK+ionpair coulddiffuse back andforthbetween1,3 and 2,4 configurations(bottompathway), oralternatively an ioncouldenter thefilter fromonesideof the

membraneas theion-water queuemovesand a K+ionexitsat theoppositeside(thetoppathway). Thetwopathscompleteacycle: in onecompletecycleeachionmovesonlya fractionof thetotal distancethroughthefilter, but theoverall electricaleffectisto moveonechargeall theway.Electrostatic repulsionbetweentheK+-ionsshouldfavor high conductionratesby

loweringK+affinity.

Hypothetical mechanism for ionconduction


16/28


Opening of the pore is effected by

helix-bending

Whentheporeopens, theinner helicesarebentat aglycine hinge.

Thisglycine residueisconservedamongdifferentpotassiumchannels.


Alpha/Beta Structures

The most frequent of the domain structures are the/ domains, which consist of acentral parallel or mixed sheet surrounded by helices. All the glycolytic enzymesare/ structures as are many other enzymes as well as proteins that bind andtransport metabolites. In/ domains, binding crevices are formed by loop regions.

These regions do not contribute to the structural stability of the fold but participate inbinding and catalytic action.

There are three main classes of/ proteins:

TIM barrel, first observed in triosephosphate isomerase.

Rossmanfold, first observed in lactate dehydrogenase.

Horseshoe fold, formed by leucine-rich motifs and first observed in aribonucleaseinhibitor


17/28


The three main classes

of/proteins

TIM-barrel Rossman-fold Horseshoe-fold


Two -- motifs can be joinedinto a four-stranded parallel

sheet in two different ways

(a) The last strand of motif1 is adjacent to the first

strand of motif 2, giving thestrand order 1 2 3 4. (barrelstructures and horseshoefold).

(b) The first strands of bothmotifs are adjacent, givingthe strand order 4 3 1 2(Open twisted sheets).In both cases the motifs are

joined by an helix (green).

Note that this implies that all -- motifs are right-handed, which has been observed empirically.


18/28


Alpha/beta barrels occur in many

different enzymesIn / structures where the strand order is 1 2 3 4, all connections are on the sameside of the sheet. An open sheet of this sort with four or more parallel strandswould leave one side of the parallel sheet exposed to the solvent and the other side

shielded by the helices. Such a domain structure is rarely observed. Instead, aclosed barrel of typically 8 twisted strands is formed. It has the helices on theoutside of the barrel, with strand 8 hydrogen-bonded to strand 1. There are a fewbarrels that contain ten parallel strands and some that contain eight parallel and twoantiparallel strands.

A minimum of about200 residuesare required to form the eight-stranded/-barrel.It occurs in many different proteins, most of them enzymes, withcompletely differentamino acid sequences and different functions. About 160 residuesform the strands

and helices. The remaining residues form the loop regions that connect thestrands with the helices. These loops have different lengths and conformations inthe different proteins.Thus the barrel forms the structural framework of theenzyme, whereas the loops contain the amino acids responsible for its catalyticchemistry.


The active site is formed by loops

at one end of the / barrelThe active site in all / barrels is in a pocket formedby the loop regions that connect the carboxy ends of

the strands with the adjacent helices

A view from the top of the barrel of the activesite of the enzyme RuBisCo (ribulosebisphosphatecarboxylase), which is involved inCO2fixation in plants. A substrate analoguebinds across the barrel with the two phosphategroups, on opposite sides of the pocket. Anumber of charged side chains (blue) from

different loops as well as a Mg2+ion (yellow)form the substrate-binding site and providecatalytic groups.


19/28


Branched hydrophobic side

chains usually dominate the core

of barrelsIn most/-barrel structures the eight strands

of the barrel enclose a tightly packedhydrophobic core of side chains from thestrands. The core is arranged in three layers, witheach layer containing four side chains from

alternate strands.The core is tightly packedwith hydrophobic residues.

In barrels the hydrophobic side chains of thehelices are packed against hydrophobic side

chains of the sheet. The helices are

antiparallel to the strands that they connect.The packing interactions between helices and strands are dominated by the residuesVal , Ileand Leu, which comprise about 40% of the

residues of the strands in parallel sheets.


Alpha/beta twisted open-sheetstructures contain helices on

both sides of the sheetIn the this class of/ structures there are helices on both sides of the sheet. Therefore a closed barrel cannot be formed, and the strands arearranged into an open twisted sheet.

There are always two adjacent strands in the interior of the sheetwhose connections to the flanking strand are on opposite sides of thesheet. This creates a crevice outside the edge of the sheet between thesetwo loops. Almost all binding sites in this class of/ proteins are locatedin crevices of this type at the carboxy edge of the sheet.

In open-sheet structures the helices are packed against both sides of the

sheet. Each strand thus contributes hydrophobic side chains to packagainst helices in two similar hydrophobic core regions, one on eachside of the sheet.


20/28


Alpha/beta twisted open-sheet

structures

The active site in open twisted/ domains is in a crevice outside thecarboxy ends of the strands. This crevice is formed by two adjacent loop

regions that connect the two strands with helices on opposite sides ofthe sheet. The rod represents a bound molecule in the binding crevice


The positions of active sites can

be predicted in / structuresIn almost every one of the more than 100 different known / structures ofthis class the active site is at the carboxy edge of the sheet.

Functional residues are provided by the loop regions that connect the

carboxy end of the strands with the amino end of the helices, like in the/-barrel structures.

Open/-sheet structures form crevices at the edge of the sheet whenthere are two adjacent connections that are on opposite sides of the sheet.

The active site is usually found in such a crevice. The positionof suchcrevices can be predicted from a topology diagram: they occur when the

strand order is reversed at the place where connections from thecarboxyends of two adjacent strands go in opposite directions, one to the left andone to the right.


21/28


Hydroxynitrile lyase from Hevea

brasiliensisHbHNL


Beta Structures

Antiparallel -structures comprise another large group of protein domainstructures. Functionally, this group is the most diverse; it includes enzymes,transport proteins, antibodies, cell surface proteins, and viruscoat proteins. The

cores of these domains are built up by predominantly antiparallel strands thatcan vary in number from four to over ten.

The strands usually form two sheets that are joined together and packedagainst each other.

The sheets have the usual twist, and when two such twisted sheets arepacked together, they form a barrel-like structure.

Antiparallel structures in general have a core of hydrophobic side chains inside

the barrel provided by residues in the strands. The surface is formed byresidues from the loop regions and from the strands.


22/28


23/28


The retinol-binding protein

A retinol molecule is bound

inside the barrel, between thetwo sheets, such that itsonly hydrophilic part (an OHtail) is at the surface of themolecule. The binding site islined with hydrophobicresidues, which provide ahydrophobic surrounding forthe hydrophobic part of the

retinol molecule.

RBP


RBP: amino acid sequencereflects structure

On a large part of the surface of RBP, side chains from residuesin thestrands are exposed to the solvent. This is achieved by alternatinghydrophobic with polar or charged hydrophilic residues in theamino acid

sequences of the strands. The amino acid sequence of strands 2 3 4 of

RBP clearly illustrate this arrangement. The sequences are listed in such away that residues which point into the barrel are aligned.


24/28


Membrane spanning-structures

Porinchannels are made by up and

down-barrels Sixteen strands form

an antiparallel barrel that traversesthe membrane. The loops at the top ofthe picture are extracellular, the shortturns at the bottom face the periplasm.


Globular proteins are onlymarginally stable

Proteins are quite unstable: Changes in pH or temperature can convert asolution of native protein molecules to a denatured state. The free energydifference between these two states is about 515 kcal/mol (the energycontribution of a single hydrogen bond is of the order of 25 kcal/mol).

This small free energy difference is a difference between two largenumbers, the enthalpy difference and the entropy difference.

Second law of Thermodynamics

HG T S =

Free Energy Enthalpy Entropy


25/28


Energy contributors between

folded and denatured state

Enthalpy: energy of the non-covalent interactions within the polypeptide chain(hydrophobic interactions, hydrogen bonds and ionic bonds) plus disulfide bonds.

The enthalpy favors the native state, the enthalpy difference between native anddenatured states can reach several hundred kcal/mol.

Entropy denotes the energy required to create order. Proteins in the native state arein one compact conformation, whereas the denatured state is disordered, with eachprotein molecules in a different conformation. Entropically the denatured state istherefore more favorable by several hundred kcal/mole, but in the opposite directionto the enthalpy difference.


The marginal stabil ity of the native

state is biologically and

functionally important.

Living cells need globular proteins in correct quantities at appropriatetimes. It is therefore equally important to be able to degrade proteins as itis to be able to synthesize them. Globular proteins in living cells usuallyhave a rapid turnover and their native states have therefore evolved to beonly marginally stable.

The catalytic activities of enzymes, and other important functions ofproteins, generally require structural flexibility, which would beinconsistent with a rigidly stabilized structure.


26/28


Anfinsen-Postulate

High resolution x-ray structure determinations of several hundred proteins

have shown that in each case the specific sequence of a polypeptide chainappears to yield only a single, compact, biologically active fold in thenative state. This fold generally has many sub-states with minor structuraldifferences between them, but all of these sub-states have the same generalfold. Comparisons with structure determinations in solution by NMR showthat the same fold also prevails in solution.

In other words: under physiological conditions there appears to be oneconformation for a given amino acid sequence that has a significantly

lower free energy than any other.


Kinetic factors are important for

protein folding

Intuitively one might imagine that all protein molecules search through allpossible conformations in a random fashion until they are frozenat thelowest energy in the conformation of native state. However, a simplecalculation shows that this is impossible: if each peptide grouphas only

three possible conformations (corresponding to the allowed regions, and loop in the Ramachandran diagram), a polypeptide chain of 150residues would then have 3150 =1068 possible conformations. If thetransition between conformations took only one pico-second (10-12

seconds), it would take 1048 years to search all these conformations - anastronomical number compared with the actual folding time of 0.1-1000seconds.To occur on this short time scale, the folding process must bedirected in some way through akinetic pathway of unstable

intermediates to escape sampling a large number of irrelevantconformations


27/28


Molten globules are intermediates

in foldingThe first observable event in the folding pathway is a collapse within a fewmilliseconds into apartly organized globular state, which is called themoltenglobule. It has most of the secondary structure of the native state, but is less compact

than the native structure and lacks the proper packing interactions in the interior of theprotein. The interior resembles more closely a liquid than the solid-like interior of thenative state. The molten globule should, therefore, not be viewed as a single structuralentity but as an ensemble of related structures that are rapidly interconverting.

In a second step, which can last up to 1second, persistent native-like elementsof tertiary structure begin to develop,possibly in the form of subdomains that

are not yet properly docked. The singlenative form is reached in the final stageof folding, which involves thehydrophobic packing in the interior aswell as the fixation of surface loops.


Burying hydrophobic side chains is akey event for the formation of the

molten globuleSecondary structure formation cannot be the thermodynamic driving force ofprotein folding, since stable hydrogen bonds can also be formed to watermolecules in the unfolded state.

However, there is a large free energy gain by bringing hydrophobic side chains outof contact with water and into contact with each other in the interior of a globularentity very early in the folding process. This reduces the number of possibleconformations that need to be searched. In addition, some of thepolar backbone -NH and -CO groups get buried in a hydrophobic environment, which enhances the

formation of helices and sheets. The formation of secondary structure early inthe folding process is therefore a consequence of burying hydrophobic side chains.

Hydrophobic side chains are usually scattered along the entire sequence in a

globular protein. About half of these side chains end up buried in the interior andthe rest scattered on the surface of the protein. This leaves the (unanswered)question what causes these residues to be selectively buried during the formation ofthe molten globule.


28/28


Folding proceeds

through many

intermediate states

The unfolded state consists of manyrapidly interconvertingconformationallydifferent molecules,U1...Un. The molten globule is anensemble of structurally relatedmolecules, M1...Mm, which are rapidlyinterconvertingand which slowlychange to a single unique conformation,the folded state F. The conversion from

the molten globule state to the foldedstate is slow and passes through a highenergy transition state, T.

Gruber Kap 3

Documents

Transcript of Gruber Kap 3