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The World of Nano-Biomechanics, Second Edition Atsushi Ikai

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THEWORLDOF NANO-BIOMECHANICS

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THEWORLDOF NANO-BIOMECHANICS

SECONDEDITION

Editedby

ATSUSHIIKAI

TokyoInstituteofTechnology,Yokohama,Japan

Elsevier

Radarweg29,POBox211,1000AEAmsterdam,Netherlands TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates

Copyright 2017ElsevierB.V.Allrightsreserved.

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PrefacetotheSecondEditionxiii PrefacetotheFirstEditionxvii

1.ForceinBiology1

A.Ikai

1.1 WhatAreWeMadeOf?1

1.2 HumanBodyandForce3

1.3 MacroscopicBiomechanics5

1.4 MolecularBasisforStructuralDesign7

1.5 SoftVersusHardMaterials9

1.6 BiologicalandBiomimeticStructuralMaterials14

1.7 ThermodynamicsandMechanicsinNanometer-scaleBiology15 Bibliography15

2.IntroductiontoBasicMechanics17

A.Ikai

2.1 ElasticandPlasticDeformationofMaterials17

2.2 StressandStrainRelationship18

2.3 MechanicalBreakdownofMaterials19

2.4 Viscoelasticity21

2.5 FluidandViscosity26

2.6 AdhesionandFriction27

2.7 WearandTearofBiologicalStructures29

2.8 MechanicallyControlledSystems32 Bibliography34

3.ForceMeasurementandMechanicalImagingApparatuses35

A.Ikai

3.1 Mechanical,Thermal,andChemicalForces35

3.2 LaserTrap36

3.3 AtomicForceMicroscope40

3.4 SurfaceForceApparatus51

3.5 BiomembraneForceProbe51

3.6 MagneticBeads53

3.7 GelColumns53

3.8 CantileverForceSensors54

3.9 Loading-RateDependence54

3.10 ForceClampMethod57

3.11 SpecificVersusNonspecificForces57 Bibliography59

4.InteractionForces63

A.Ikai

4.1 CovalentVersusNoncovalentBonds63

4.2 BasicsofElectrostaticInteraction64

4.3 VariousTypesofNoncovalentInteractions66

4.4 ApplicationofExternalForce72

4.5 InteractionForceBetweenMacromolecules72

4.6 WaterattheInterface74 Bibliography75

5.PolymerChainMechanics77

A.Ikai

5.1 PolymersintheBiologicalWorld77

5.2 PolymerChains78

5.3 End-to-EndDistance80

5.4 PersistenceLength85

5.5 PolymersinSolution87

5.6 PolymersontheSurface89

5.7 PolymersAsBiomimeticMaterials90

5.8 PolymerPullout90 Bibliography92

6.AnalysisofDataGleanedbyAtomic-ForceMicroscopy95

S.Kasas,G.Dietler

6.1 Introduction95

6.2 GeneralProcessingofTopographicAFMImages96

6.3 Specimen-Speci ficAnalysisProcedures100

6.4 ProcessingofForceSpectroscopyData104

6.5 Conclusions108 Bibliography109

7.Single MolecularInteraction111 A.Ikai

7.1 Ligand ReceptorInteractions112

7.2 Sugar LectinInteractions115

7.3 Antigen AntibodyInteractions117

7.4 GroELandUnfolded-ProteinInteractions118

7.5 Lipid ProteinInteractions120

7.6 AnchoringForceofProteinstotheMembrane122

7.7 ReceptorMapping123

7.8 ProteinUnanchoringandIdentification125

7.9 MembraneBreaking126 Bibliography130

8.Single-MoleculeDNAandRNAMechanics133

A.Ikai

8.1 StretchingofDouble-StrandedDNA133

8.2 UnzippingofdsDNA137

8.3 ChainDynamicsandTransitionofDNAandRNA138

8.4 DNA ProteinInteraction140

8.5 DNAMachine142

8.6 ProspectforSequenceAnalysis142 Bibliography143

9.Single-MoleculeProteinMechanics147 A.Ikai,R.Afrin

9.1 IntroductiontoProteinManipulation147

9.2 Protein-StretchingExperiments148

9.3 IntramolecularCores150

9.4 StretchingofModularProteins152

9.5 DynamicStretching154

9.6 CatchBonds154

9.7 Protein-CompressionExperiments157

9.8 InternalMechanicsofProteinMolecules166

9.9 MechanicalControlofProteinActivity168

9.10 ComputerSimulationofProteinDeformation169

9.11 CaseStudies:ProteinsandPolypeptidesofNotableStructural Characteristics170 Bibliography181

10.NanomechanicsofMotion-SupportingMolecularSystems187

A.Ikai

10.1 CellMovementandStructuralProteins187

10.2 MuscleandMotorProteins189

10.3 SingleMolecule/FilamentMeasurements191

10.4 FlagellaforBacterialLocomotion192

10.5 Mycoplasma Gliding192

10.6 MechanicsandEfficiencyofMotorProteins194

10.7 VideoViewofMotorProteinsinActionbyHigh-SpeedAFM195 Bibliography195

11.Finite-ElementAnalysisofMicrobiologicalStructures199 S.Kasas,T.Gmur,G.Dietler

11.1 Introduction199

11.2 ABriefHistoryoftheFinite-ElementMethod200

11.3 TheFinite-ElementMethod201

11.4 ApplicationoftheFinite-ElementMethodtoMicrobiologicalStructures202

11.5 Conclusions215 Bibliography216

12.NanomechanicalBasesofCellStructure219

A.Ikai

12.1 RedBloodCell:ModelCellinBiomechanics219

12.2 HelfrichTheoryofMembraneMechanics221

12.3 Deformationof2DMembrane223

12.4 MembraneandCytoskeleton226

12.5 AssociationofMembraneProteinsWithCytoskeleton227

12.6 Nano-IndentationExperimentsonLiveCells231

12.7 StiffnessTomographyandCellResponseStudies241 Bibliography243

13.NanorheologyofLivingCells249 T.Okajima

13.1 CellRheology249

13.2 AFMMeasurementsofCellModulus251

13.3 High-ThroughputMeasurementsofCellRheologicalProperties257

13.4 ElasticModulusofNormalandCancerCells260

13.5 AFMImagingModeforMeasuringViscoelasticPropertiesofCells261 Bibliography264

14.MolecularandCellularManipulationsforFuture Nanomedicine267

A.Ikai

14.1 ProspectsforUsefulApplicationsforNanomedicine267

14.2 BioconjugationofMaterials268

14.3 NanomechanicalManipulationofCellsAimingatNanomedical Applications269

14.4 CellSurgery270

14.5 ChromosomalSurgeryandGeneManipulation270

14.6 TissueSurgery271

14.7 LiposomalTechnology271

14.8 DrugDelivery273

14.9 DNAandRNARecoveryFromtheChromosomeandtheCell274

14.10 WoundHealing276 Bibliography280

AppendixOne:BeamBending283

AppendixTwo:V-ShapedCantilever297

AppendixThree:PersistenceLengthVersusKuhnLength299

AppendixFour:HertzModel301

AppendixFive:DerivationoftheLoading-RateDependenceoftheMeanRuptureForce309 Index 313

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LISTOFCONTRIBUTORS

R.Afrin

TokyoInstituteofTechnology,Yokohama,Japan

G.Dietler

EcolePolytechniqueFédéraledeLausanne,Lausanne,Switzerland

T.Gmur

EcolePolytechniqueFédéraledeLausanne,Lausanne,Switzerland

A.Ikai

TokyoInstituteofTechnology,Yokohama,Japan

S.Kasas

EcolePolytechniqueFédéraledeLausanne,Lausanne,Switzerland; UniversitédeLausanne,Lausanne,Switzerland

T.Okajima

HokkaidoUniversity,Sapporo,Japan

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PREFACETOTHESECONDEDITION

LifeemergedontheEarthseveralbillionsyearsagothroughthe self-assemblingtendencyofprebioticmolecules,inparticular,proteins, lipids,andpolynucleotides.Moleculesaremadeofcovalentlylinkedatoms andhavedifferentphysical/chemicalproperties.Dependingontheir differentmakeups,theyeitherattractorrepeleachotherwhentheycome intocloseproximity.Intheabsenceofspecialcatalysts,interactionsbetween suchmoleculesaremostlylimitedtononcovalenttypes,whichareessentiallyelectrostaticinnatureandweakerthancovalentbonds,especiallyin water.Emergenceoflifewasbasedonnoncovalentself-assemblyofcovalentlybondedprebioticmolecules.

Anuncontrolledattractiveself-assemblageleadstomeaninglessaggregate formationbut,inaconstructiveself-assembly,molecules findspecifictargets formutualassociationandformhigher-orderstructureswithsomenotable functions.Oneofsuchhigher-orderstructuresontheprimordialEarthwasa living “cell.” Allthelifeformswe findtodayonthisplanetcantracetheir lineagebacktosuchprimordialcells.Wearetheproductsofalongand uninterruptedhistoryofevolutionfromoriginalprimitivecells.Weaknoncovalentinteractionsareresponsibleforsupportingourlivesasdistinguished frominanimatecongregationsofatomsandmolecules.

Inourdailyenvironment,noncovalentassociationofmoleculesisweak, andresultingstructurescanbeeasilydeformedorevendisassembled.Importantly,thelife-supportingmoleculesandassembledstructuresaresmalland floatinginwater.Waterparticipatesinmanyaspectsoflife-supportingreactions.Oneparticularroleofwateristosurroundindividualmoleculesand keepthenoncovalentinteractionundereasycontrol.

Biomechanicsatmicro-tonanoscalestructuresandmaterialsdealswith themeasurementofmechanicalpropertiesofbiologicalobjectsandinterpretstheexperimentallyobtainedresultsaccordingtothebasicmechanics developedinphysicsandengineering fields.Biologicalsamplesviolate almostalltheassumptionsroutinelymadetoderivebasicequationsinmechanicsforasimplifiedinterpretationofexperimentalresults.Notonlyare biologicalsamplesathousandtimessofterthansteelorrocks,thusallowinga largedeformationunderasmallappliedforce,butalsotheyaremadeof complexlayeredstructuresmadeofamixtureofdifferentmaterials(inhomogeneous)andmanifestdirection-dependentmechanicalproperties

(anisotropic)inadditiontoanextremelycomplexsurfacetopography. Currently,mainlybecauseofalackofappropriatetheoreticaltreatmentof sampleswithsuchcomplexity,mostoftheexperimentalresultsareinterpretedassumingthatsamplesarehomogeneous,isotropic,andhavea flatsurface.Difficultyofpreparingatestspecimensatisfyingtheassumptionsof moreadvancedtheoreticaltreatmentisanotherobstacle.Despitesuchdifficultiesandproblems,measurementsandinterpretationsofmechanicalpropertiesofsmall-scalebiologicalspecimensareproducinginteresting findings fortherevelationofbasicbiologicalprinciples.Newtechnologiestomanipulateindividualcells,proteins,andDNA/RNAareemergingfromsuch studies.Whatwelearnedabouttheprinciplesofself-assemblingsystems arewidelyappliedintechnological fieldstoproducenewmaterialsandsystemshavingadvancedfunctions.Bylearningaboutthemechanicalnatureof livecells,newmedicaltechnologiesforsingle-cellmanipulationareunder intensiveinvestigations.

Inasense,weliketoask,onwhatmechanicalprinciplesareourbodies constructed?Fromastructuralpointofview,ourbodyisconstructedon multiplexednetworksof fi brousproteinslocallycompartmentalizedby cellmembranes.Compartmentalizationisnecessarymainlybecausebiologicalinformationtransferisdependentondiffusionofsignalmolecules inwater.Withinasmallcompartment,signaltransductionbymolecular diffusionisfastenoughtosustainlifeonameaningfultimescale,buton alargescale,itisfatallyslow.Cellssenseandrespondtoenvironmental stresses,mechanicallyandbiochemic ally.Theysenseexternallyapplied forcethroughlocaldeformationoftheiroutermoststructure,i.e.,cell membrane.Thissensationisconveyedtootherpartsofthecellbyway ofthe fi brousnetworksystemmentionedbefore.Thestrengthand deformabilityofthemechanicalconnectionswithinthe fi brousnetwork thatextendstoallovertheentirecellbodyformsimportantbackground forthebiologicalinformationtransduction.Itcouldbefasterthandiffusiondependentinformationtransfer.Propertiesofproteinmoleculesresponsiblefornetworkfunctionsareatthefrontlineofthecurrentresearch. Thuswebelievethatmechanicalpropertiesofcellularstructuresarefundamentalfortheadvancementofourknowledgeinbiologyandbiotechnologicalmanipulationoflivingcells.

Thisbookdescribesbasicnotionsofmechanicstobiologicallyoriented studentsandtothoseresearchersconsideringmakingmechanicalmeasurementsontheirsamplesusingnanotechnologicalmethods,inparticular

atomicforcemicroscopy.Wetriedtobuildaneasyaccesstobasicmechanics forthosenotfamiliarwiththemethodinthe field.

The firstversionofthisbookappeared2008.Inthelasteightyearsthere hasbeenremarkableprogressinthe fieldsofnanotechnologyandnanobiology.Itisagoodtimingtorevisethe firstedition,addingnewexperimental andtheoreticalresultsfrompublishedliteraturetofamiliarizereaderswith somefundamentalnotionsofbiologicalnanomechanicstothereaders.At thesametime,asmuchaspossiblewecorrectederrorsfoundinthe firstedition.Wehopethebookwillbefriendlyguideforstudentsandresearchers interestedinmechanicalpropertiesofthosenanometer-sizedmembersof thebiologicalworld.

Thisbookhasgreatlybenefitedfromgenerouscontributionsof Dr.R.Afrin(Chapter9),Dr.G.Dietler,Dr.T.GmurandDr,SandorKasas (Chapters6and11),andDr.TakaharuOkajima(Chapter13).Publicationof thebookhasbeenmadepossiblebytheuntiringeffortsandcollaborations ofthoseconcernedatElsevier,inparticular,Ms.AnitaKoch,Ms.Amy ClarkandMr.PaulChandramohan.Wethanktheauthorsandjournals ofpublishedworkforgivingpermissionsforustocitetheartworks fromtheoriginalliterature.

Iwouldliketoexpressmysinceregratitudetoallthepeoplewhohave madepublicationofthisbookpossible.

October2016

AtsushiIkai(principalauthor) TokyoInstituteofTechnology

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PREFACETOTHEFIRSTEDITION

Nano-biomechanics,thetitle fieldofthisbook,iscurrentlyemergingasa newandattractiveareaofscientificresearchbridgingbiologicaland mechanicalsciencesatthemolecularlevel.Biomechanicswithoutthe prefixof nano hasbeenaquiteactive fielddealingmainlywithmacroscopic bodilymovementsandespeciallywiththedynamicsofblood flow.In nano-biomechanics,avarietyofnewlydevelopeddeviceswiththecapabilityofobservingandmanipulatingindividualatomsandmoleculesare ambitiouslyappliedtoelucidatetheprinciplesoflife-supportingmolecular interactions.Imyselfamnotaphysicistormechanistbutabiochemist workinginthisexciting fieldandaminterestedinthematerialnatureof biomoleculesandbiostructuresthattriggeredtheemergenceoflifesome fourbillionyearsago,andsincethen,havebeensupportingproliferation oflifesosuccessfully.

SinceIstartedworkingwithatomicforcemicroscopesalmost20years ago,however,Ihaveexperiencedsomeproblemsinbridgingbiochemistry andmechanicsofmaterialsinmyownwork.Irealizedthatusingequations thatrelatedthemeasuredquantitiestothemechanicalparametersofthe materialfortheinterpretationofexperimentallyobtaineddatawasonething butitwasquiteanothermattertounderstandthebackgroundofthoseequations.Inanapplied fieldsuchasthis,provedequationsarepickedupfrom variousdifferentsourcesofmechanics,presentingdifficultyforanewcomer to findtherighttextbookseverytimeheorsheencountersnewequations. Thisbookismeanttobeofsomehelpinsuchoccasionsanddealsexclusivelywiththeprovenresultsofclassicalmechanicscurrentlyusedinthe measurementofmaterialpropertiesofproteinsandcellsatthesinglemolecularandsingle-cellularlevels.

Thankstoarecentinstrumentaldevelopmentthatisnurturinganenormousenthusiasmamongscientistsandengineerstocreateanew fieldof nanotechnology,someofthetraditionalbarriersthatexistedbetween biologicalandphysicalsciencesarenowrapidlydisappearing,atleast,at themolecularlevel.Itisnaturallytruethattheultimategoalsofphysicalscientistsandthoseofbiologicalscientistsaredifferent,butallofushaveshared interestsinthebehaviorofmolecules,smallorlarge,andinanewpossibility ofmanipulatingthembydirectlytouchingeachoneofthem.

Sincebiologicalmacromoleculesarenotelectricallyconductive,biologicalinformationtransferisperformedmechanically,notelectronicallyasin thecaseofcomputertechnology,throughdirectcontactsofparticipating atomsandmolecules.Theactivityofanenzyme,forexample,iscommonly modulatedthroughbindingandunbindingofeffectormoleculestothe enzyme.Atthecellularlevel,aligandmoleculeasacarrierofextracellular physiologicalinformationbindstoamembrane-associatedreceptor,triggeringarelayofmechanicalinformationtransferfromtheoutsidetothe insideofthecell.

Examplessuchasgivenhereinaboundinbiology,promptingusto considermechanicsasanimportantandindispensabletoolinunderstandingthebasicsofbiologyanddevelopinganewengineeringmethodology forhandmanipulatingproteins,DNA,andcells.Oneofthediverse purposesofmanipulationisinthedevelopmentofnewbiomedical technologies.

Usingmechanicsasamanipulationtoolatthemolecularlevelrequiresus tounderstandatleasttheessenceofthemechanicsofmaterials,whichhasa longandoutstandinghistoryinphysicsandengineering.Formanyofus withbackgroundsinbiology,molecularbiology,biochemistry,orchemistry,thelevelofundergraduateeducationinmechanicsisratherlimited,and anextraeffortisrequiredtounderstandtheworkingprinciplesofinstrumentsforthemeasurementofmechanicalpropertiesofmaterialsandto interprettheresultsofsuchmeasurements.Mostoftheresearchpapersin the fieldsrelevanttonano-biomechanicsarewrittenontheassumption thatthereadersarefamiliarwithelementarymechanicsaswellaswiththe backgroundofderivationsofmanyoftheequationsvitaltotheinterpretationofdata.Itistime-consumingatleast,thoughnotimpossible,to findreferencestotherequiredknowledgefromavastarrayoftextbookson mechanicsandtounderstandthebackgroundofthe finalequationstobe usedinthemeasurementandinterpretationofdata.

Thisbookisessentiallyacollectionofbasicequationsinmacroscopic continuummechanicsthatarenecessarytounderstandingresearchpapers inbiomechanicsatthenanometerandnanonewtonlevels.Itriedtoexplain howsuchequationswerederivedfromthebasicprinciplesoflinearmechanics,hopingthatthisbookwillsavetimeforthosewhoarecoming intothisnew fieldandlookingforaconcisecompilationofnecessary knowledgefromvariousdisciplinesofclassicalmechanics.Thesubjectof thisbookismainlystaticmechanicsand,asaresult,suchotherwise

importantsubjectsinnano-biomechanicsasviscoelasticity, fluiddynamics, andnonlinearmechanics,forexample,arenottreatedoronlybrieflyintroduced.Readersarerecommendedtoconsultwithpopulartextbooksin appropriate fields.Somereadersmay findthisbooktooelementaryor filled withtoomanyequationsbecauseItriedto fillinthebackgroundderivations,evenelementary,asmuchaspossiblesothatthosewhohavehitherto notbeenfamiliarwithmechanicscanseethemeaningofequationsand enjoytheprocessofdevelopingamechanicalwayofthinking.

Asstatedearlier,sincetheemphasisofthebookisintheexpositionof basicmechanics,examplesofapplicationworkarenotatallexhaustive andnotmeanttobe.Myapologyisdueheretomanyauthorsofimportant workthatIcouldnotrefertointhisbook.Examplesaretakentoilluminate basicideasofapplyingmechanicalprinciplestothestudyofbiologicalmacromoleculesandstructuresbuiltuponthem,andmanyofthemarefromthe workdoneintheLaboratoryofBiodynamicsofTokyoInstituteofTechnologywhereIwork.Myspecialthanksgotothepublishersandindividual authorswhogenerouslygrantedmetherighttoreproducecited figures fromtheirpublications.

Iwouldliketothankmanyfriendsandcolleagueswhosupportedmein writingthisbook;amongthem,specialthanksareduetoDr.R.Afrinfor hercontributionofthesectionofCaseStudyoncarbonicanhydraseIIin Chapter8andDrs.S.Kasas,T.Gmur,andG.DietlerwhokindlycontributedChapter12ontheapplicationofthe fi niteelementmethod.MyspecialthanksarealsoextendedtoDrs.H.SekiguchiandI.Haradafor preparingsomeofthe fi gures,Dr.M.Miyataforprovidinganoriginal photographof Mycoplasma,Drs.R.Afrin,A.YersinandH.Sekiguchifor proofreadingoftheoriginalmanuscriptandMr.A.Itohforpreparingthe coverdesign.Iam,however,solelyresponsibleforanymistakesandinappropriateexplanationsthatmaybefoundinthebook,andexpectto receivekindlycommentsfromawidespectrumofreadersthrough e-mail(ikai.a.aa@m.titech.ac.jp).

IalsoexpressmysinceregratitudetoDrs.O.Nishikawa,S.Morita,and M.Tsukada,amongmanyothers,forintroducingmetothe fieldofnanomechanicsofatomsandmoleculesandencouragingmetocontinuethe workinthe field.Inaddition,Iextendmyheartfeltthankstomypast andpresentcolleaguesandgraduatestudentswithwhomIhadandcurrently haveexcellentopportunitiestoworktogetherelucidatingtheexquisitenaturesofbiomacromoleculesandbiologicalstructures.

Finallybutnotintheleast,IwouldliketothankMs.KristiGreen,Ms. DonnadeWeerd-Wilson,Mr.EzhilvijayanBalakrishnan,andMr.Erik OosterwijkatElsevierwhoparticipatedintheproductionofthisbook andwereofgreathelptome.

AtsushiIkai

September,2007

ForceinBiology

A.Ikai

TokyoInstituteofTechnology,Yokohama,Japan

Contents

1.1 WhatAreWeMadeOf?1

1.2 HumanBodyandForce3

1.2.1 GravityandHydrodynamicForce3

1.2.2 FrictionalCoefficients5

1.3 MacroscopicBiomechanics5

1.4 MolecularBasisforStructuralDesign7

1.5 SoftVersusHardMaterials9

1.6 BiologicalandBiomimeticStructuralMaterials14

1.7 ThermodynamicsandMechanicsinNanometer-scaleBiology15 Bibliography15

1.1WHATAREWEMADEOF?

Themainthemewearegoingtoexploreinthisbookisthequestion of “ Whatkindsofmaterialsarewemadeof? ” Ourbodiesaresoftandfragilecomparedwithmanyinanimateobjectsinthisworld,man-madeor not.Can ’ twehavearock-hardbodysothat,inacaraccident,thecar istheonethatiscrashedandwearetheonestosurvive?Ifitwereso, cardriversincongestedcrossingswouldbemoreapprehensive.Ourother dailyactivitiesmightbeseverelycompromised,though.Otherwise,since ourbodyissaidtobetheresultofself-assemblyofalargenumberofmolecules,canwe,inthefuture,controltheassemblyanddisassemblyprocessesofourmoleculessothatwemay ,atleast,reassemblethemafter injurieshavedysfunctionalizedourbody?Arti fi cialmanipulationof atoms,molecules,cells,andtissuesofourbodyisessentiallythesubject of nano-biomechanics .Forthemanipulationofsuchbodilyobjectsina distantfuture,weneedtoknowthephysicalpropertiesofthematerials thatmakeupourbody.Asidefromanimportantexceptionofthe nerve-brainsystem,ourbodyfunctionsmorelikeamechanicaldevice ratherthananelectroniccomputer.We,therefore,investigatethemechanicalpropertiesofthebodilycomponents,namely,proteins,nucleic

TheWorldofNano-Biomechanics

ISBN:978-0-444-63686-7

http://dx.doi.org/10.1016/B978-0-444-63686-7.00001-8

acids,polysaccharides,lipidassemblies,biomembranes,cells,andsoonby usingthestate-of-the-arttechnologiesavailabletousatthepresentstage. Itisespeciallyimportanttorealizethatthemostabundantbodilycomponents,proteins,areelectricallynonconductive,andthereforetheinformationtransferwithinandbetweenprotein-basedstructuresismainly conductedthroughtheirmechanicalcontactsandensuingdeformations. Sincemechanicalmanipulationisperformedwithanapplicationofforce tothesampleobjects,wewillexplore,inthe fi rstchapter,themeaningof forcefromourdailyexperience.Forceissomethingthatcanbefeltandis amorefamiliarconceptthanthermo dynamicfunctionssuchasenthalpy orentropy.Thisbookdealswiththeeffectofforceonverysmallscale becausewewillbetalkingaboutatomsandmolecules,andeventually aboutlivingcellsthatarestilllessthan1mminsize.Atomsarevery stronglybuiltofprotons,neutrons,andelectronsandwillnotbreak downinourbody,exceptforatinyfractionofradioisotopesifthere areany.Moleculesareclustersofatomsbondedtogetherbycovalent bonds,whicharealsoquitestronganddif fi culttobreak,butaremuch weakercomparedwiththeforceoperatingatthenuclearlevel.Molecules canbeconvertedfromoneformtoanotherbycreating,breaking,and/or exchangingcovalentbonds,oftenwiththehelpofacatalyst.Catalysts, whenusedinindustry,convertnitro gengasintoammoniainonenotable example,andinlivingorganismstensofthousandsofthemareatwork, convertingfoodstuffintovariousp artsofourbodyandintotheenergy consumedinourdailyactivities.

Catalystsinourbodyarecalledenzymes.Oneofthem,calledinvertase, forexample,bindswithasugarmoleculeandconvertsitintoglucoseand fructosebybreakingacovalentbondintheoriginalmolecule.Selectively bindingaspecificsubstratemoleculefromamongmillionsofsimilarlylookingmoleculesisthemostimportant firststepforanyenzyme.Bindinginthis caseispromotedbyweakerforcesassociatedwith “noncovalentinteractions” or “noncovalentbonds.” Ourbodilymovementsandactionsare theresultofthesenoncovalentinteractionsbetweenandamongtensof thousandsofmoleculesinourbody.Inthenextfewchapters,wewillinvestigatetypesofinteractionsoperatingatthemolecularlevelinlivingorganisms.Tolivemeanstoperformdailyactivities,andtodosoandtoimprove theperformance,organismshavedevelopedmanyexoticdevicesbuiltof proteins,nucleicacids,lipids,andcarbohydratesasthemajorsourcematerials.Wewillinvestigatethebasicphysicalpropertiesofdevicesthuscreated aswellasthoseofthesourcematerials.

1.2HUMANBODYANDFORCE

1.2.1GravityandHydrodynamicForce

Wefeelforcebythesensationtothemusclesofourbodywhenwelift aweightagainstgravity,orwhenwesuddenlyaccelerateourcar,for example.Sinceforceistheproductofmass(m)andacceleration(a),we feelitwhenthecarisacceleratingbutdonotfeelitwhenthevehicleis glidingataconstantspeed(i.e.,when a ¼ 0).Whenweridearollercoaster, wefeelgravitationalaswellascentrifugalforceasthecoastingtrainrushes forwardconqueringtheupsanddownsandsharpcornersonthetrack. Wealsofeelforceasashockwhensomeonesuddenlypushesusfrom behind.Asuddenapplicationofaforcesuchasthisiscalledan “impact.” Forceis,thus,somethingthatcanbefeltasabodilysensationwhenwe arepushedaround.Whenyoubreakormovesomething,youneedforce, anditisbettertohavearoughestimateoftherequiredforceforthetask.

WefeelthegravityoftheEarthbecausewe,withabigandheavybody, liveundertheair,whichhasmuchlessdensitythanourbody.Ifyoulivein waterlikewhalesand fishesdo,youprobablydon’tfeelthegravitythat muchbecausethegravitationalforceislargelycompensatedbythe flotation effectinwater.Asyougodownthescaletomuchsmallerlevels,you encounterthelifewithlesssensationofgravitationalpull.Bacteria,for example,swimaroundupanddown,leftandrightwithoutfeelingmuch ofthegravitationalpull.Theyfeelaratherstrongeffectofviscosityofwater. Asthescaleofyourbodybecomessmall,theviscosityeffectbecomespredominantcomparedwiththeinertialeffectofmass.Thedimensionless Reynoldsnumber(Ry)givesaroughestimateoftherelativeratioofinertial forceversusviscousforce.

where, R and v arethecharacteristicsize(forexample,thelength)and velocityofthemovingbodyand r, h arethedensityandviscositycoefficient ofwater(¼ 0.001Pas ¼ 0.001Ns/m2),respectively.Fora2-m-longanimalswimmingunderwaterataspeedof10m/s, Ry ¼ 1000(kg/m3) 2(m) 10(m/s)/0.001(kg/ms) ¼ 2 107,averylargevalue.Whereas foramicroorganismoflength1 mmandswimmingat1 mm/s, Ry isvery small: Ry ¼ 1000(kg/m3) 10 6 10 6/0.001 ¼ 1 10 6.IftheReynoldsnumberislessthan w2000,the flowpatternaroundthemovingbody issmoothwithoutanyturbulenceandcalledalaminar flow,whereaswhen

Ry > 2000,the flowtendstobeturbulentofteninvolvingvortices.Inboth casesof flow,themovingbodyexperiencesaninertialresistanceasitpushes abodyofwaterasideandviscousresistancefromthewaterstucktothe entirebodysurface.Inaturbulent flow,themovingbodymustexperiencea dragforceduetoaneddy(Fig.1.1).

Viscosityisameasureofresistanceofaliquidwhenitisforcedto flow overasolidsurface(seeChapterTwo).Foramovingbodyofmolecular scale,the flowarounditisasmoothlaminar flow,notaturbulentone. Theforceactingonasphericalobjectofradius a inalaminar flowisgiven bytheStokes’ lawaswillbementionedbelow,where f, h,and v are,respectively,thefrictionalcoefficient, fluidviscosity,andvelocityofthesphere.

Forabacteriumofanapproximatediameterof1 m mandswimming inwaterataspeedof1 m mpersecond,thefrictionalforceisapproximately0.02pN ¼ 0.02 10 12 N( ¼ 6 p 0.001N/m 2 s 10 6 m 10 6 m/s),whichisovercomebytheforcegeneratedbythebacterial fl agellasystem.Inamicro-to-nanometer-scaleworld,viscousforce

Figure1.1 TheReynoldsnumberistheratioofinertialforcestoviscousforcesina fluid flow.Itinfluences,forexample,swimmingpatternsoflargeandsmallorganisms. Photo courtesyofPatakuso.

predominatesovertheinertialresistance,andthemagnitudeoftheviscous dragcanbecalculatedaccordingtotheStokes ’ lawasdescribedabove.

1.2.2FrictionalCoefficients

Intheirdailyactivityin fluid,smallorganismsareunderstronginfluenceof thefrictionaldragforce.Thusitisimportanttohaveanestimateofthefrictionalcoefficient.Thecoefficientmustbechangeddependingonthesize andshapeofthemovingbody.Thefrictionalcoefficientfornonspherical bodiescanbeapproximatedbythemethoddevelopedbyGarcia [1,2] or by fittingtoanalyticalexpressionsforprolateoroblateellipsoids [3,4].AccordingtoRef. [1],anapproximatevalueofthefrictionalcoefficientofan objectofanyshapecanbeobtainedbymodelingtheshapeoftheobjectby anassemblyofsmallspheresofradiusawiththecenter-to-centerdistance rij fromanothersphereofthesameradius.Asimplifiedequationforthecaseof identical N subunitsisasfollows.

where fN and f1 arethefrictionalcoefficientof N-merandthatofa monomer,respectively.ThemethodwasappliedbyIkaitoobtainthe frictionalcoefficientofcomplexproteins [5].Hydrodynamicforceactingon smallobjectssuchasmicroorganismsormoleculesisquitesmall.Itis, however,animportantfactorforunderstandingtheirbehavior.

Exactformulasforthefrictionalcoefficientsofprolate(long)andoblate (flat)ellipsoidofrevolutionaregivenin Table1.1[3,6]

Theeffectofvarioustypesofforceactingonmacroscopicbiological structureshasbeenstudiedinthe fieldofbiomechanics.Sophisticatedanalysisbasedonmathematicalformulationofmechanicsisappliedtounderstandtheresponseofthebiologicalstructurestowardexternallyapplied forces.Comprehensivetreatisesarefoundintheliterature [7,8].

1.3MACROSCOPICBIOMECHANICS

Thedisciplineofbiomechanicsitselfhasalonghistory.Thisbranchof sciencefocusesonmechanicalprinciplesofthefunctionandmovement ofourbody,andthusdealsmainlywithmacroscopicmechanics.Thebasis ofthedisciplineisthehighlydevelopedtheoreticalandexperimental

Table1.1 FrictionalCoefficientsofProlateandOblateEllipsoidinLaminarFlow

ShapeFrictionalratioa

Prolateellipsoid

Radiusof equivolume sphere

Axialratio (a and b are semiaxesof ellipsoid)

aTheseareaveragedexpressionsfortheparallel(jj)andperpendicular(t)movementagainstthemajor semiaxis.

CompiledafterS.Hansen,Translationalfrictioncoefficientsforcylindersofarbitraryaxialratios estimatedbyMonteCarlosimulation,J.Chem.Phys.121(2004)9111 9115andK.E.vanHolde, C.Johnson,P.S.Ho,PrinciplesofPhysicalBiochemistry,seconded.,Prentice-Hall,EnglewoodCliffs, NJ,1988.

mechanicswithalonghistoryofbrilliantworkandwithhighlyusefulapplicationstotheconstructionandmaterialsinindustry,tonameonlya few.Thoughitisabranchofengineeringmedical,muchworkhasalso beendoneinrelationtoabroadspectrumofmedical fieldsincludingsports andrehabilitationmedicine,andpeoplewithavarietyofbackgroundsare concernedwiththisdiscipline.Thereisadeepscientificandindustrial commitmenttobiomechanicsincludingthemechanicsofmusclecontraction,blood flow,organdevelopment,effectofinjuries,artificiallimbs,sports medicine,cellularmechanics,and,asarecentaddition,themolecularand cellular-levelmechanicsinourbody.Nano-biomechanicscanbeconsidered asanoffspringofbiomechanicsinthesensethatitdealswiththeeffectof forceonbiomoleculesandbiostructureshavingdimensionsofnanometer. Theprincipleofmechanicsisthesameinbiomechanicsandnanobiomechanics,butthemethodstomeasureasmallforceanditseffecton biosystemsaredifferentfromthoseusedinmacroscopicbiomechanics.Classicalmechanicsdealsmainlywithmaterialsthatarehomogeneousin compositionandlargeintheirscalecomparedwiththesizeofatestprobe. AcomprehensivetreatmentofbiomechanicsisgivenbyFung [7].

Recentdevelopmentofvariousphysicalmethodstomeasuresmallforces andsmalldisplacementshasencouragedresearchersinterestedinbiomacromoleculesandcellularstructurestoelucidatetherelationshipbetween themagnitudeofanappliedforceonasampleandtheextentofitsdeformation(stress strainrelationship)atthemolecularlevel.Byexperimentally establishingsuchrelationsandbyapplyingtheoreticalpredictions,wecan extractmechanicalparametersinherenttothematerialpropertiesofthe

sample.Thankstothetechnologicalandtheoreticaladvancementsinnanoscienceandnanotechnology fields,itisnowpossibletopushand/orpulla singlemoleculeofproteinstoobtaintheforcetounfolditfromacompact globuletoalinearlyextendedstring.Resultingresponsecurvestellusabout therigidityandtensilestrengthoftheinter-andintramolecularsegmental interactionsresponsibleformaintainingafunctionalfoldedstructure.A similarexperimentisnowalsopossibleonasingle-strandDNA,thebasic geneticmaterial.Themethodhasbeenappliedtoelucidatethemechanistic principleoffoldingDNAofatotallengthapproximately1mintothe cellularnucleusofanapproximatediameterofafewmicrometers.

Inthisbook,thematerialpropertiesofbiologicalmacromoleculesand structuresthatareself-assembledfromthemareexpoundeduponinlater chapters.

1.4MOLECULARBASISFORSTRUCTURALDESIGN

Thebasicprincipleofthestructuraldesignofbiologicalsystemsisto buildeverythingbottomupfrommolecules.Macroscopiccomponentsof thebodyareallbuiltdirectlyfrommoleculesthatareweaklybutmostspecificallyinteractingwitheachother.Sincetherearenoconstructionworkers around,ourbodyisbuiltontheprincipleofself-assemblyofconstituent molecules.Incontrast,acraneataconstructionsite,forexample,ismade ofarelativelysmallnumberofmacroscopicmembersofexplicitdesigns tomakethemrigidandunbending.Thehumanarmexecutesasimilar taskasacrane’sbutonasmallerscale.Themotionofanarmiscontrolled directlybyacollectionofmicroscopicmusclecells(myocytes),andtheforce isgenerateddirectlyfromthemolecularmotionofprotein filamentsinthe cell.Thus,biologicalsystemsarebuiltonthedynamicinteractionsofalarge numberofmolecules,mainlyproteins.Proteinsarelinearpolymersof20 kindsofaminoacidsandaretightlyfoldedintospecific3Dconformations thatareprogrammedtomeettheiruniquefunctionsinmostspecificways. Onanindividualbasis,proteinsfunctionasenzymes,antibodies,receptors,channels,inhibitors,andhormones.Whenorganized,theyfunctionas microtubules,muscle filaments,tendons,bones,teeth,hair,andsilk fibers,to nameafew.Thousandsofenzymesareknown,forexample,eachcatalyzing aspecificreactioninaconcertedway,sothatthousandsofbiochemicalreactionsproceedinacontrolledfashiontokeepthehostorganismhappily alive.Enzymesarefunctionallyhighlyspecialized.Anenzymehasthecapacitytobindonlyasinglekindofmolecule,calledasubstrate,toitsactivesite

andcarryoutanecessarytransformationofthemolecule.Bindingofasubstratetotheactivesiteisthe firststepofanenzymecatalysis.Suchbindingis basicallyamechanicalprocessinthesensethatthesubstrateisattractedand directedtotheactivesitethroughmechanicallyguidedpathways.Whenthe substrateis finallyclosinginontotheenzyme’sactivesite,the3Dconformationoftheenzymeisalteredtoaccommodateitintoitsactivesite.The boundsubstrateisforcedtochangeitsconformationintoadifferent,somewhatdistortedonefromitsmoststablestate.Thedistortedconformationis similartotheactivatedstateofthesubstrateinthereactionpathwaythat leadstoaspecificproduct.Theboundsubstratethussits “activated” in theactivesiteoftheenzyme.Thisactivationisdonewithoutraisingtemperaturebutattheexpenseoftheenergyofbindingtotheactivesite.The activesiteofanenzymehasstrategicallydeployedfunctionalaminoacids toholdthesubstrateinarightgeometry.Theconformationofthebound substrateresemblesitsactivatedstate,thusfacilitatingtheconversionfrom reactanttoproductunderambientconditions.Initsactivatedstate,thesubstrateisinamechanicallystrainedconformationandtheenzymemustbe rigidenoughtosustainthestrainforalongenoughtimeforthereaction toproceedinthedirectionofproductformation.

Binding tobeprecise,specificbinding isanimportantcentralissuein biochemistry.Manyproteinsworkinassociationwithothermolecules, endlesslyrepeatingmutualbindingandunbindingprocesses.Whenbinding moleculesaresmall,theyarecalledligandsandproteinsarereceptors,but veryoftenligandsarespecificpartsofmacromoleculessuchasDNA,proteins,andpolysaccharides.Insuchcases,themacromoleculesarealsocalled ligands.Antibodiesareagoodexampleofproteinsthatarespecializedin bindingratherthancatalysis.Theyconstituteacloselyrelatedfamilyofproteinswithacommon3Dstructurebuteachhavingadifferentaffinitytoa selectedligandmoleculecalledanantigen.Antigenbindingtoanantibody issimilartosubstratebindingtoanenzyme,butantibodiesneitheractivate theirligandsnorcatalyzetheirtransformationtoothermolecules.Ithasbeen anticipatedthatsomeantibodiesmightactasacatalystafterbindingaspecific antigen [9].Infact,geneticengineeringtechnologyhelpedconversionofan antibodytoanenzyme.Anattempttomodifythearrangementofamino acidresiduesinthebindingsiteofanantibodysothatitwouldactivate theboundligandwassuccessfullyperformedbyLerner [10].The “antibody turnedenzyme” iscalledacatalyticantibodyor “abzyme.” Reviewsonthe catalyticantibodiesarefoundinRefs. [11 13]

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practical sense of Master Ambrose, a new industry was started—that of candying fairy fruit, and exporting it to all the countries with which they trafficked, in pretty fancy boxes, the painted lids of which showed that art was creeping back to Dorimare.

As for Ranulph, when he grew up he wrote the loveliest songs that had been heard since the days of Duke Aubrey—songs that crossed the sea and were sung by lonely fishermen in the far North, and by indigo mothers crooning to their babies by the doors of their huts in the Cinnamon Isles.

Dame Marigold continued to smile, and to nibble marzipan with her cronies. But she used sometimes sadly to wonder whether Master Nathaniel had ever really come back from beyond the Debatable Hills; sometimes, but not always.

And Master Nathaniel himself? Whether he ever heard the Note again I cannot say. But in time he went, either to reap the fields of gillyflowers, or to moulder in the Fields of Grammary. And below his coffin in the family chapel a brass tablet was put up with this epitaph:

NATHANIEL CHANTICLEER

PRESIDENT OF THE GUILD OF MERCHANTS

THREE TIMES MAYOR OF LUD-IN-THE-MIST TO WHOM WAS GRANTED NO SMALL SHARE OF THE PEACE AND PROSPERITY HE HELPED TO BESTOW ON HIS TOWN AND COUNTRY.

An epitaph not unlike those he used to con so wistfully in his visits to the Fields of Grammary.

And this is but another proof that the Written Word is a Fairy, as mocking and elusive as Willy Wisp, speaking lying words to us in a feigned voice. So let all readers of books take warning! And with this final exhortation this book shall close.

Columbine

"And can the physician make sick men well,

And can the magician a fortune divine Without lily, germander, and sops in wine? With sweet-brier, And bon-fire, And strawberry-wire, And columbine."

"Within and out, in and out, round as a ball, With hither and thither, as straight as a line, With lily, germander, and sops in wine. With sweet-brier And bon-fire And strawberry-wire And columbine."

"Any lass for a Duke, a Duke who wears green, In lands where the sun and the moon do not shine, With lily, germander, and sops in wine. With sweet-brier And bon-fire And strawberry-wire And columbine."

"When Aubrey did live there lived no poor, The lord and the beggar on roots did dine With lily, germander, and sops in wine. With sweet-brier And bon-fire, And strawberry-wire, And columbine."

"There are windfalls of dreams, there's a wolf in the stars, And Life is a nymph who will never be thine, With lily, germander, and sops in wine. With sweet-brier, And bon-fire, And strawberry-wire, And columbine."

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Issuu converts static files into: digital portfolios, online yearbooks, online catalogs, digital photo albums and more. Sign up and create your flipbook.