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ffifumryKffi#'ffi"Protein-ligand interactions andtheir analysisBabur Z. ChowdhrySchool of Chemical and Life Sciences, University of Greenwich, WellingtonStreet, Woolwich, London SE18 6PF, UK.

Stephen E. HardingNCMH Physical Biochemistry Laboratory, University of Nottingham, School ofBiosciences, Sutton Bonington, Leicestershire LE12 sRD, UK.

% lnfioductionIn each cell of an organism, a myriad of reactions, covalent and non-covalent,occur at any given point in time. These reactions are co-ordinated and regulated,both spatially and temporally. Each reaction has a speciflc purpose, occurs as aresult of finely-tuned inter- and intramolecular recognition mechanisms, andforms part of an intricate network of interdependent multi-component linear/non-linear reactions in interconnected compartments (organelles etc.) of thecell. Moreover the ffontiers of viability of such reactions-and the living organ-isms that depend on them-are marked by extreme conditions: 1-12 for pH,-5-110'C for temperature, 0.7-120 MPa for hydrostatic pressure, and 0.6-1.0 forwater activity. Amongst the many molecules that participate in such reactions inthe complex-and, as yet hardly understood, milieu of the cell-are proteins.

Proteins play a pivotal, indeed essential role, in cellular (and in multicellularorganisms, extracellular) activity. Their numerous biological functions togetherwith the molecular basis of their biophysical propertiesftehaviour are thereforeof multidisciplinary interest within the framework of what are generally calledthe biological (biochemistry, pharmacology, physiology, immunology, etc.) andphysical (physics, chemistry, mathematics, computing, etc.) sciences.

Both our intrinsic curiosity-driven philosophical knowledge base and our needor desire to modi$r (or use), fiom an applied perspective (medical, agricultural,biotechnological), the properties or behaviour of proteins are increasingly de-pendent on the ability to modulate the physico-chemical, and hence biologicalbehaviour of these molecules. Because protein-ligand interactions play akey role incellular metabollsm, detailed knowledge of such interactions, at both a microscopicand mactoscopic level, is also required. Just two examples, which immediatelystand out from the many, are antigen-antibody interactions and proteins which

BABUR Z . CHOWDHRY AND STEPHEN E . HARDING

actaSleceptors(membraneornon-membranebound):neurotransmissionde-pendsontheabil ityofsmallandlargemolecularweightmoleculestorecognizeand bind to specific sites on large membrane bound receptors' Another example:

enzyme-ligand interactions form a very large group of important complexes'

whichhavebeeninvestigatedformanyyears.Inthesesystemsnotonlyisthesubstrate(s)ofobviousimportance,butsoareothermolecules,whichregulateenzymeactivitysuchasto""'y-"'andpositiveandnegativemodulatorsinboth allosteric and non-allosteric proteins. other important systems, which are

nowbeingstudiedatamolecularlevel,includeligandbindingtostructuralpro-. teins, protein-DNA binding, protein-saccharide, protein-protein, and protein-

peptideinteractions.Theformation-andmaintenance_ofthequaternaryStructure of multisubunit proteins, the self-assembly of large Structures such as

microtubules or chromatin and transcription factor-promoter interactions are

other examples; although the list is actually almost endless'

Theterm.ligand'inbiologicalsystemscanhavemanydifferentmeanings.Itisusually,initsbroadestsensE,usedtomeananymoleculewhichinteractswithagivenmolecule( inth iscaseaprote in) .Theterm. l igand, thusinc ludesothermacromolecules(peptides/proteins/nucleicacids/lipids/carbohydtatesotmixedmolecularspeciesttrereof)aswellas,small 'molecularmass(arbitrati|y<-7-2kDa)molecules.Ligandsthereforecompriseaverylargeandstructurallydiversegroupofmoleculeswhich,unsurprisinglyalsodisplayawidevarietyofphysico.chemicalcharacteristics.Thismakesitdiff,culttounderstand,delineate'anddrawgeneralizedconclusionsconcerningtheirbiophysicalproperties.Perhapsthe major criterion, in the context of the discussion that follows, is that ligands

caninteractina(potentially)reversible,non-covalentmannelwithaproteinand thereby modJate its biological role in a controllable way (i.e. without the

requirement to make or break covalent bonds)'

Tofullyunderstandprotein-ligandinteractionsrequiresthat'ataminimum'thefollowingcriteriabemet.Firstthatthebiophysicalpropertiesofboththeploteinandthe..l igandunderinvestigationareexaminedindependentlyandthat their biophysicai behaviour be fully understood: this requirement applies

tobothexistingand/ornewlydesignedand(bio)synthesizedpeptides/proteinsandligands.Currently,and.contrwytopopularoptnion,wearefarfromachievingsuchartann,exceptatonextremelysryerfiaallwel'Knowledgeofthestructure/conformation'i fpossib le,at theatomic levelof theprote inandthel igandintheunboundformisaprerequisiteforprotein-l igandinteractionstudies.Althoughitmayappeartrivialtothereader,thehighlevelresolutionstructulesofmostproteins(either by X-ray crystallography or high resolution NMR) have not' to date' been

establ ished. I t rscurrent lyest imatedthatsuchdataexis ts foronlyabout30peptides/proteins (1) but it is seriously arg'able, in the sense of the generally

acceptedmeaningofthetermhigh(atomic)resolution(i.e.<0.8A1,thateventhistargethasnotbeenmet'Additionallytheprocessofformingacomplexbe-tweenasmal l l igandmoleculeandaprote in isacompl icatedequi l ibr iumprocess. Both theligand and the protein in the solvated state probably exist as

an ecuilibrium mixture of several conformers. Admittedly for many'small mol-

P R O T E I N _ L I G A N D I N T E R A C T I O N S A N D T H E I R A N A L Y S I S

Table 1 Parameters which have to be defined for the protein, the ligand, and the protein-l igand complex

chemical composit ion, concentrat ion/purity (contaminants) /stabi l i ty/specif ic act ivl ty orassay for, e.g. structural proteins and ligand/state of structural integrity. Ability to freezeor air dry and pre-history of protein and l igand samples may be important.

Solubi l i ty ( in dif ferent solvents: aqueous, organic, and mixed phase); state of aggregation.Struciure: size. shape. geometry, topology: protein sequence. and post-translat ionai

modif icat ions must be known; abi l i ty to use molecular biology techniques (strategies forover-expression and mutagenesis of recombinant protein are cri t ical); combinatorialmelhods for over production of ligand.

Dynamics of protein/ l iganOT protein-t igand'complex'.

ecular weight ligands' high resolution x-ray and/or NMR structures do exist. Thefact that our knowledge base is deflcient in this area has signiflcant implicationsfor understanding protein-tigand interactions, which unfortunately will not betouched upon, in this brief overview. In addition a myriad of other properties ofthe reactants also requile to,be established (see Table 1).

Secondly the protein-ligand complex must also be fully characterized. Norm-ally, at a simplistic level, the non-covalent interaction between a protein (p) anda ligand (L) is often represented as follows:

P + L e P L t 1 lHowever as williams and westwell have pointed out (2) the interaction shouldactually be written in the following form:

n * r.-- r'r' l2lEquahon 2 may, at flrst glance appear identical to Equahon 1. However theformulism of Equation 2 recognizes, or is taken to mean, that once p and L haveundergone an interaction or association, they no longer exist. They have, insteadbeen replaced by the modifled entities p' and L'. (Tight complexes often resultwhen protein-ligand interactions are significantly stronger than ligand-solventand protein-solvent interactions.)

The well described equilibrium molar association or binding constant, I("(other popular symbols are I(5 or just IQ corresponding to Equation 2 is thendescribed by:

11" = p'�L'l / [P] tll) t3lwhere the square brackets means the molar concentration (M, or rnol.l-i), whichof course is not an SI unit. For the system described by Equation 2 (or Equahon 1)the unitsl of K" will be M-1 (l.mol-i). The more popularly used 'molar dissociationconstant' Ka is simply the reciprocai of I(,,, so for the system of Equation 2..

Kd = [P].El/ [P'L']

rIn strict thermodynamic terms K^ and I(a are both dimensionless. Dimensions are howevernormally added, but only to indicate the dimensions of the quantities used to calculate thern.See Price, N. c., Dwek, R. A., wormald, M. R. and Ratcliffe, R. G., princtples and prob'Lems in physicalChemistry,3rd edition, Oxford Universiry press. 2001.

l4l

BABUR Z . CHOWDHRY AND STEPHEN E . HARDING

with the units of Ka, M or mo1.l-1. Traditionally, Kas of < 5 pM are regarded as

strong interactions, and > 50 pM as 'weak'. Any technique chosen to measule

Ka (or IQ has to cope with a concentration/concentration range where all species

(P, L, and P'L') are present: this means to probe the K4 for strong interactions,

low concentrations are required and for weak interactions, high concentrations.

Of course there are other reactions mole complicated than that described by

Equations 1 or 2. Amorg generalized form of Equation 2 for a binary system is:

nP + mL <-+ P'rL'-

and the corresponding I(" will be:

6" : [P',L'*l/ ([P]".p1*)

Other variants of Equations 3-6 include the use of weight concentrations C (g/l or

g/ml: again, not SI units) rather than molar concentrations: (the K", K4 notation

is then replaced by, e.g.X", XJ.

Establishing the stoichiomeitry and associationidissociation equilibrium con-

stants (or the corresponding rate constants) is only one step: the goal ofresearch

into protein-ligand interactions is to understand, again it must be emphasized,

in minute molecular detail the relationship between function and molecular

recognition, structure, kinetics, energetics, and dlmamics of as many deflned

systems as possible. Use can tfien be made of this plethora of knowledge such

that either the behaviour of previously unknown systems can be hypothesized

in advance of experimental information being gained or that completely new

systems can be designed. The pursuit of this goal is a time-consuming and

difficult task! Especially when it is realized that the inter-relationship between

all of the foregoing parametels (intermolecular recognition-structule-dynamics-

kinetics-thermod;mamics) need to be assessed and need to be further ascer-

tained over a wide variety bf environmental solution conditions. Table 2 cites the

most commonly used experimental variables (but of course, there are countless

others) and Table 3 provides an overall summary of the factors that need to be

taken into account when applying a technique (or, preferably, a collection of

Table 2 Experimental variables in the analysis of the protein, the ligand' and theprotein-ligand complex

Reagent variablesProtein/l igand concentrationLigand structural variants (analogues/homologues)Protein structural variants (site/group specific, conservative/non-conservative mutants;

denatuled fo-T/partlally unfolded folms, prese_nce ol absence of cofactors)

Environmental solut ion condit ion variablesTemperature, pH, buffers (ionic strength and nature of buffers)Osmolytes, solvents including co-solvents, salt /ion concentrationDenaturants (urea /GdnCl (and other lyotropic /chaotropic agents)/detergents'sufactants), stabilizers (azide, glycerol etc.); mercaptoethanol, DTT; gaseous phase

used fo1 experiments, et :Combinations of the above

tsl

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P R O T E I N - L I G A N D I N T E R A C T I O N S A N D T H E I R A N A L Y S I S

Table 3 Factors to be taken into account in technique(s) used for investlgatingprotein/ l igand /protein-l igand complexes

Design of expelimental protocol

Intormation content

Availabi l i ty. cost

Complementary/alternative tectrniques"

Analytical parameters associated with the technique(s): concentration of material required,compared to other techniques, sensitivity, detection limit, accuracy, precision,reproducibi l i ty/repeatabi l i ty, use or necessity for isotopic or non-isotopic label l ing, ease ofuse, computing resources, methods/complexity and different methods of data analysis,errors/errcr propagation/statistical analysis of results (if possible); time scales ofexperimental (spectroscopic) techniques used.

Abil i ty to use the technique in the presence of buffer addit ives and/or environmental solut ionconditions eiven in T3ble 2

The experimentalist should insure ifrat at every stage of the experiment and (particularly) inthe data analysis, the correct (appropriate) theoretical models are used. In usinginstrumental techniques the'black box data analysis'syndrome should be avoided.Critically compare and contrast With work on other related systems. Each method will haveits own advantages and drawbacks for a given system. The abilityto interpret experimentaldata both intrinsically and in terms of a theoretical basis is very important. Thesystematic/accumulated instrumental errors are often important, but usually ignored.

uTry, if possible to use more than one technique (method) to verify the overall results or the values foroarticular Darameters.

techniques) to the analysis of protein-ligand interactions. Itis extremely irnportantto cross-reference parameters inTables 1,2, and 3.

Within the requirement that the changes in the conversion of P to P' and L toL' (i.e. changes in the reacting species in the 'complex' compared to their individual s(ates) need to be ascertained as a step towards a fuIl understanding ofprotein-ligand interactions, the following factors include some of the import-ant parameters which require consideration. The changes in structure, in eitherspecies, may range ffom extremely subtle (i.e. an 'insignificant' structural re-organization; 'simply' a tightening or loosening of the internal structure) to largescale changes in both local and global conformational properties. The list ofchanges which occur not only include changes in structure (secondary/tertiary/quaternary), conformation, size, shape geometry, and topology but also includechanges in the charge distribution, the state ofhydration and protonation, andthe partial molar volume as well as changes in the surface accessible surfacearea, polarity (hydrophobicity), and intra- and intermolecular enffopy factors (e.g.rotational and translational motional properties of molecules: and the d;mamicsof water molecules). The molecular nature of the binding interface, the identi-flcation and quantiflcation of the (individually) 'weak' cooperative molecularforces holding the protein-ligand complex together (such as hydrogen bonding,dipole-dipole interactions, van der Waals forces-induction and dispersion forcesalone or combined-hydrophobic, electrostatic interactions, etc.) and the number/role, if any, of water molecules at the binding interface are other importantfactors which have to be considered. Association of other oroteins or other

BABUR Z. CHOWDHRY AND STEPHEN E. HARDING

ligand species subsequent to the interaction of the initial species may also beinvolved; thus making the problem even more complicated! Furthermore ifmultisite ligand binding to either a monomeric or multimeric (homomeric/heteromeric subunit) protein is involved then cooperativity effects and linkagephenomena (allosteric effects) have also to be taken into account and examinedby the appropriate experimental/theoretical techniques. Williams and Westwell(2) have also emphasized the important, but often overlooked, point that thebinding energy betr,yeen two reacting species is not only a propeffy of theinterface between them, but also depends on the modiflcations (as alluded toabove) of the internal structures of one or both of the reacting partners.

Obviously in order to achieve the objectives of investigating protein-ligandinteractions a multidisciplinary technique/methods approach has to be adoptedas we stress inTable 3. Some of the methods which are used to examine proteinand/or protein-ligand interactions have been compared and contrastedinTable 4which has been based to a certain extent on a recent review by Philo (ref. 3), andthe contributions to this book (other useful sources can be found in refs. 4 and 5).

ffi A btief comparison of the 'high resolution'methodsSince structural elucidation is so important in protein-ligand interactions itmakes sense to compare and contrast the application of the two most powerfuland commonly used methods for attaining this goal, namely X-ray crystallo-graphy and high resolution NMR. Both methods complement each other. Thereis no doubt, however that, cuffently at least, X-ray techniques can give a better-defined structure in less time compared to NMR techniques. However the twomethods should be considered convergent (they do after all use similar softwaretools)'in that for molecules (that are amenable to NMR analysis) the X-ray struc-ture, if available, can be a substantial aid in the elucidation of the NMR structure.In addition both methods require, as a general rule of thumb, the same amountof material for analysis; approximately 10 mg per 10 kDa of protein. Howeverthe fact that NMR can be used for obtaining information on the dynamics of asystem, the association constant for a protein-ligand interaction and, for smallmolecular weight proteins and/or protein-ligand complexes, the structure ofdifferent conformations in equilibrium should also be taken into account.

The advantages of X-ray crystallography, for structure determination of proteins,ligands, and their complexes are as follows:

(a) It is a well-established technique.

(b) More mathematically direct image construction is required, compared toNMR.

(c) Objective interpretation of data (usually) easier than NMR.

(d) Raw data processing highly automated.

(e) Quality indicators available (resolution, R-factor).

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BABUR Z . CHOWDHRY AND STEPHEN E . HARDING

(f) Mutant proteins, different ligands, and homologues structures (as low as 25%

sequence identity) may be extracted by using molecular replacement and

subsequent use oielectron densityielectron density difference methods as an

aid for comParison.

(g) Large molecules and assemblies can be determined' e'g' virus particles

(howeverorderingbetweensuchassembliesisnotusuallyasgoodas-i.e.

theyoftendiffractlesswellthan_smallmolecularweightmolecules).

(h) Surface water molecules relatively well defi'ned'

(i) often produces a single struchrral model that is easy to visualize and interpret'

fi)Useofsynchrotronradiationcombinedwithcryo.conditionsusuallyspeedsup data acquisition, improves resolution and stability of crystals'

ThedisadvantagesofX-raycrysta|tographicmethodsincludethefollowing:

(a)Protein/protein-ligand--complexhastoformstablecrystalsthatdiffractwelland X-ray crystaG$aptry does not directly yield hydrogen atom positions'

(Neutron diftaction t".irrriqu"t make this possible but usually only for small

molecules and large size crystals are required')

(b) Need heavy atom derivatives that form isomorphous crystals' unless molecu-

lar rePlacement methods are used'

(c)Crystalproductioncanbedifficultandtime-consumingandoftenimpossible.

(d) Unnatural, non-physiological environment' i'e' may not wholly represent

structure as it exists in solution'

(e)Difficultyinapportioninguncertaintybetweenstaticanddyrramicdisorder.

(fl Surface residues may be influenced by crystal packing'

(g) Large molecular weight flexible modular proteins can be problematic and

the final strucftlral model lepresents a time-averaged structure, where

details of molecular mobility remain unresolved'

The ad'vrmtage.s ofNMR for structural elucidation are as follows:

(a)Experimentsaleconductedundersolutionconditionswhichare.closertobiological conditions' than X-ray methods (i.e. free from artefacts resulting

from crystallization).

(b)Canprovideinformationondynamics(relaxation,rotational'andtransla-tionaldiffirsionmeasurements,prctonexchangerates,andthevibrationalmotionofatoms)andidentifyindividualsidechainmotions.

(c)SecondaryStructulecanoftenbederivedfromlimitedexperimentaldata.

(d)Increasinglyusedtomonitorconformationalchangeonligandbindingto

Protein or to fragments thereof'

(e)Goodforcompar ingandcheckingthecorrect fo ldofmutants(usefu l forprotein folding/time resolved studies)'

( f )Solut ioncondi t ionscanbeexpl ic i t lychosenandreadi lychanged,e 'g.pH'temperature, etc' (but high concentrations of buffers may be a problem)'

l 0

PROTEIN_L IGAND INTERACTIONS AND THEIR ANALYSIS

(g) 'Internal' water molecules can be detected (as in X-ray), but surface watermolecules can be problematic because of fast exchange (lifetimes can still beascertained).

(h) H-bonds can now be detected (rather than inferred from distance constraintsas in X-ray).

(i) The increasing availability of high resolution instruments (e.g. 700 MHz andabove) combined with new data acquisition/analysis techniques and greatercomputing power is and will continue to allow significant improvements inthe application of NMR techniques.

The disadvantages of NMRfor shactural elucidahon include the following:

(a) Usually require concentrated solutions, which may present a danger ofaggregation: this problem is manifested in e.g. many antibody systems.

(b) Currently limited to determination of relatively small proteins (< 20 kDa),but there are exceptions.

(c) Surface residues generallyrless well deflned than in X-ray crystallography be-cause of mobility of surface residues and experimentally fewer interactions(NOEs).

(d) Often produces an ensemble of possible structures rather than one model (thiscan be advantageous) but conformational variability can make data interpre-tation difficult and complete structure determination required if proteinsequence homology is less than - 60%.

(e) Labelling often used, e.g. 2H, 13C, and 1sN and may cause problems in termsofprotein yield and expense.

It has to be emphasized that true high resolution structures sf 1[a raertino

species and the complex are sorely needed in order for example to:

(a) Obtain statistically unbiased data on both protein stereochemistry/precisegeometry and the validity of the parameters used to obtain the data (i.e. theirrefi.nement).

(b) Check the application of 'normal mode' calculations.

(c) Calculate charge density distributions.

(d) Analyse hydration shells around protein molecules and also for a moreaccurate view ofhydrogen bonds.

(e) Obtain precise information on the direction of amino acid side chain/domainmovement.

(f) Model alternative conformations/multi-conformational species.

(g) Help to quantitate the physico-chemical parameters involved in the inter-action, especially weak non-covalent interactions.

(h) Obtain precise surface areas.

Not only are these parameters required for intrinsic reasons but also for help-ing to analyse data obtained from other techniques and thereby improve thetheoretical underpinning of experimental observations.

1 1

BABUR Z . CHOWDHRY AND STEPHEN E ' HARDING

ffi Othet sPectroscoPic techniques

oneof theur tuesofspectroscopictechniquesusedinexaminingprote ins,ligands,andprotein-ligandinteractionsisthattheyareusuallynon-destructiveandsometimesnon-invasive,howeversomeproceduresdorequiretheattach.ment of labels at speciflc sites on one or both of the interacting species'

Relatively soft ionization methods introduced in mass spectromew (MS) allow

agent lephasef fansfer f romsolut ionintothegasphaseof themassSpectro-metersuchthatevenweaklyboundnon.covalentcomplexescanbedetectedintactandthei rmassanalysed.MSmethodssuchasEsl-Mshavebeguntoberecentlyusedfbrthedeterminationofassociationconstantsofnon-covalentinteractions. one of the advantages of these methods is that signals due to pro-

tein,ligand,andprotein-ligandionsignalscanbedetectedseparately'Inadditionhigher order structure formation' e'g' dimerization may also be detected by MS

methods,althoughanalyticalultracentrifugationisprobablybettelsuitedfolthis PurPose

Fluorescence specvoscow is iL technique that is very sensitive (picoglam quanti-

t iesofmater ia l . " , 'u"d" t " . ted)andWel l -su i tedformeasurement intherealdme domain. tts principal shortcomings' however' are that only the structure of

thefluorescentprobeandtheimmediateenvironmentisreportedandthedataobtained is not easy to interplet. Nonetheless, fluorescence spectroscopy has

foundwiderrr"irmtodyi"gthephysico-chemicalpropertiesofproteins'protein-ligand interactions, utta plot"it' dynamits' This is because almost all proteins

containnaturallyfluorescentaminoacidresiduessuchastyrosineandtrypto.phan. In addition, a large number of fluorescent dyes have been developed that

can be used to speciicatty probe the function and/or structure of macro-

molecules'Incaseswhereonlylimitedquantit iesofproteinsareavailable(e.g.arecentlyexpressedrecombinantproteinorapreciousproteinpharmaceutical),fluorescence spectroscopy is often the method of choice for studying properties

such as stability, trydroiynamics' kinetics' or ligand binding' because of its

exquisitesensitivtty.Eve,,.i.'.",",wherethestructureofproteinsis.well.known'(eitherfromx-raydiffractionorNMR),elec\onparamagneticresonance(EPR)'time.resolved Jluorescence spectroscopy ' and stopped-flow methods can be particularly useful

for investigating their dlmamic behaviour'

CirqiqrdtcWoism(CD)iSanimportant,widelyusedandcommonlyavailabletechniqueforsecondaryStructuledetermination.Itrequiresexpertiseindatacollection/analysistobeusedtoitsfullpotential 'whichisnotalwaysthecase.Nevertheless it has the following advantages in that relatively small amounts of

material are required (0'1-1 mg/ml can often suffice) and results can be obtained

quickly (in hours rather than weeks or months as in the case of NMR and X-ray)'

Theaminoacidsequenceoftheproteinneednotbeknown_althoughobviouslyithelps.CDcanbeusedtoexaminetheeffectsofenvironmentalconditionssuchaspH,tempelatufe,etc.,ontheoverallproteinconformation,inthepresenceandabsenceofaligand.It isinfactaveryusefultechniquepriortoX-raycrystallo.graphy and/or NMR for screening mutant proteins' obtained by molecular

l 2

PROTEIN-L IGAND INTERACTIONS AND THEIR ANALYSIS

cloning methods, in relation to their secondary structure characteristics. CD,used in the stop-flow mode is particularly useful for studying protein-ligandinteractions in real time. In order to minimize the signal to noise ratio, in CDstudies, solution components should, ideally, be UV transparent; this limits theuse of certain buffers, salts, and solvents. Unfortunately because of theoreticalproblems, the technique cannot be easily/reliably used to determine and inter-pret tertiary structure or changes therein.

Fourier transform infrared spectrosclpy (FT-fR) is also very useful for secondarystructure determination of proteins, where the information content is verysimilar to CD techniques. However there is greater flexibility in FT{R, at leastfrom the viewpoint of the type of samples that can be investigated: tissueslices, cells, solid state (powder; freeze dried) samples, crystals, thin fllms, andaqueous proteins and protein-ligand samples. FT-IR can be used to investigatethe structure of proteins in the presence and absence of ligands, solubilized inDrO by using the protein amide 1 band for analysis, provided that the absorp-tion bands of the ligand do not interfere. Use of HrO is a problem. Other prosand cons of this techniqueiare similar to those for CD and Raman spectroscopy.It is, for example, rseful for examining the effect of variations in solution con-ditions on proteins and protein-ligand interactions. In addition the presenceand absence of hydrogen bonds can also be investigated. FT-IR is limited to theuse of short path length cells and research grade instruments are not com-monly available.

Raman spectroscopy has been used for many years to study structural andenzymatic proteins as well as protein-ligand (particularly enzyme-substrate/inhibitor) interactions, albeit in laboratories specializing in the technique. Againit is an excellent method for the study of variable solution state conditions. Theuse of resonance Raman is advantageous, since speciflcity and sensitivity areimproved relative to off-resonance Raman. Information about electronic statescan also be gained fiom resonance Raman spectra. Fluorescence effects maypose a problem but the FT-Raman technique can very often eliminate these. Itsuse is not limited by cell path lengths and as in FT{R different sample srates,e.g. cellular tissue can be examined.

& Non-spectral methodsEEtilibrium dialysis and pH tihation represents the traditional approach-whichdates back to the classical Scatchard analysis-to the study of ligand binding.Another titration probe, ITC (sothermal titrahon calorimetry) together w'ith therelated DSC (drfferential scanning calorimetry) technique are now widely and in-creasingly, used to examine proteins and protein-ligand interactions. The twotechniques are complementary and are the only methods currently availablewhich allow the direct determination of the enthalpy of an intra- or inter-molecular reaction. Other thermodlmamic parameters can also be determineddirectly, indirectly, or by using model-based assumptions. Both techniques

t 3

BABUR Z . CHOWDHRY AND STEPHEN E . HARDING

provide invaluable information in relation to the energetics of a system and

significant advances have been made, especially over the last two decades, in

the theoretical basis of interpretation of experimental results. It has to be

emphasizedhoweverthat theyaremacloscopictechniqueswi that tendantadvantagesanddisadvantageswhich,shouldnotbeovel lookedintermsofexperimentaldesignandinterpretationoflesults.Asimilarcomplementarypair of techniques are sednnentahon velocitry and sedimentahon eEilibntnn analyhcal

ittracenwifuganon. The latter, like calorimetry' provides an absolute thermo-

dynamicprobeofinteractions(intermsofthestoichiometryandassociationordissociation constants, Ka' and the related Gibbs free energy AG : RT.lnKJ and

molecular mass (oligomeric structure). The former (sedimentation velocity) is a

goodtool for theseparat ionandanalys isofheterogeneousmixturesof thevarious reaction components, as well as a highly useful solution conformation

probeoftheleactantsandproducts.Anotherusefulprocedure-alsobasedonaseparation principle (but requiring a separation medium as opposed to a centri-

fugalfleld)isaffinilychromalography(fiontalandzonalprocedures).Interactionstoichiometries can also be obtained by the technique of coupling size exclusion

chromatography to multi-angle laser light scattering. This SECIMALLS procedure

(Tab\e4)isverymuchcomplementarytoanalyticalultracentrifugationandisconsidered in the earlier book by creighton in the Practical Approach series (see

Chaptergofref.6).AnalyticalultracentrifugationandSECarejusttwoexamplesof lt-ydrodynamic procedures. others, based on the electrical properties of pro-

teins and ligands, are eTeclo-ophcs (which can provide valuable information on

the effect of ligand binding on solution conformation of a protein) based-like

fluorescence depolarization-on the measurement of rotational diffirsion be-

haviour, and the rapidly emerging probe (with its great resolving powel on Very

small quantiti es) of capillary electrophoreis. solutionx-ray andneutron scattenng' aTso

provideaverysensitivehandleontheeffectsofl igandbindingonthesolutionionformation of a protein, and the surface probe of atomic force microscopy pro-

vides the potential for individual complexes to be visualized (although very

difficult to use and data interpretation can be problematic)''

ffi Computational methods/molecular modelling

contemporary computing facilities and capacity have revolutionized virtuallv

every aspect of scientiflc investigations related to protein-ligand interactions'

This includes experimental protocol design as well as collection and analysis of

data.Mostpeople,unfortunately,associatemolecularmodell ingwiththenowcommonplace colourful complex two- and three-dimensional protein or

protein-iigand docking images produced via graphic workstations' Howevet

this gives a totally erfoneous picture of the importance of molecular rnodelling

techniquesforanalysingandpredictingthephysicalpropertiesofmolecules.Itisperhaps,impossibletounderestimatetheusefulnessofsuchtechniquesbothcurrently and in the future.

t 4

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BABUR Z . CHOWDHRY AND STEPHEN E ' HARDING

ffi lnlotmation sourcesuseful sources of information include the protein structural databases given ir

Tabte5.Inadditionmanyscientif icl iteraturereferencedatabasesnowexist'These include, but are not limited to' the following:

(a) Mimas (ISI@, Citation Indexes & ISTP@ hffp://wos'mimas'ac'uk/)' Access re-

quires institutional affi liation'

(b) Chemical abstracts'

(c) Medline.

(d)PubMed (www'ncbi'nlm'nih'gov/)' A new facility called PubMed Central is

due at the time of going to Press:

? Additional lemarl{sTo date we have obtained' *h"t -"y appgar at first sight' an incredible amount

ofdataandt}reoreticaiinsightintothepropertiesandbehaviourunderpinningthe function of proteins in biological systerns' However in

-reality it could' nay

should, be argued tfrat we have merely scratched the surface in our quest to

understand and predict the intricate and interwoven complexities of protein-

l igandinteract ions.Thereisnodoubt thatamul t id isc ip l inaryapproachisrequired which entalts intra- and/or inter-laboratory collaborations which in

turn necessitate the collaborator or non-expert to have some level of under-

standing of the techniques/methods in which they thernselves are not experts'

It is to be hoped that the subject matter'included in this volume will help' at

least in Part, to achieve this aim'

However tne discerning reader will note that the foregoing overview has not

dealt with any topic in aelail uut has merely attempted to wet their appetite for

questioning currerlt achievements ind future exploration' T:i" T

an infinite

numberofunansweredquest ionscomparedtoanswels. 'Forexample,non-covalently controlled ph"r'o*""" are still very poorly understood' It has not yet

been possible to design, /ro m first prinnpl"' "t"t a srr-rall'molecular weight mol-

ecule that binds with a ,designed' affinily and speciflcity to a binding site in a

protein of known *"t'o"of,it structure' despite widespread interest in' e'g'

rational drug design' Again at the expense of repetition' qu-estions should be

asked, above all uy novile researchers as to the applicability of'test-tube' experi-

ments to biological systems' What are the effects for example' of the fact that

biologicalsystemsao"otoperateunderequilibriumthermodynamicconditions?Further what are trr" ffii."tions of the concentrations at which molecules

occur, the packing density (as well as membrane association) of reactions and

the activity of water, l" t"it' compared to the manner an{.;ollltrons under

which mvitroexperiments are conducted? In truth we are still at the first stage

of a long quest. Nonetheless the techniques described in this volume are

helping us at Ieast make some inroads in the right direction' ;

T6

PROTEIN-L IGAND INTERACTIONS AND THEIR ANALYSIS

References1. Longhi, S., Czjzeck, M., and Cambillau, C. (1998). Curr. Opin. Struct. Biol.,8,73O.2. Williams, D. H. andWestwell, M. S. (1998). Chem.Soc.Rev.,27,57.3. Philo,J.S.(1999). InCtmentProtocolsinProteinScience(ed.P.T.Wingfield),

pp.20.7.7.-20.1.13. J. Wiiey & Sons, NY.4. Hensley, P. (1996). Structure, 4, 367.5. Harding, S. E. (1993). Biotech.Gen.Eng.Rev.,a1.,317.6. Creighton, T. E. (1997). Proteinstructure: aPracticalApproach.OxfordlJniversityPress.

T 7