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Protein-ligand interactionsand
their analysis
BaburZ. Chowdhry
Schoolof Chemicaland LifeSciences,University
of Greenwich,
Wellington
Street,Woolwich,LondonSE18 6PF,UK.
StephenE. Harding
NCMHPhysicalBiochemistry
Laboratory,
University
of Nottingham,
Schoolof
Biosciences,
SuttonBonington,Leicestershire
LE12sRD, UK.
% lnfioduction
In 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 a
result of finely-tuned inter- and intramolecular recognition mechanisms, and
forms part of an intricate network of interdependent multi-component linear/
non-linear reactions in interconnected compartments (organelles etc.) of the
cell. 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,0.7-120MPafor hydrostaticpressure,and 0.6-1.0for
water activity. Amongst the many molecules that participate in such reactions in
the complex-and, as yet hardly understood, milieu of the cell-are proteins.
Proteins play a pivotal, indeed essential role, in cellular (and in multicellular
organisms, extracellular) activity. Their numerous biological functions together
with the molecular basis of their biophysical propertiesftehaviour are therefore
of multidisciplinary interest within the framework of what are generally called
the biological (biochemistry, pharmacology, physiology, immunology, etc.) and
physical (physics,chemistry, mathematics, computing, etc.) sciences.
Both our intrinsic curiosity-driven philosophical knowledge baseand our need
or 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 biological
behaviour of these molecules. Becauseprotein-ligandinteractionsplay akey role in
cellularmetabollsm,detailed knowledge of such interactions, at both a microscopic
and mactoscopic level, is also required. Just two examples, which immediately
stand out from the many, are antigen-antibody interactions and proteins which
E. HARDING
B A B U RZ . C H O W D H RAYN DS T E P H E N
actaSleceptors(membraneornon-membranebound):neurotransmissionde-
pendsontheabilityofsmallandlargemolecularweightmoleculestorecognize
receptors' Another example:
and bind to specific sites on large membrane bound
large group of important complexes'
enzyme-ligand interactions form a very
whichhavebeeninvestigatedformanyyears.Inthesesystemsnotonlyisthe
substrate(s)ofobviousimportance,butsoareothermolecules,whichregulate
enzymeactivitysuchasto""'y-"'andpositiveandnegativemodulatorsin
important systems,which are
both allosteric and non-allosteric proteins. other
nowbeingstudiedatamolecularlevel,includeligandbindingtostructuralpro-
. teins, protein-DNA binding, protein-saccharide, protein-protein, and protein-
peptideinteractions.Theformation-andmaintenance_ofthequaternary
of large Structures such as
Structure of multisubunit proteins, the self-assembly
interactions are
microtubules or chromatin and transcription factor-promoter
endless'
other examples; although the list is actually almost
Theterm.ligand'inbiologicalsystemscanhavemanydifferentmeanings.Itis
usually,initsbroadestsensE,usedtomeananymoleculewhichinteractswitha
givenmolecule(inthiscaseaprotein).Theterm.ligand,thusincludesother
macromolecules(peptides/proteins/nucleicacids/lipids/carbohydtatesotmixed
molecularspeciesttrereof)aswellas,small'molecularmass(arbitrati|y<-7-2
kDa)molecules.Ligandsthereforecompriseaverylargeandstructurallydiverse
groupofmoleculeswhich,unsurprisinglyalsodisplayawidevarietyofphysico.
chemicalcharacteristics.Thismakesitdiff,culttounderstand,delineate'and
drawgeneralizedconclusionsconcerningtheirbiophysicalproperties.Perhaps
that follows, is that ligands
the major criterion, in the context of the discussion
caninteractina(potentially)reversible,non-covalentmannelwithaprotein
way (i.e. without the
and thereby modJate its biological role in a controllable
requirement to make or break covalent bonds)'
Tofullyunderstandprotein-ligandinteractionsrequiresthat'ataminimum'
thefollowingcriteriabemet.Firstthatthebiophysicalpropertiesofboththe
ploteinandthe..ligandunderinvestigationareexaminedindependentlyand
this requirement applies
that their biophysicai behaviour be fully understood:
tobothexistingand/ornewlydesignedand(bio)synthesizedpeptides/proteinsand
ligands.Currently,and.contrwytopopularoptnion,wearefarfromachievingsuchart
ann,exceptatonextremelysryerfiaallwel'Knowledgeofthestructure/conformation'
ifpossible,attheatomicleveloftheproteinandtheligandintheunbound
formisaprerequisiteforprotein-ligandinteractionstudies.Althoughitmay
appeartrivialtothereader,thehighlevelresolutionstructulesofmostproteins
have not' to date' been
(either by X-ray crystallography or high resolution NMR)
established.Itrscurrentlyestimatedthatsuchdataexistsforonlyabout30
sense of the generally
peptides/proteins (1) but it is seriously arg'able, in the
acceptedmeaningofthetermhigh(atomic)resolution(i.e.<0.8A1,thateven
thistargethasnotbeenmet'Additionallytheprocessofformingacomplexbe-
tweenasmallligandmoleculeandaproteinisacomplicatedequilibrium
state probably exist as
process.Both theligand and the protein in the solvated
for many'small mol-
an ecuilibrium mixture of several conformers. Admittedly
P R O T E I N _ L I G A NIDN T E R A C T I O NA
SN D T H E I R A N A L Y S I S
chemicalcomposition, concentration/purity
(contaminants) activltyor
/stability/specific
assayfor, e.g. structuralproteinsand ligand/stateof structuralintegrity.Abilityto freeze
or air dryand pre-history
of proteinand ligandsamplesmaybe important.
(in
Solubility differentsolvents:aqueous,organic,and mixedphase);stateof aggregation.
Struciure:size.shape.geometry, topology:proteinsequence. and post-translationai
modifications must be known;abilityto use molecular biologytechniques (strategies for
over-expression and mutagenesis of recombinantproteinare critical);
combinatorial
melhodsfor over productionof ligand.
Dynamics of protein/liganOT
protein-tigand'complex'.
ecular weight ligands' high resolution x-ray and/or NMR structures do exist. The
fact that our knowledge base is deflcient in this area has signiflcant implications
for understanding protein-tigand interactions, which unfortunately will not be
touched upon, in this brief overview. In addition a myriad of other properties of
the reactantsalso requile to,be established(seeTable1).
Secondlythe protein-ligand complex must also be fully characterized. Norm-
ally, at a simplistic level, the non-covalent interaction between a protein (p) and
a ligand (L) is often represented as follows:
P+LePL t1l
However as williams and westwell have pointed out (2) the interaction should
actually be written in the following form:
n * r.-- r'r' l2l
Equahon2 may, at flrst glance appear identical to Equahon 1. However the
formulism of Equation2 recognizes, or is taken to mean, that once p and L have
undergone an interaction or association,they no longer exist. They have, instead
been replacedby the modifled entities p' and L'. (Tight complexesoften result
when protein-ligand interactions are significantly stronger than ligand-solvent
and protein-solvent interactions.)
The well described equilibrium molar association or binding constant, I("
(other popular symbols are I(5 or just IQ corresponding to Equation2 is then
describedby:
= p'L'l / [P]tll)
11" t3l
where the squarebracketsmeansthe molar concentration(M, or rnol.l-i),which
of course is not an SI unit. For the system described by Equation2 (or Equahon1)
the unitsl of K" will be M-1 (l.mol-i).The more popularly used 'molar dissociation
constant' Ka is simply the reciprocai of I(,,,so for the system of Equation2..
of the protein,
in the analysis
variables
Table2 Experimental the ligand'andthe
protein-ligand
complex
( a ) Reagentvariables
Protein/ligandconcentration
Ligandstructuralvariants(analogues/homologues)
mutants;
Proteinstructuralvariants(site/groupspecific,conservative/non-conservative
denatuledfo-T/partlallyunfoldedfolms, prese_nce ol absenceof cofactors)
solutionconditionvariables
(b) Environmental
Temperature,pH, buffers(ionicstrengthand natureof buffers)
Osmolytes,solventsincludingco-solvents, salt /ion concentration
Denaturants(urea/GdnCl (andother lyotropic/chaotropicagents)/detergents'
DTT;gaseousphase
sufactants),stabilizers(azide,glyceroletc.); mercaptoethanol,
used fo1experiments,et :
(c) Combinationsof the above
PROTEIN-LIGAND
I N T E R A C T I O NA
SN D T H E I RA N A L Y S I S
Designof expelimentalprotocol
Intormation
content
Availability.
cost
Complementary/alternative tectrniques"
Analyticalparametersassociatedwith the technique(s):concentrationof materialrequired,
comparedto other techniques,sensitivity,detectionlimit, accuracy,precision,
reproducibility/repeatability,use or necessityfor isotopicor non-isotopic labelling,
easeof
use, computingresources,methods/complexity and differentmethodsof data analysis,
errors/errcrpropagation/statistical analysisof results(if possible);time scalesof
experimental (spectroscopic)techniquesused.
Abilityto use the techniquein the presenceof bufferadditivesand/orenvironmental solution
conditionseivenin T3ble2
The experimentalistshouldinsureifrat at everystageof the experimentand (particularly)in
the data analysis,the correct(appropriate) theoreticalmodelsare used. In using
instrumental techniques the'blackboxdata analysis'syndrome shouldbe avoided.
Criticallycompareand contrastWithwork on other relatedsystems.Eachmethodwill have
its own advantagesand drawbacksfor a givensystem.The abilityto interpretexperimental
data both intrinsicallyand in terms of a theoreticalbasis is veryimportant.The
systematic/accumulated instrumentalerrorsare often important,but usuallyignored.
uTry,if possibleto use more than one technique(method)to verify
the overallresults or the valuesfor
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9
E. HARDING
B A B U RZ . C H O W D H RAYN DS T E P H E N
(aslow as 25%
(f) Mutant proteins, different ligands, and homologues structures
replacement and
sequence identity) may be extracted by using molecular
density difference methods as an
subsequentuse oielectron densityielectron
aid for comParison.
virus particles
(g) Large molecules and assemblies can be determined' e'g'
(howeverorderingbetweensuchassembliesisnotusuallyasgoodas-i.e.
theyoftendiffractlesswellthan_smallmolecularweightmolecules).
(h) Surface water molecules relatively well defi'ned'
interpret'
(i) often produces a single struchrral model that is easyto visualize and
fi)Useofsynchrotronradiationcombinedwithcryo.conditionsusuallyspeeds
up data acquisition, improves resolution and stability of crystals'
ThedisadvantagesofX-raycrysta|tographicmethodsincludethefollowing:
(a)Protein/protein-ligand--complexhastoformstablecrystalsthatdiffractwell
atom positions'
and X-ray crystaG$aptry does not directly yield hydrogen
(Neutron diftaction t".irrriqu"t make this possible but usually only for small
molecules and large size crystals are required')
unless molecu-
(b) Need heavy atom derivatives that form isomorphous crystals'
lar rePlacement methods are used'
(c)Crystalproductioncanbedifficultandtime-consumingandoftenimpossible.
represent
(d) Unnatural, non-physiological environment' i'e' may not wholly
structure as it exists in solution'
(e)Difficultyinapportioninguncertaintybetweenstaticanddyrramicdisorder.
(fl Surfaceresidues may be influenced by crystal packing'
problematic and
(g) Large molecular weight flexible modular proteins can be
structure, where
the final strucftlral model lepresents a time-averaged
details of molecular mobility remain unresolved'
ofNMR for structural elucidation are as follows:
Thead'vrmtage.s
(a)Experimentsaleconductedundersolutionconditionswhichare.closerto
resulting
biological conditions' than X-ray methods (i.e. free from artefacts
from crystallization).
(b)Canprovideinformationondynamics(relaxation,rotational'andtransla-
tionaldiffirsionmeasurements,prctonexchangerates,andthevibrational
motionofatoms)andidentifyindividualsidechainmotions.
(c)SecondaryStructulecanoftenbederivedfromlimitedexperimentaldata.
(d)Increasinglyusedtomonitorconformationalchangeonligandbindingto
Protein or to fragments thereof'
(e)Goodforcomparingandcheckingthecorrectfoldofmutants(usefulfor
protein folding/time resolved studies)'
(f)Solutionconditionscanbeexplicitlychosenandreadilychanged,e'g.pH'
be a problem)'
temperature, etc' (but high concentrations of buffers may
l0
P R O T E I N _ L I G AINNDT E R A C T I O A
NNS DT H E I RA N A L Y S I S
(g) 'Internal' water molecules can be detected (as in X-ray), but surface water
molecules can be problematic becauseof fast exchange (lifetimes can still be
ascertained).
(h) H-bonds can now be detected (rather than inferred from distance constraints
as in X-ray).
(i) The increasing availability of high resolution instruments (e.g.700 MHz and
above) combined with new data acquisition/analysis techniques and greater
computing power is and will continue to allow significant improvements in
the application of NMR techniques.
Thedisadvantages
of NMRfor shacturalelucidahoninclude the following:
(a) Usually require concentrated solutions, which may present a danger of
aggregation: 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 generallyrlesswell deflned than in X-ray crystallography be-
cause of mobility of surface residues and experimentally fewer interactions
(NOEs).
(d) Often producesan ensemble of possiblestructures rather than one model (this
can be advantageous)but conformational variability can make data interpre-
tation difficult and complete structure determination required if protein
sequencehomology is lessthan - 60%.
(e) Labelling often used, e.g. 2H, 13C,and 1sNand may causeproblems in terms
ofprotein yield and expense.
It has to be emphasized that true high resolution structures sf 1[a raertino
speciesand the complex are sorely needed in order for example to:
(a) Obtain statistically unbiased data on both protein stereochemistry/precise
geometry and the validity of the parameters used to obtain the data (i.e.their
refi.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 more
accurate view ofhydrogen bonds.
(e) Obtain precise information on the direction of amino acid side chain/domain
movement.
(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 reasonsbut also for help-
ing to analyse data obtained from other techniques and thereby improve the
theoretical underpinning of experimental observations.
11
E' HARDING
B A B U RZ . C H O W D H RAYN DS T E P H E N
ffi OthetsPectroscoPictechniques
oneoftheurtuesofspectroscopictechniquesusedinexaminingproteins,
ligands,andprotein-ligandinteractionsisthattheyareusuallynon-destructive
andsometimesnon-invasive,howeversomeproceduresdorequiretheattach.
both of the interacting species'
ment of labels at speciflc sites on one or
in massspectromew(MS)allow
Relatively soft ionization methods introduced
agentlephaseffansferfromsolutionintothegasphaseofthemassSpectro-
metersuchthatevenweaklyboundnon.covalentcomplexescanbedetected
intactandtheirmassanalysed.MSmethodssuchasEsl-Mshavebeguntobe
recentlyusedfbrthedeterminationofassociationconstantsofnon-covalent
methods is that signals due to pro-
interactions. one of the advantagesof these
tein,ligand,andprotein-ligandionsignalscanbedetectedseparately'Inaddition
may also be detected by MS
higher order structure formation' e'g' dimerization
methods,althoughanalyticalultracentrifugationisprobablybettelsuitedfol
this PurPose
(picoglam quanti-
Fluorescence specvoscowis iLtechnique that is very sensitive
t i e s o f m a t e r i a l . " , ' u " d " t " . t e d ) a n d W e l l - s u i t e d f o r m e a s u r e m e n t i n t h e r e aof
l
however' are that only the structure
dme domain. tts principal shortcomings'
thefluorescentprobeandtheimmediateenvironmentisreportedandthedata
fluorescence spectroscopy has
obtained is not easy to interplet. Nonetheless,
foundwiderrr"irmtodyi"gthephysico-chemicalpropertiesofproteins'protein-
This is because almost all proteins
ligand interactions, utta plot"it' dynamits'
containnaturallyfluorescentaminoacidresiduessuchastyrosineandtrypto.
dyes have been developed that
phan. In addition, a large number of fluorescent
function and/or structure of macro-
can be used to speciicatty probe the
molecules'Incaseswhereonlylimitedquantitiesofproteinsareavailable(e.g.a
recentlyexpressedrecombinantproteinorapreciousproteinpharmaceutical),
of choice for studying properties
fluorescence spectroscopyis often the method
or ligand binding' because of its
such as stability, trydroiynamics' kinetics'
exquisitesensitivtty.Eve,,.i.'.",",wherethestructureofproteinsis.well.known'
(eitherfromx-raydiffractionorNMR),elec\onparamagneticresonance(EPR)'time.
' andstopped-flow
spectroscopy methodscanbe particularly useful
resolvedJluorescence
for investigating their dlmamic behaviour'
CirqiqrdtcWoism(CD)iSanimportant,widelyusedandcommonlyavailable
techniqueforsecondaryStructuledetermination.Itrequiresexpertiseindata
collection/analysistobeusedtoitsfullpotential'whichisnotalwaysthecase.
in that relatively small amounts of
Neverthelessit has the following advantages
suffice) and results can be obtained
material are required (0'1-1 mg/ml can often
as in the caseof NMR and X-ray)'
quickly (in hours rather than weeks or months
Theaminoacidsequenceoftheproteinneednotbeknown_althoughobviously
ithelps.CDcanbeusedtoexaminetheeffectsofenvironmentalconditionssuch
aspH,tempelatufe,etc.,ontheoverallproteinconformation,inthepresenceand
absenceofaligand.ItisinfactaveryusefultechniquepriortoX-raycrystallo.
proteins' obtained by molecular
graphy and/or NMR for screening mutant
l2
PROTEIN-LIGAN
I NDT E R A C T I O A
NNS DT H E I RA N A L Y S I S
& Non-spectralmethods
EEtilibrium dialysisand pH tihation represents the traditional approach-which
dates back to the classical Scatchard analysis-to the study of ligand binding.
Another titration probe, ITC (sothermaltitrahon calorimetry)together w'ith the
related DSC(drfferentialscanningcalorimetry)technique are now widely and in-
creasingly, used to examine proteins and protein-ligand interactions. The two
techniques are complementary and are the only methods currently available
which allow the direct determination of the enthalpy of an intra- or inter-
molecular reaction. Other thermodlmamic parameters can also be determined
directly, indirectly, or by using model-based assumptions. Both techniques
t3
E. HARDING
B A B U RZ . C H O W D H RAYN DS T E P H E N
a system and
provide invaluable information in relation to the energetics of
especially over the last two decades,in
significant advanceshave been made,
experimental results. It has to be
the theoretical basis of interpretation of
emphasizedhoweverthattheyaremacloscopictechniqueswithattendant
advantagesanddisadvantageswhich,shouldnotbeovellookedintermsof
experimentaldesignandinterpretationoflesults.Asimilarcomplementary
pair of techniques are sednnentahon eEilibntnn analyhcal
velocitryand sedimentahon
like calorimetry' provides an absolute thermo-
ittracenwifuganon.The latter,
dynamicprobeofinteractions(intermsofthestoichiometryandassociationor
: RT.lnKJ and
dissociation constants, Ka' and the related Gibbs free energy AG
(sedimentation velocity) is a
molecular mass (oligomeric structure). The former
goodtoolfortheseparationandanalysisofheterogeneousmixturesofthe
conformation
various reaction components, as well as a highly useful solution
probeoftheleactantsandproducts.Anotherusefulprocedure-alsobasedona
to a centri-
separation principle (but requiring a separation medium as opposed
fugalfleld)isaffinilychromalography(fiontalandzonalprocedures).Interaction
size exclusion
stoichiometries can also be obtained by the technique of coupling
SECIMALLS procedure
chromatography to multi-angle laser light scattering. This
(Tab\e4)isverymuchcomplementarytoanalyticalultracentrifugationandis
series(see
considered in the earlier book by creighton in the Practical Approach
Chaptergofref.6).AnalyticalultracentrifugationandSECarejusttwoexamples
properties of pro-
of lt-ydrodynamic procedures. others, based on the electrical
(which can provide valuable information on
teins and ligands, are eTeclo-ophcs
of a protein) based-like
the effect of ligand binding on solution conformation
rotational diffirsion be-
fluorescence depolarization-on the measurement of
powel on Very
haviour, and the rapidly emerging probe (with its great resolving
solutionx-rayandneutronscattenng'aTso
small quantiti es)of capillaryelectrophoreis.
provideaverysensitivehandleontheeffectsofligandbindingonthesolution
microscopypro-
ionformation of a protein, and the surface probe of atomicforce
to be visualized (although very
vides the potential for individual complexes
difficult to use and data interpretation can be problematic)''
ffi Computationalmethods/molecularmodelling
virtuallv
contemporary computing facilities and capacity have revolutionized
related to protein-ligand interactions'
every aspect of scientiflc investigations
analysis of
This includes experimental protocol design as well as collection and
data.Mostpeople,unfortunately,associatemolecularmodellingwiththenow
protein or
commonplace colourful complex two- and three-dimensional
Howevet
protein-iigand docking images produced via graphic workstations'
rnodelling
this gives a totally erfoneous picture of the importance of molecular
techniquesforanalysingandpredictingthephysicalpropertiesofmolecules.It
isperhaps,impossibletounderestimatetheusefulnessofsuchtechniquesboth
currently and in the future.
t4
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tl
I
E' HARDING
B A B U RZ . C H O W D H RAYN DS T E P H E N
ffi lnlotmationsources
given ir
the protein structural databases
useful sources of information include
Tabte5.Inadditionmanyscientificliteraturereferencedatabasesnowexist'
the following:
These include, but are not limited to'
Access re-
(a) Mimas (ISI@,Citation Indexes & ISTP@hffp://wos'mimas'ac'uk/)'
quires institutional affi liation'
(b) Chemical abstracts'
(c) Medline.
facility called PubMed Central is
(d)PubMed (www'ncbi'nlm'nih'gov/)' A new
due at the time of going to Press:
? Additionallemarl{s
appgar at first sight' an incredible amount
To date we have obtained' *h"t -"y
ofdataandt}reoreticaiinsightintothepropertiesandbehaviourunderpinning nay
systerns' However in -reality it could'
the function of proteins in biological our quest to
scratched the surface in
should, be argued tfrat we have merely
the intricate and interwoven complexities of protein-
understand and predict
l i g a n d i n t e r a c t i o n s . T h e r e i s n o d o u b t t h a t a m u l t i d i s c i p l i n a r y a p p rwhich
o a c h i sin
inter-laboratory collaborations
required which entalts intra- and/or
non-expert to have some level of under-
turn necessitate the collaborator or
in which they thernselves are not experts'
standing of the techniques/methods
in this volume will help' at
It is to be hoped that the subject matter'included
least in Part, to achieve this aim'
that the foregoing overview has not
However tne discerning reader will note for
merely attempted to wet their appetite
dealt with any topic in aelail uut has an infinite
exploration'
questioning currerlt achievements ind future T:i" T
n u m b e r o f u n a n s w e r e d q u e s t i o n s c o m p a r e d t o a n s w e l s . ' F o r eItx ahas
m pnot
l e , nyet
on-
covalently controlled ph"r'o*""" are still very poorly understood'
mol-
prinnpl"' "t"t a srr-rall'molecularweight
been possible to design,/ro m first
,designed' affinily and speciflcity to a binding site in a
ecule that binds with a in' e'g'
protein of known *"t'o"of,it structure' despite widespread interest
of repetition' qu-estions should be
rational drug design' Again at the expense experi-
as to the applicability of'test-tube'
asked, above all uy novile researchers that
are the effects for example' of the fact
ments to biological systems' What
biologicalsystemsao"otoperateunderequilibriumthermodynamicconditions?
Further what are trr" ffii."tions of the concentrations at which molecules
and
membrane association)of reactions
occur, the packing density (as well as under
to the manner an{.;ollltrons
the activity of water, l" t"it' compared
conducted? In truth we are still at the first stage
which mvitroexperiments are are
techniques described in this volume
of a long quest. Nonetheless the
in the right direction' ;
helping us at Ieast make some inroads
T6
PROTEIN-LIGAN
I NDT E R A C T I O A
NNS DT H E I RA N A L Y S I S
References
1. 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.
T7