0RINTED WITH PERMISSION OF 4HE )NSTITUTE OF 0URE AND !PPLIED 0HYSICS )0!0*!0!.%3% */52.!, /& !00,)%$ 0(93)#3
6OL .O!  PP  ;0ART=)NVITED 2EVIEW 0APER.ANOMECHANICS OF
0ROTEIN "ASED "IOSTRUCTURES!TSUSHI )+!))N THIS ARTICLE WE REVIEW RECENT STUDIES ON
NANOMECHANICS OF BIOSTRUCTURES PERFORMED IN
THE ,ABORATORY OF "IODYNAMICS AT 4OKYO )NSTI
TUTE OF 4ECHNOLOGY 7E EMPLOYED THE FORCE
SPECTROSCOPY MODE OF THE ATOMIC FORCE MICROSCOPE TO DETERMINE THE HIDDEN MECHANICALPROPERTIES OF PROTEIN BASED BIOSTRUCTURES THATHAVE MADE LIFE ON THE EARTH SO SUCCESSFUL 7E
INVESTIGATED THE MECHANICAL HETEROGENEITY OF
THE INTERNAL STRUCTURE OF GLOBULAR PROTEINS AND
CELL MEMBRANES 3INGLE MOLECULES OF GLOBULAR
PROTEINS WERE STRETCHED FROM THEIR TWO ENDS
AFTER BEING SANDWICHED BETWEEN THE PROBE
OF THE ATOMIC FORCE MICROSCOPE AND THE SUBSTRATE THROUGH A COVALENT CROSSLINKING SYSTEM
4HE RESULTING FORCE EXTENSION CURVE REVEALEDMECHANICAL HETEROGENEITIES IN THE CONFORMA
TION OF GLOBULAR PROTEINS 4HE COVALENT CROSS
LINKING SYSTEM WITHSTOOD A TENSILE FORCE OF UP
TO  ¢  N. LOADING RATE   N.S
WHILE MOST OF THE NONCOVALENTLY FOLDED PRO
TEIN SUB STRUCTURES WERE COMPLETELY STRETCHED
OUT WITH LESS FORCE 4HE RESULT OF FORCE SPEC
TROSCOPY SUPPORTED A LONG STANDING CONJECTURE THAT AN ENZYME CANNOT SIMPLY BE A SOFT
MATERIAL BECAUSE IT MUST CATALYZE CHEMICALREACTIONS INVOLVING THE FORMATION AND BREAK
DOWN OF MECHANICALLY RIGID COVALENT MOL
ECULES .EXT THE !&- FORCE SPECTROSCOPYWAS APPLIED TO DETERMINE THE FORCE NEEDEDTO DISRUPT NONCOVALENTLY ASSEMBLED BIOSTRUC
TURES SUCH AS COMPOSITE BIOMEMBRANES COM
POSED OF LIPIDS AND PROTEINS 7E WERE ABLE TO
SHOW THAT INTRINSIC MEMBRANE PROTEINS THATARE SECURELY ANCHORED TO A LIPID BILAYER COULD
BE PULLED OUT OF THE MEMBRANE WITH A SIGNIFICANTLY LESS FORCE THAN THAT REQUIRED FOR COVALENT BOND BREAKDOWN BUT WITH A FORCE IN THE

COMPARABLE RANGE REQUIRED FOR THE DISRUPTION
OF THE INTERNAL STRUCTURES OF GLOBULAR PROTEINS&ROM THE AVAILABLE RESULTS FROM OUR GROUP AND
OTHER GROUPS A NEW CONCEPT OF FORCE BASED
BIOSTRUCTURE ASSEMBLY IS EMERGING

 )NTRODUCTIONLIBRIUM STATES COMPARED WITH SYSTEMS MADE
OF RIGID MATERIALS SUCH AS METALS OR CONCRETE
7E AIM AT DETERMINING HOW SOFT SOFT BIOLOGICAL STRUCTURES ARE AND AT DEVELOPING APPROPRIATE METHODS OF MEASURING THE OVERALL AND
LOCAL STIFFNESS OF PROTEIN BASED BIOSTRUCTURES
USING RECENT NANOMECHANICAL MEASUREMENTTECHNOLOGIES 4HIS IS WHERE THE THEORIES AND
EXPERIMENTAL TECHNIQUES DEVELOPED AND USEDIN APPLIED PHYSICS AND ENGINEERING FIELDS FOR
THE MECHANICAL CHARACTERIZATION OF MACROSCOPIC MATERIALS FIND THEIR APPLICATIONS IN BIO
MEDICAL INNOVATIONS)N THIS ARTICLE WE REVIEW RECENT STUDIES ON
NANOMECHANICS OF BIOSTRUCTURES PERFORMED IN
THE ,ABORATORY OF "IODYNAMICS AT 4OKYO )NSTI
TUTE OF 4ECHNOLOGY 7E LIKE TO SUMMARIZE THE
IMPORTANCE OF STUDYING THE NANOMECHANICS OF
BIOSTRUCTURES TO UNDERSTAND THE BASIC ARCHITECTURE OF LIFE AND ITS PROCESSES !T THE MAC
ROSCOPIC LEVEL GRAVITATIONAL AND HYDRODYNAMICAL FORCES ARE THE TWO MAJOR EXTERNAL STRESSESACTING ON BIOLOGICAL STRUCTURES "ECAUSE OF
THE SMALL SIZE OF SUCH STRUCTURES HOWEVER
THE GRAVITATIONAL EFFECT BECOMES OF MINOR IM
PORTANCE AS ONE GOES DOWN TO THE CELLULAR
AND MOLECULAR LEVEL AND THE EFFECT OF HYDRODYNAMIC STRESS TENDS TO BE RESTRICTED TO SPECIAL CIRCUMSTANCES WHERE CELLS AND MOLECULESARE SUBJECTED TO A STRONG SHEAR FLOW AS IN THE
BLOOD VESSELS OF ANIMALS 4HAT IS ELEMENTAL
BIOLOGICAL STRUCTURES ON THE MICROMETER AND
NANOMETER SCALES ARE MORE OR LESS IN EQUILIB
RIUM STATES MOST OF THE TIME INDICATING THATMECHANICAL STRESSES AND THEIR CORRESPONDINGSTRAINS DO NOT PERSIST FOR LONG PERIODS IN THESE
SYSTEMS 4HIS IS BECAUSE BIOLOGICAL SYSTEMS ARESOFT AND CAN RELAX RATHER RAPIDLY TO THEIR EQUI!S STATED ABOVE THE BEHAVIOR OF NANO
METER SCALE BIOLOGICAL SYSTEMS CAN BE DESCRIBED BY MECHANISMS BASED ON EQUILIBRIUM
THERMODYNAMICS BUT THIS DOES NOT MEAN THATBIOMOLECULES AND RELATED SYSTEMS ARE NOT UN
DER MECHANICAL STRESSES IN ANY CIRCUMSTANCES
)N FACT THEY ARE LIKELY SUBJECTED TO A STRONGMECHANICAL STRESS BE IT FOR A VERY SHORT TIME
DURING ACTIVATION FROM ONE EQUILIBRIUM STATETO ANOTHER  !N ENZYME REACTION IS EFFECTIVE
IN ILLUMINATING THIS SITUATION "EFORE AND AF
TER THE REACTION OF CONVERTING THE SUBSTRATE 3
TO THE PRODUCT 0 AS CATALYZED BY THE ENZYME% BOTH % 3 AND % 0 STATES ARE IN CHEMICAL EQUILIBRIUM AND NO MECHANICAL STRESS AC
CUMULATES IN ANY OF THE CHEMICAL SPECIES IN
VOLVED (OWEVER AS THE REACTION PROGRESSES% BINDS 3 TO FORM AN ACTIVATED COMPLEX WHERE
BOTH % AND 3 ARE IN DIFFERENT CONFORMATIONSFROM THEIR EQUILIBRIUM ONES SUGGESTING THATSOME CHEMICAL BONDS IN THE COMPLEX ARE DISPLACED FROM THEIR EQUILIBRIUM LENGTHS ANDOR
ANGLES THEREBY GENERATING MECHANICAL STRESSES IN THE COMPLEX 4HE TIME SCALE OF THIS ME+%97/2$3ATOMIC FORCE MICROSCOPY !&FORCE SPECTROSCOPY PROTEINSTRETCHING RECEPTOR MAPPING'RO%, AND PROTEIN INTERACTIONMEMBRANE PROTEIN EXTRACTION,ABORATORY OF "IODYNAMICS 'RADUATE 3CHOOL OF "IOSCIENCE AND "IOTECHNOLOGY 4OKYO )NSTITUTE OF 4ECHNOLOGY 9OKOHAMA   *APAN
2ECEIVED *ULY   ACCEPTED !UGUST   PUBLISHED .OVEMBER  ¥  4HE *APAN 3OCIETY OF !PPLIED 0HYSICS*3!0 )NTERNATIONAL .O *ULY % MAIL ADDRESS AIKAI BIOTITECHACJP    
    
  
&IG 3CHEMATIC VIEW OF OPERATING PRINCIPLE OF
TYPICAL COMMERCIAL !&- 4HE LASER BEAM REmECTEDFROM THE BACK OF THE GOLD COATED CANTILEVER IS
RECEIVED BY A BISECTED OR QUADRASEPTED PHOTO
DIODE DETECTOR 4HE DIFFERENCE BETWEEN THE OUT
PUTS OF THE TOP AND BOTTOM PHOTODIODES IS KEPT
AT NEAR ZERO LEVEL BY CHANGING THE HEIGHT OF THE
SAMPLE STAGE THROUGH THE FEEDBACK CONTROL SYS
TEM 4HE INPUT VOLTAGE FROM THE FEEDBACK SYSTEM
TO THE PIEZO SCANNER BELOW THE SAMPLE STAGE IS
RECORDED AND INTERPRETED TO REPRESENT THE TOPOGRAPHY OF THE SAMPLE SURFACECHANICAL STRESS CANNOT BE READILY ESTIMATED
BUT IS LIKELY TO BE MUCH LONGER EG FROMMICRO TO MILLI SECOND RANGE THAN THE TIMESCALE OF MOLECULAR RELAXATION PROCESSES WHICH
TAKE PLACE IN THE PICOSECOND RANGE )T HAS
INDEED BEEN ARGUED THAT SINCE AN ENZYMEACTS AS A CATALYST ITS INTERNAL STRUCTURE MUSTHAVE SUFFICIENT RIGIDITY TO SUSTAIN THE MECHANICAL STRESS IMPOSED ON IT DURING ACTIVATION 
BUT THERE IS NO APPROPRIATE METHOD TO DETECTTHE PRESENCE OR ABSENCE OF RIGID SUBSTRUC
TURES WITHIN A SINGLE MOLECULE OF PROTEIN UNTIL
THE RECENT ADVENT OF ATOMIC FORCE MICROSCOPE
!&-  4O ADDRESS THIS PROBLEM WE EMPLOYEDTHE FORCE MEASUREMENT MODE OF THE !&- !S
ILLUSTRATED IN &IG  THE !&- IS COMPOSED OFA SMALL CANTILEVER WITH AN INTEGRATED PROBE AT
ITS FREE END AN OPTICAL LEVER SENSING SYSTEMFOR CANTILEVER DEFLECTION AND A SAMPLE STAGE
ON TOP OF A PIEZOMOTOR THAT DRIVES THE SAM
PLE STAGE HORIZONTALLY X AND Y AND VERTICALLYZ  7HEN THE !&- IS OPERATED IN THE FORCECURVE MODE THE PIEZOTUBE UNDER THE SAMPLE
STAGE MAKES ONLY A VERTICAL Z MOVEMENT AT
A FIXED LATERAL POSITION SO THAT IT IS POSSIBLE
TO STRETCH A POLYMER CHAIN FROM ITS TWO ENDS
WHILE CONSTANTLY MONITORING TENSILE FORCE AS A
FUNCTION OF CHAIN EXTENSIONn     



4O ENSURE THE CORRECT ANALYSIS OF THE
FORCE VERSUS EXTENSION RELATIONSHIP OF A PRO
TEIN MOLECULE IT MUST BE ESTABLISHED AT LEAST
TO A REASONABLE LEVEL THAT THE OBSERVATION IS
MADE ON A SINGLE CHAIN OF THE SAMPLE PROTEINAND NOT ON MULTIPLE CHAINS 7E AND MOST
OTHER RESEARCHERS IN THIS FIELD USE THE FOLLOWING CRITERION 4HE PERCENTAGE OF SUCCESSFUL
RECORDING OF CHAIN ELONGATION MUST BE LESS
THAN n OF THE TOTAL NUMBER OF TRIALSTHE REST SHOWING NO POSITIVE INDICATION OF THE
PRESENCE OF TENSILE MATERIALS BETWEEN THE!&- TIP AND THE SUBSRATE )F THE SUCCESS RATEOF FORMING CONNECTIONS BETWEEN THE SAMPLEPROTEIN AND THE TIP IS SMALL THE PROBABILITY OF
DOUBLE OR EVEN TRIPLE CAPTURE EVENTS WILL BE
NEGLIGIBLY SMALL IE LESS THAN nA GLOBULAR PROTEIN COULD BE STRETCHED OUT
WITH A FORCE IN THE RANGE OF A FEW HUNDREDS
OF P.S 4ITIN EXTENSION WAS FIRST REPORTED
BY 'AUB AND HIS COLLEAGUES AS CITED ABOVESHOWING A TRAIN OF FORCE PEAKS CORRESPONDING
TO THE SUCCESSIVE BREAKDOWN OF THE MODULAR SUBUNITS OF THE GIANT PROTEIN WHICH HAS
HUNDREDS OF SUCH SUBUNITS CONNECTED BY VERYFLEXIBLE LINKER PEPTIDES 4ITIN TURNED OUT TO BE
A CONVENIENT SAMPLE IN MANY RESPECTS FOR USE
IN NEW TYPES OF FORCE SPECTROSCOPY METHODSBECAUSE OF ITS MODULAR STRUCTURE THAT ALLOWSRESEARCHERS TO STRETCH ANY PART OF THE PROTEIN
GIVING THE SAME RESULTS 4HE GLOBULAR MODULES OF TITIN WAS SHOWN TO BREAK DOWN WITH
A FORCE OF A FEW TENS AND A FEW HUNDREDS OF
P.S DEPENDING ON LOADING RATE

4HE MECHANICAL UNFOLDING OF GLOBULAR
PROTEINS USING THE !&- WAS INITIATED BY -ITSUI ET AL  AND LATER SUBSTANTIALLY EXTENDED BY

4HE FORCE CURVE MODE OF THE !&- WAS
ALSO APPLIED FOR HARVESTING PROTEIN MOLECULESEMBEDDED IN THE CELL MEMBRANE 4HESE PROTEINS HAVE HYDROPHOBIC SEGMENTS THAT SPAN
THE HYDROPHOBIC INTERIOR OF THE LIPID BILAYER
AND FUNCTION AS MEDIATORS OF CHEMICAL IN
FORMATION FROM THE OUTSIDE TO THE INSIDE OF
THE CELL AND VICE VERSA )N ADDITION TO THESEMEMBRANE SPANNING SEGMENTS SUCH PROTEINSUSUALLY HAVE HYDROPHILIC EXTRACELLULAR AND CYTOPLASMIC DOMAINS AND APPEAR TO BE SECURELY

SEVERAL GROUPS USING THE GIANT MOLECULE TITIN
CONNECTIN   -ITSUI ET AL STRETCHED AN IM
MOBILIZED PROTEIN MOLECULE ON A GOLD SURFACETHROUGH n3n!Un BONDS BY FORMING ANOTHERSET OF SIMILAR BONDS WITH A GOLD COATED CANTILEVER THAT APPROACHED THE PROTEIN FROM THE
TOP 4HEY REPORTED THE AVERAGE SLOPE OF THE& n% CURVES THAT SHOW FOR THE FIRST TIME THAT

*3!0 )NTERNATIONAL .O *ULY 

ANCHORED TO THE MEMBRANE )F THE PROTEIN
PROTEIN INTERACTION IS STRONGER THAN THE PROTEIN LIPID INTERACTION PROTEIN SHOULD FORM
AGGREGATES IN THE MEMBRANE OTHERWISE
PROTEINS WOULD BE STRONGLY SOLVATED BY LIPID
MOLECULES -ANY IF NOT ALL OF THE MEMBRANE
PROTEINS ARE IN ASSOCIATION WITH SUB MEMBRANOUS PROTEIN LAYERS CALLED THE CYTOSKELETON
THROUGH NONCOVALENT INTERACTIONS AND MEDI
ATE THE TRANSFER OF EXTRACELLULAR INFORMATION
TO THE INTRACELLULAR ENZYME SYSTEMS )N ORDERTO HARVEST MEMBRANE PROTEINS BY EXTRACTIONFROM THE CELL MEMBRANE BOTH LIPID PROTEIN
AND PROTEIN CYTOSKELETON INTERACTIONS MUST BE
DISRUPTEDCOPHORIN THE PROTEIN ON THE RED BLOOD CELLMEMBRANE )F BOUNDARY LIPIDS ARE EXTRACTEDTOGETHER WITH GLYCOPHORIN THE FORCE WOULD
BE IN THE RANGE OF  P. 7E WANTED TOTEST "ELLS ESTIMATE OF THE ANCHORING FORCE OF
A MEMBRANE PROTEIN TO THE CELL SURFACE USING
AN !&-!NOTHER PROMISING APPLICATION OF NANO
MECHANICAL MEASUREMENT AT THE MOLECULARLEVEL IS THE DETECTION OF SPECIFIC RECEPTOR MOL
ECULES ON VIABLE CELL SURFACE USING AN !&PROBE MODIFIED WITH COVALENTLY IMMOBILIZEDSPECIFIC LIGAND MOLECULES 4HE PROBE MAKES
A $ SURVEY OVER THE CELL SURFACE AND RECORDS
THE INTERACTION FORCE BETWEEN THE PROBE AND
THE SURFACE AS THE INDICATION OF THE PRESENCEOF RECEPTOR MOLECULES WITH BIOCHEMICAL SPECIFICITY TO THE LIGAND ON THE PROBEn 3UCH AN
APPLICATION OF THE !&- FOR RECEPTOR MAPPINGIS BASED ON THE ASSUMPTION THAT THE UNBINDING FORCE BETWEEN LIGANDS AND RECEPTORS IS
SUBSTANTIALLY LESS THAN THE FORCE REQUIRED TO
EXTRACT OR UPROOT PROTEINS FROM THE CELL SUR)N HIS PIONEERING WORK ON THE ADHESIVEINTERACTIONS BETWEEN CELLS IN  "ELL EX
TENDED HIS THEORETICAL STUDY ON CELL ADHESIONTO THE ESTIMATION OF THE FORCE REQUIRED TODISRUPT THE INTERACTION BETWEEN AN INTRINSICMEMBRANE PROTEIN AND A LIPID BILAYER AND
PREDICTED THAT A FORCE OF ABOUT  P. IS RE
QUIRED TO UPROOT A SINGLE MOLECULE OF GLY       

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FACE )F NOT THE FORCE MEASURED EVERY TIME
THE PROBE MAKES CONTACT WITH THE CELL SURFACEAND IS RETRACTED IS NOT THE UNBINDING FORCE OF
THE LIGAND FROM THE RECEPTOR BUT THE UPROOTING FORCE OF THE LATTER FROM THE MEMBRANE
AND MAPPING WILL NOT BE POSSIBLE !LL THE LI
GAND MOLECULES ON THE !&- TIP WILL BE EVEN
TUALLY COVERED WITH RECEPTOR MOLECULES AND NORESPONSE IS EXPECTED FROM THE LIGAND RECEPTOR
INTERACTIONS )T IS THEREFORE IMPORTANT TO ESTABLISH THE RANGE OF FORCE REQUIRED TO EXTRACTMEMBRANE PROTEINS FROM THE VIABLE CELL SUR
FACE IN COMPARISON TO THE UNBINDING FORCE OF
LIGAND RECEPTOR PAIRS OF INTEREST 7E ALLOWEDBIFUNCTIONAL COVALENT CROSSLINKERS IMMOBILIZEDON THE !&- TIP TO FORM COVALENT BONDS WITH
CELL SURFACE PROTEINS AND THEN PULLED OUT THE
MEMBRANE PROTEINS TAKING ADVANTAGE OF ON
THE STRENGTH OF A COVALENT BONDn -ETHODS AND -ATERIALS!&- PROBES AND SILICON SUBSTRATES WERECHEMICALLY FUNCTIONALIZED SO THAT THE MACRO
MOLECULES CAUGHT BETWEEN THEM ARE COVALENTLY CROSSLINKED TO THE TIP ON ONE END AND
TO THE SUBSTRATE ON THE OTHER 4HE CHEMICALMODIFICATION METHOD OF CANTILEVERS IS GIVEN
IN &IG  4HE SUBSTRATE USED WAS MODIFIEDIN A SIMILAR MANNER 4HE SAMPLE MOLECULE
THUS SANDWICHED BETWEEN THE TIP AND THE
SUBSTRATE WILL THEN BE PUT UNDER A CONTINUOUS TENSILE STRESS BY INCREASING THE TIP STAGE
DISTANCE 4HE SANDWICHED SAMPLE WILL THEN
BE STRETCHED UNTIL ONE OR MORE OF THE WEAKER

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  
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&IG #OVALENT CROSSLINKING SYSTEM USED FOR THE MEASUREMENT OF NONCOVALENT INTERACTION FORCES CITED FROM REF  WITH PERMISSION *3!0 )NTERNATIONAL .O *ULY &IG &ORCE CURVE MODE OPERATION OF !&- 4HE RELATIONSHIP GIVEN IN THE TEXT IE%$nD IS EXPLAINED)NVITED 2EVIEW 0APER   
 

BONDS IN THE SYSTEM ARE BROKEN $URING THE
STRETCHING OF THE PROTEIN MOLECULE THE ME
CHANICAL RESPONSE OF THE SAMPLE AGAINST THE
TENSILE STRESS PRODUCES A DOWNWARD DEFLEC
TION OF THE CANTILEVER WHICH HOLDS THE PROBE
AT ITS FREE END 4HE EXTENT OF CANTILEVER DE
FLECTION D IS DETECTED BY A BISECTED PHOTODIODE DETECTOR AND BY KNOWING THE CANTILEVER
FORCE CONSTANT K THE TENSILE FORCE APPLIED TO
THE SAMPLE IS CALCULATED USING &nK  D  4HE
EXTENSION OF THE SAMPLE % DURING THE PROCESSOF INCREASING THE PROBE STAGE DISTANCE $ IS
CALCULATED USING %$nD  4HE EXPERIMENTAL
PARAMETERS OBTAINED FROM A FORCE SPECTROSCOPY EXPERIMENT ARE SCHEMATICALLY SHOWN IN&IG  AND EXPLAINED IN THE LEGEND

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 #HEMICALS AND PROTEINS
4HE SILANIZATION REAGENT USED  AMINOPROPYLTRIETHOXY SILANE !04%3 WAS OBTAINEDFROM 3HIN %TSU #HEMICAL #O 4OKYO *APAN $ISUCCINIMIDYL SUBERATE $33 AND . SUCCINIMIDYL   PYRIDYLDITHIO PROPIONATE 30$0
WERE PURCHASED FROM 0IERCE 2OCKFORD ), 
4HEY WERE STORED UNDER A HIGHLY DEHYDRATED
AND LOW TEMPERATURE CONDITIONS AND USEDWITHOUT FURTHER PURIFICATION 0HOSPHATE BUFFERED SALINE AS A LIQUID MEDIUM FOR CELLS DUR
ING ATOMIC FORCE MICROSCOPY EXPERIMENTS WASOBTAINED FROM 'IBCO ,ANGLEY /+  /THERCHEMICALS WERE BOUGHT FROM 3IGMA 3T ,OUIS-/  4HE PROTEINS USED IN THE STRETCHING EX
PERIMENTS WERE PRODUCED BY THE RECOMBINANT
METHOD WITH THE INSERTION OF CYSTEINE RESIDUES OUTSIDE THE . AND # TERMINI TOGETHERWITH . TERMINAL HISTIDINE TAGS TO FACILITATE PU
RIFICATION BY AFFINITY CHROMATOGRAPHY #ELLS-OUSE 4 FIBROBLAST CELLS WERE GROWN
IN $ULBECCOgS MODIFIED %AGLESg MINIMUM ME
DIUM $-%- FOR SEVERAL DAYS AND IN THE FINAL
STAGE OF !&- EXPERIMENTS THE MEDIUM WAS
CHANGED TO PHOSPHATE BUFFERED SALINE !TOMIC FORCE MICROSCOPE
3EVERAL COMMERCIALLY AVAILABLE !&- IN
STRUMENTS WERE USED INCLUDING A .ANOSCOPE)))A $IGITAL )NSTRUMENTS 3ANTA "ARBARA #!
AN .6!  !&- /LYMPUS 4OKYO *APAN
AND A "IO3COPE 6EECO 7OODBURY .9  #AN
TILEVERS WERE PURCHASED FROM /LYMPUS AND &IG 3CHEMATIC VIEW OF /SP!
STRETCHING ON !&- SAMPLE STAGE
CITED FROM REF  WITH PERMISSION 

6EECO 4HE CANTILEVERS AND CRYSTALLINE SILICON
SUBSTRATES WERE SILANIZED WITH !04%3 AND
FURTHER MODIFIED WITH 30$0 TO MAKE THEIRSURFACE REACTIVE WITH 3( GROUPS ON THE EN
GINEERED PROTEINS WITH CYSTEINE RESIDUES &OR
THE EXTRACTION OF MEMBRANE PROTEINS !&TIPS WERE MODIFIED WITH $33 SO THAT THE PROTEINS COULD BE IMMOBILIZED THROUGH THEIR AMINO GROUPS TO THE TIPS 'OLD COATED CANTILEVERS
/LYMPUS WERE ALSO USED AFTER DIRECT MODIFICATION WITH 30$0 ,OADING RATE DEPENDENCE)T HAS BEEN POINTED OUT THAT THE MEA
SURED FORCE SHOULD BE DEPENDENT ON HOW FAST
ONE LOADS THE TENSILE FORCE ON THE SAMPLE
AND THE EXPONENTIAL DEPENDENCE OF THE MEAN
FORCE F ON THE LOADING RATE HAS BEEN ESTABLISHED BOTH THEORETICALLY AND EXPERIMENTALLY

AS

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WHERE K " 4 AND X` ARE THE "OLTZMANN CON
STANT THE TEMPERATURE + AND THE ACTIVATIONDISTANCE OF THE BOND TO BE BROKEN
#ANTILEVER FORCE CONSTANT WAS CALIBRATEDBASED ON THE EQUI PARTITION THEOREM OF THE

THERMAL ENERGY AND THE KINETIC ENERGY OF THE
CANTILEVER 2ESULTS
 0ROTEIN STRETCHING EXPERIMENTS&IRST WE DESCRIBE THE STRETCHING EXPERIMENT OF SEVERAL GLOBULAR PROTEINS WHERE WE
FOUND THE PRESENCE OF HIGHLY RESISTANT SUBSTRUCTURES AGAINST TENSILE FORCE WITHIN A SINGLEPROTEIN MOLECULE 7E EXPECT THAT THIS FINDING
WILL PROVIDE EXPERIMENTAL EVIDENCE OF WHAT
HAS BEEN ANTICIPATED FROM THE THEORETICALPOINT OF VIEW AS DESCRIBED IN )NTRODUCTION
7HEN THE GLOBULAR PROTEIN /SP! WAS
SANDWICHED BETWEEN THE !&- TIP AND THE
SUBSTRATE USING BIFUNCTIONAL COVALENT CROSS
LINKERS AS ILLUSTRATED IN &IG  AND SUBSEQUENTLY STRETCHED FROM THE TWO ENDS AS THE
DISTANCE BETWEEN THE TIP AND THE SUBSTRATEWAS INCREASED WE OBSERVED & n% CURVES WITHTWO FORCE PEAKS SUCH AS THOSE GIVEN IN &IG 3INCE THIS PROTEIN OUT OF ITS TOTAL 
AMINO ACID RESIDUES HAS TWO TERMINAL GLOBULAR DOMAINS CONNECTED BY THE CENTRAL ` SHEETAS SHOWN IN &IG  IT IS MOST NATURAL TO PRESUME THAT THE TWO FORCE PEAKS CORRESPOND TO*3!0 )NTERNATIONAL .O *ULY 

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&IG &n% CURVE OF /SP! 7ILDTYPE /SP! A /SP! &! B AND /SP! "( C  )N D SUPERIMPOSITION OF CURVES OF WILD TYPE /SP! TOP PANEL /SP! &! MIDDLE PANEL AND /SP! "(BOTTOM ARE GIVEN !BBREVIATIONS /SP! &! AND /SP! "( ARE GIVEN IN THE TEXT CITED FROM
REF  WITH PERMISSION 

   
   

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&IG &n% CURVES OF APO AND HOLO CALMODULIN TOGETHER WITH lTTING CURVES BASED ON THE WORM
LIKE CHAIN WITH DIFFERENT VALUES OF PARAMETERS CITED FROM REF  WITH PERMISSION *3!0 )NTERNATIONAL .O *ULY &IGURE  SHOWS THE RESULTS OF THE EXPERIMENT ON CALMODULIN IN TWO FORMS ONE WITH
#A AND BIOLOGICAL ACTIVITY THE OTHER WITH
NEITHER #A NOR ACTIVITY 4HE & n% CURVE IN THE
PRESENCE OF #A SHOWED A SINGLE FORCE PEAK
AND THE ONE WITHOUT #A DID NOT SHOW ANY
FORCE PEAKS 4HE $ CONFORMATIONS OF THE
TWO FORMS OF CALMODULIN ARE _ HELICAL COUNTERPART STRUCTURES OF /SP! IN WHICH TWO GLOBULAR SUBDOMAINS ARE CONNECTED BY A SINGLEROD OF _ HELIX BUT THE & n% CURVE IN THIS CASE
SHOWED ONLY ONE PEAK
4O UNDERSTAND THE & n% CURVES OF PROTEINSTRETCHING WE OBTAINED SIMILAR CURVES FOR THE
STRETCHING OF SUBSTANTIALLY HELICAL POLY , GLU
TAMIC ACID AT ACIDIC P( AND RANDOMLY COILEDONES AT NEUTRAL AND ALKALINE P(S !S GIVEN

THE BREAKDOWN OF THE TWO DOMAINS AND THE
LOW STRESS STRETCHING IN THE INITIAL nNM
CORRESPONDS TO THE UNZIPPING OF THE CENTRAL` SHEET 4O ESTABLISH THE CORRESPONDENCE BE
TWEEN THE SUBSTRUCTURES OF THE PROTEIN AND
THE TWO FORCE PEAKS TWO TYPES OF AMINO ACIDSUBSTITUTION WERE CONSTRUCTED 4HE INTRODUCTION OF AN AMINO ACID REPLACEMENT IN THE
MIDDLE OF THE PRIMARY STRUCTURE &! THATIS PHENYLALANINE AT THE  POSITION WAS RE
PLACED BY AN ALANINE RESIDUE ABOLISHED ONE
OF THE TWO FORCE PEAKS SEE &IG   /SP!
"( A MUTANT /SP! PROTEIN HAVING AN EXTRA` HAIRPIN INSERTED BETWEEN STRANDS  AND 
ALSO ABOLISHED ONE OF THE TWO FORCE PEAKS )TIS THUS APPARENT THAT THE CENTRAL FLAT REGIONIS MECHANICALLY STRONGLY ASSOCIATED WITH THE
APPEARANCE OF ONE OF THE PEAKS !N INTERPRETATION BASED ON A SIMPLE VISUAL INSPECTION
NEEDS TO BE AUGMENTED BY OBTAINING MORERESULTS ON AMINO ACID REPLACEMENT IN OTHER
AREAS

IN &IG  THE STRETCHING AND RETRACTION CURVESOF BOTH HELICAL AT P(  AND RANDOMLY
COILED AT P(  POLY , GLUTAMIC ACID WERECOMPLETELY REVERSIBLE AND REPEATABLE FOR MORE
THAN  TRACES )N THE HELICAL CONFORMATIONSEXPECTED THE POLYMER WAS STIFFER THAN THATIN THE RANDOMLY COILED STATE /UR RESULT ON A
POLY , ALANINE BASED HELICAL POLYMER SHOWED
SIMILAR FORCE CURVES WHEN IT IS STRETCHED 3UCH
RESULTS HELP US UNDERSTAND THE VERY SMOOTH& n% CURVES OF APO CALMODULIN WITHOUT #A
AS GIVEN ABOVE 2 !FRIN TO BE PUBLISHED )NVITED 2EVIEW 0APER

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&IG &n% CURVES OF POLY , GLUTAMIC ACID AT P(S  TOP AND  BOTTOM  4HE LEFTPANELS REPRESENT STRETCHING CURVES WHILE THE RIGHT PANELS RETRACTING CURVES CITED FROM
REF  WITH PERMISSION  -OLECULAR DYNAMICS SIMULATION)N THE APPLICATION OF COMPUTER SIMULATIONTO PROTEIN STRETCHING THE STEERED MOLECULAR
DYNAMICS 3-$ HAS BEEN USED IN SEVERAL CAS
ES WITH SOME SUCCESS  )N 3-$ SIMULATIONA VIRTUAL SPRING WAS PLACED BETWEEN PROTEIN
AND THE PULLING POINT !S THE TIP IS MOVED
AT A CONSTANT VELOCITY THE PROTEIN UNFOLDING
MAY NOT TAKE PLACE AT THE SAME VELOCITY 4HE
DIFFERENCE BETWEEN THE TWO VELOCITIES WILLSTRETCH THE HYPOTHETICAL SPRING WHOSE EXTEN
SION WILL BE INTERPRETED AS THE TENSILE FORCE
ON THE PROTEIN 7E APPLIED THIS METHOD TO
THE UNFOLDING OF CARBONIC ANHYDRASE AND
OBTAINED THE RESULT SHOWN IN &IG  )N THE
TOP PANEL OF THE FIGURE THE EXPERIMENTAL & n%
CURVES CITED FROM !LAM ET AL  ARE GIVEN AND
THE SIMULATION RESULT IS GIVEN IN THE BOTTOMPANEL 4HE LARGE FORCE PEAK OBSERVED AFTERNM STRETCHING IS REPRODUCED IN THE SIMULA
TION 3IMULATION RESULTS WITH MANY SMALL AND
MEDIUM FORCE PEAKS SEEM TO DISCLOSE MORE
DETAILS OF THE STRETCHING MECHANICS AND SOMETIMES UNDER SPECIAL EXPERIMENTAL CIRCUM
STANCES FINE STRUCTURES OF FORCE SPECTROSCOPYHAVE BEEN OBSERVED


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&IG %XPERIMENTAL TOP AND 3-$ SIMULATION BOTTOM RESULTS OF
CARBONIC ANHYDRASE STRETCHING SHOWING THE APPEARANCE OF A VERY LARGEFORCE PEAK MARKED WITH AN ARROW SIGNIFYING A MAJOR STRUCTURAL TRANSITION CITED FROM REF  AND  WITH PERMISSION !NOTHER INTERESTING ASPECT OF THE ME
CHANICAL STRETCHING OF CARBONIC ANHYDRASE WAS
THAT THE PROTEIN IN ITS NATIVE HAS A KNOTTED
CONFORMATION AS REVEALED BY 8 RAY CRYSTALLOGRAPHY  !S A MECHANICAL CONSEQUENCE OF
THIS KNOT STRUCTURE IT WAS VERY DIFFICULT TO
STRETCH THE PROTEIN DUE TO KNOT TIGHTENING AT
THE MOLECULAR LEVEL  4HE RESULT IN &IG 
TOP WAS OBTAINED FOR A MUTANT PROTEIN IN
WHICH KNOT TIGHTENING WAS AVOIDED 7E
FOUND THAT THIS PROTEIN HAS A MECHANICALLYVERY TIGHT INTERNAL CORE IN ADDITION TO THE KNOTSTRUCTURE 4HIS FINDING WAS IN AGREEMENT WITH
THE PREVIOUS WORK ON THIS PROTEIN IN SOLU
TION  5SING THE SAME PROTEIN -ITSUI ET

AL  AND /KAJIMA ET AL  STUDIED ITS DYNAMIC RESPONSE AGAINST A SLOWLY OSCILLATING TENSILEFORCE AND REPORTED THAT IN A CERTAIN RANGE OFEXTENSION PHASE INVERSION WAS OBSERVED BE
TWEEN THE SINUSOIDAL INPUT SIGNAL AND THE
OUTPUT SIGNAL FROM THE PROTEIN MOLECULE )T
WAS INTERPRETED THAT THE PROTEIN BECOMESPARTICULARLY VISCOUS IN A CERTAIN EXTENSIONRANGE IMPLYING THAT THE DRIVING FORCE FOR RE
FOLDING OR MISFOLDING INVERTS THE PHASE "RITTLE FRACTURE
7HEN FORCE PEAKS APPEAR IN THE & n %CURVES OF GLOBULAR PROTEIN EXTENSION WE INTERPRETED THEM AS REFLECTING BRITTLE FRACTURESOF SUBMOLECULAR STRUCTURES )N MACROSCOPICMECHANICS A SUDDEN BREAKDOWN OF GLASSOR CERAMICS HAS BEEN TREATED IN THE THEO
RETICAL FRAMEWORK FIRST LAID OUT BY 'RIFFITH
IN   !CCORDING TO 'RIFFITHgS THEORYTHE SIZE OF A SMALL CAVITY IS ELONGATED UN
DER STRESS AND REACHES A CRITICAL LENGTH OVER
WHICH A CRACK STARTS PROPAGATING )N THE FOL
LOWING EQUATION A C % K3 AND m ARE THE
CRITICAL LENGTH OF THE CAVITY FOR CRACK PROPAGATION 9OUNGgS MODULUS OF THE PROTEIN THE
SURFACE ENERGY OF THE NEWLY CREATED SURFACEAFTER FRACTURE AND THE THRESHOLD STRESS FOR THE
FRACTURE TO PROPAGATE 3INCE FORCE PEAKS HAVEBEEN OBSERVED FOR PRIMARILY ` SHEET PROTEINSWE APPLY THE SIMPLE THEORY ON A PIECE OFSHEET MATERIAL

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K 3 MAY BE TAKEN AS THE HYDROPHOBIC'IBBS ENERGY OF THE SURFACE AND m MAY BE*3!0 )NTERNATIONAL .O *ULY 

APPROXIMATED BY THE PEAK STRESS OBSERVED IN
THE & n% CURVE OF PROTEIN STRETCHING )F WE IN
SERT AS A TRIAL % -0A K3*MOLNM
AND m-0A WE OBTAIN A CNM WHICHWAS OBTAINED 5NDER A SIMILAR LOADING RATERANGE THE UNBINDING FORCE OF THE PAIR IS NOTAS LARGE AS EXPECTED FROM ITS EXCEPTIONALLYHIGH THERMODYNAMICAL STABILITY AND AROUSEDINTEREST AMONG CONCERNED RESEARCHERSIS NOT AN UNREASONABLE VALUE !NOTHER INTERPRETATION HAS ALSO BEEN PUT FORWARD )T HAS
BEEN PROPOSED ON THE BASIS OF EXPERIMENTAL
OBSERVATION THAT THE APPEARANCE OF A FORCEPEAK IN THE CASE OF STRETCHING THE ALL ` SHEETPROTEIN %LIP DEPENDS ON THE PULLING DIREC
TION OF THE POLYPEPTIDES IN A GIVEN ` SHEET
7HEN TWO STANDS WERE SEPARATED DIAGONALLY
TO THE CHAIN AXIS AS IN A FASHION OF UNZIPPINGNO FORCE PEAKS WERE OBSERVED BUT WHEN THEYWERE PULLED ALONG THE CHAIN AXIS IN A SHEARING
MODE A FORCE PEAK APPEARED

4HIS ASPECT OF FORCE SPECTROSCOPY WASSTUDIED FOR EXAMPLE IN ANTIBODY ANTIGEN SYSTEMS 4HE FORCE REQUIRED TO SEPARATE A SINGLE
ANTIGEN ANTIBODY COMPLEX HAS BEEN OF GREAT
INTEREST AND DETERMINED FOR VARIOUS DIFFERENT COMBINATIONS )T IS NOW ACCEPTED THAT
THE FORCE REQUIRED TO MECHANICALLY DISRUPT A
MOLECULAR SYSTEM DOES NOT DIRECTLY CORRELATEWITH THE FREE ENERGY FOR THE STABILIZATION OF
THE SYSTEM BUT RATHER IT MORE CLOSELY COR
RELATES TO THE ENTHALPY OF THE SYSTEM 4HIS
CONCLUSION WAS OBTAINED BY COMPARING THE
OBSERVED FORCE REQUIRED TO DISRUPT LIGAND PRO
TEIN INTERACTION WITH THE RESULT OF A THERMODY
NAMIC STUDY ON VARIOUS MUTANT PROTEINS WITH
DIFFERENT AFFINITIES TO SPECIFIC LIGANDS $ETAILSARE FOUND IN THE LITERATURE CITED ABOVE 0ROTEIN PROTEIN INTERACTIONS
4HE MEASUREMENT OF PROTEIN PROTEIN IN
TERACTION IS ANOTHER POPULAR APPLICATION OF
FORCE SPECTROSCOPY !S THE FIRST EXAMPLE OF
SUCH AN EXPERIMENT THE WELL CHARACTERIZEDLIGAND RECEPTOR SYSTEM OF BIOTIN AVIDIN HAS
BEEN INVESTIGATED 4HIS LIGAND RECEPTOR PAIRFORMS A VERY STABLE COMPLEX AND IS WIDELYUSED IN BIOLOGICAL EXPERIMENTS AS A TOOL FORIMMOBILIZING BIOMOLECULES ON SOLID SURFACES
OR ON OTHER MOLECULES 7HEN THE EXPERIMENTAL RESULT OF THE FORCE MEASUREMENT WAS
ANALYZED IN TERMS OF THE LOADING FORCE DEPEN
DENCE AN ACTIVATION DISTANCE OF nNM#HAPERONINES ARE KNOWN TO HELP NEWLYSYNTHESIZED PROTEINS CORRECTLY FORM BIOLOGICALLY ACTIVE SPECIFIC $ STRUCTURES FROM THEIRDISORDERED CONFORMATION IN THE INITIAL STAGE
'RO%, A CHAPERONINE HAS BEEN SHOWN TO EN

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 #HAPERONINE SYSTEM STUDIED BY NONCOMPRESSIVE FORCE SPECTROSCOPY

ON THE SUBUNIT PLAYS A CRUCIAL STEP IN THE INTERNALIZATION OF UNFOLDED PROTEINS INTO THE'RO%, CAVITY IT IS OF INTEREST TO DETERMINE THE
MAGNITUDE OF UNBINDING FORCE AT THIS SITE
! MODIFIED METHOD OF FORCE SPECTROSCOPY
HAS BEEN APPLIED TO THIS SYSTEM 4HE !&- TIP
MODIFIED WITH DENATURED PROTEIN MOLECULESWAS BROUGHT IN CLOSE PROXIMITY SO THAT THE
DENATURED PROTEIN STARTS MAKING CONTACT WITHIMMOBILIZED 'RO%, ON THE SURFACE OF THE SUBSTRATE 4HE TIP AND SUBSTRATE WERE KEPT AT ANONCOMPRESSIVE DISTANCE DURING THE FORCECURVE MEASUREMENT 4HE RESULTING & n% CURVESARE REPRODUCED IN &IG A  4HEY DO NOT HAVE

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&IG .ONCOMPRESSIVE FORCE MEASUREMENT IN 'RO%, PEPSIN SYSTEM IN THE ABSENCELEFT AND PRESENCE RIGHT OF !40 CITED FROM REF  WITH PERMISSION 

TRAP IN ITS CAVITY AN UNFOLDED FORM OF A PRO
TEIN AND TO KEEP IT ISOLATED UNTIL THE PROTEINSPONTANEOUSLY FORMS THE CORRECT $ STRUCTUREWITHOUT BEING SIDETRACKED TO ACCUMULATE IN
CORRECTLY FOLDED STRUCTURES BY FOR EXAMPLEASSOCIATING WITH OTHER PROTEIN MOLECULES )T
HAS BEEN SHOWN THAT 'RO%, HAS A SEVEN FOLDROTATIONAL SYMMETRY ARISING FROM A CIRCULAR AR
RANGEMENT OF SEVEN SUBUNITS IN ONE TIRE AND
EACH SUBUNIT HAS A SINGLE BINDING SITE FOR AN
UNFOLDED POLYPEPTIDE !FTER BINDING TO SUCHSITES THE DENATURED PROTEIN IS TRANSFERRED INTO
THE MOLECULAR CAVITY OF THE 'RO%, WITH THE
HELP OF !40 WHERE THE PROTEIN FOLDING REAC
TION TAKES PLACE "ECAUSE THE UNBINDING OF
THE DENATURED PROTEIN FROM THE BINDING SITE

*3!0 )NTERNATIONAL .O *ULY &IG 2ESULT OF FORCE MAPPING OF SPECIlC POLYSACCHARIDES ON lXED TISSUE SECTION PROBEDBY GALACTOSIDE SPECIlC LECTINS /PTICAL MICROSCOPIC IMAGE OF !&- CANTILEVER CLOSING IN ON
A SMALL AREA OF SECTIONED SAMPLE ! AND ON THE RIGHT TWO DIAGONAL ROWS OF DARK SQUARES
REPRESENTING POSITIVE RESPONSE TO LECTIN MOLECULES ON THE TIP " AND # AND AREA OF ALMOSTNO RESPONSE AS EMPTY SQUARES $  &LUORESCENCE MICROSCOPIC IMAGE OF STAINED SAMPLE WITH
THE SAME LECTIN AS THAT USED FOR MAPPING % CITED FROM REF  WITH PERMISSION 
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BELING WITH FLUORESCENT DYES WERE DISCUSSED3OME OF THE RESULTS OF THE MAPPING OF RECEPTORS ARE GIVEN IN &IG 

 -EMBRANE PROTEIN MAPPING
4HE METHOD OF MEASURING THE FORCE RE
QUIRED TO UNBIND LIGAND RECEPTOR PAIRS WASAPPLIED TO THE CELL SURFACE RECEPTORS AS A NEW
METHOD OF MAPPING THE PRESENCE OF A PARTICULAR TYPE OF RECEPTOR ON A SURFACEn )N ONE
OF SUCH APPLICATIONS THE !&- TIP WAS COV
ERED WITH THE PROTEIN _  MACROGLOBULIN AND)T IS THEREFORE NECESSARY FOR US TO DETER
MINE THE RANGE OF FORCE REQUIRED TO UNBIND A
LIGAND FROM THE RECEPTOR AND THAT FOR EXTRAC
TION OF MEMBRANE PROTEINS FROM THE CELL SUR
FACE 4HIS PROBLEM IS ADDRESSED IN THE NEXTSECTION

4HIS MAPPING METHOD WAS LATER EXTENDED
TO SEVERAL DIFFERENT MEMBRANE SYSTEMS AND
THE ADVANTAGES AND DISADVANTAGES OF THE!&- BASED MAPPING METHOD IN CONTRAST TOCONVENTIONAL METHODS BASED ON RECEPTOR LA



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&IG &ORCE CURVES OBTAINED ON THE VIABLE CELL SURFACE USING !&- TIPS MODIlED WITH BIFUNCTIONAL COVALENT CROSSLINKERS
,EFT A FORCE CURVE DEPICTING CONTACT WITH THE CELL SURFACE TOP AND RETRACTING CURVES AFTER CONTACT BOTTOM THREE
CURVES  2IGHT RETRACTING CURVES WITH DOUBLE TOP TRIPLE MIDDLE AND ZERO JUMPS 4HE GRAY ARROW IN CURVE ! SHOWS THE
APPROXIMATE POSITION OF THE TIP IN CONTACT WITH THE CELL SURFACE AND SOLID ARROWS IN OTHER lGURES REPRESENT THE POSITION
WHERE COMPRESSION TURNED TO STRETCHING OF THE SAMPLE CITED FROM REF  WITH PERMISSION )F THE FORCE WAS DUE TO SOME OTHER EVENTSSUCH AS THE UPROOTING OF RECEPTOR MOLECULESFROM THE CELL SURFACE THE FORCE MAPPING OF
RECEPTORS LOSES ITS VALIDITY )F THE UNBINDINGFORCE IS COMPARABLE OR EVEN LARGER THAN THE
FORCE REQUIRED TO EXTRACT RECEPTORS FROM THE
CELL MEMBRANE RECEPTORS WOULD HAVE BEEN
EXTRACTED WITH A FINITE PROBABILITY WHICH OB
SCURES THE RESULT OF FORCE MAPPING

SCANNED OVER THE SURFACE OF A LIVING CELL IN THE
CULTURE MEDIUM POINT BY POINT TO DETERMINETHE PRESENCE OF ITS SPECIFIC RECEPTOR )DEALLY
THE FORCE REQUIRED TO SEPARATE THE CANTILEVER
FROM THE CELL SURFACE SHOULD BE ZERO WHEN
THERE IS NO RECEPTOR AND A NON ZERO FINITEVALUE WHEN LIGAND RECEPTOR INTERACTION IS ESTABLISHED 

 

THE REPULSIVE FORCE SIGNIFYING THE NONCOM
PRESSIVE OPERATION BUT SHOW ATTRACTIVE FORCE
WITH A MORE OR LESS FLAT PLATEAU OF ABOUTNM WHICH IS TERMINATED TO THE HORIZONTALPOSITION BY A JUMP OF THE CANTILEVER 4HE AP
PEARANCE OF THE PLATEAU WAS INTERPRETED AS A
RESULT OF MULTIPLE AND SUCCESSIVE UNBINDING
EVENTS AND THE FORCE OF UNBINDING WAS CALCULATED FROM THE DEFLECTION OF THE CANTILEVER IN
THE PLATEAU REGION (ISTOGRAMS OF THE HEIGHTAND THE LENGTH OF THE PLATEAU GAVE MEAN ¢3% OF  ¢  P. AND  ¢ NM RESPECTIVELY)N THE PRESENCE OF !40 THE CONFORMATION OF'RO%, CHANGES AND THE SEVEN BINDING SITESFOR UNFOLDED PEPTIDES ARE MORE SPREAD OUT4HE FORCE CURVE OBTAINED UNDER SUCH CONDITIONS WERE CHANGED INTO THOSE SHOWN IN &IGB  4HE & n% CURVE NOW SHOWS A SHARP FORCEPEAK WITHOUT A PLATEAU 4HE RESULT IMPLIESTHAT BECAUSE THE DISTANCE BETWEEN BINDING
SITES ON A 'RO%, IS NOW LARGE THE DENATUREDPROTEIN CAN INTERACT ONLY WITH ONE OR AT MOSTTWO BINDING SITES REDUCING EACH UNBINDING
EVENT TO MORE OR LESS AN ISOLATED EVENT 4HE
NUMERICAL VALUE OF THE UNBINDING FORCE WASSIMILAR TO THAT OBTAINED IN THE ABSENCE OF !40
CONFIRMING THAT THE EFFECT OF !40 IS RESTRICTEDTO THE CHANGE IN THE GEOMETRY OF 'RO%, AND
HAS NO SIGNIFICANT INFLUENCE ON BINDING FORCEAT INDIVIDUAL SITES)NVITED 2EVIEW 0APER

 -EMBRANE PROTEIN EXTRACTION
4HE CELL MEMBRANE IS A COMPOSITE STRUC
TURE OF PHOSPHOLIPIDS AND PROTEINS !MONG
CELL SURFACE ASSOCIATED PROTEINS INTRINSIC PROTEINS ARE THOSE THAT SPAN THE LIPID MEMBRANE
FROM ONE SIDE TO THE OTHER HAVING A CENTRALHYDROPHOBIC SEGMENT CONNECTING ITS EXTRACELLULAR DOMAIN TO THE CYTOPLASMIC ONE AND
ARE THUS SECURELY ANCHORED TO THE MEMBRANE)N GENERAL THEY ARE NOT DISPLACED FROM THE
MEMBRANE UNLESS THE LATTER IS DESTROYED BY
FOR EXAMPLE TREATMENT WITH DETERGENTS !N
OTHER TYPE IS CALLED THE PERIPHERAL MEMBRANE

PROTEINS WHICH ARE BOUND TO THE LIPID SUR
FACE OR TO THE EXTRACELLULAR DOMAIN OF INTRINSICMEMBRANE PROTEINS AND THUS EASILY DISPLACED
BY THE ADDITION OF FOR EXAMPLE CHELATINGAGENTS
)T IS OF FUNDAMENTAL AND PRACTICAL INTEREST TO MEASURE THE FORCE REQUIRED TO DISLODGE
THE INTRINSIC MEMBRANE PROTEINS FROM THE CELL
SURFACE BY FORCIBLY PULLING THEM OUT USING THE!&- &OR THIS PURPOSE WE HAVE TO MAKE IT
SURE THAT THE WEAKEST BOND IN THE FORCE SPEC
TROSCOPY SYSTEM BETWEEN THE TIP AND THE SUBSTRATE IS THE TARGETED BOND OR INTERACTION 7E
THEREFORE USED THE MODIFIED !&- TIP WITH THE
AMINO REACTIVE BIFUNCTIONAL COVALENT CROSS
LINKERS SUCH AS 30$0 3ULFO 30$0 AND $33/NCE COVALENT BONDS ARE FORMED BETWEEN THE
CROSSLINKERS ON THE TIP AND THE AMINO BEARING
MOLECULES ON THE CELL SURFACE THE SUBSEQUENTSEPARATION OF THE TIP FROM THE SUBSTRATESHOULD DISRUPT THE NONCOVALENT INTERACTIONSBETWEEN THE MEMBRANE AND THE INTRINSIC PROTEIN PROVIDED THAT SUCH INTERACTIONS COULD BE
RUPTURED WITH A FORCE SIGNIFICANTLY LESS THAN
THE FORCE REQUIRED TO SEVER A COVALENT BOND
IE IN THE RANGE OF  ¢   N. 
3OME OF THE EXPERIMENTALLY OBSERVEDFORCE CURVES UNDER SUCH AN EXPERIMENTAL SET
UP ARE GIVEN IN &IG  7HEN AN UNMODIFIED
TIP WAS USED THE FORCE CURVE OFTEN SHOWED A
DOWNWARD DEFLECTION IN THE RETRACTION REGIMEWITH A GRADUAL DECREASE IN THE DEGREE OF DE*3!0 )NTERNATIONAL .O *ULY 

FLECTION WITHOUT ANY JUMPS TO THE HORIZONTALPOSITION OF THE CANTILEVER 4HIS IS PROBABLY DUETO THE ALMOST CONTINUOUS UNBINDING EVENTSOF MULTIPLE WEAK ADHESIONS BETWEEN THE TIP
AND THE CELL SURFACE &OR A MODIFIED TIP WITH
COVALENT CROSSLINKERS A SIMILAR DOWNWARD
DEFLECTION WAS PROLONGED AND TERMINATEDABRUPTLY WITH A JUMP TO THE HORIZONTAL POSITION 3OMETIMES TWO OR THREE SUCH JUMPSWERE OBSERVED BEFORE THE FINAL JUMP 3OMEOF THE FORCE CURVES EVEN ENDED WITH A LARGEDOWNWARD DEFLECTION SIGNIFYING THAT THE TIP
WAS NEVER RELEASED FROM THE CELL SURFACE
7HEN THE FORCE CHANGE ASSOCIATED WITH
THE JUMPS WAS COLLECTED AS A HISTOGRAM THE
MAJORITY OF THE FORCE VALUES CLUSTERED AT AP
PROXIMATELY THE MEAN WITH AN 3% OF  ¢ P. )T WAS SHOWN THAT THE !&- TIP USEDIN THE ABOVE EXPERIMENT ACTUALLY CARRIED EX
TRACTED PROTEINS AS TENSILE MATERIALS ON ITS SUR
FACE WHICH WAS REMOVED BY THE TREATMENT
WITH PROTEOLYTIC ENZYMES 4HE IDENTIFICATION
OF THE EXTRACTED PROTEINS WAS ATTEMPTED BY
THE IMMUNOFLUORESCENCE METHOD AND INTEGRINS AND FIBRONECTIN WERE IDENTIFIED
4HE HISTOGRAM OF THE FINAL RUPTURE FORCE
IS GIVEN IN &IG  TOGETHER WITH THE LOADING RATE DEPENDENCE OF THE MEAN FORCE VALUEOF THE HISTOGRAM IN &IG  !N ALMOST FLAT
DEPENDENCE OF THE EXTRACTION FORCE ON THE
LOADING RATE IS APPARENT AND IT CONFIRMS THE
THEORETICAL FRAMEWORK OF "ELLgS PROPOSITION
THAT THE ACTIVATION DISTANCE OF PROTEIN EXTRAC
TION FROM THE LIPID BILAYER IS AS LONG AS A FEW   

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2ESULT BETWEEN  TO P. BINS WAS APPROXIMATED BY A 'AUSSIAN CURVE IN SOLID CITEDFROM REF  WITH PERMISSION   

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&IG ,OADING RATE DEPENDENCE OF PROTEIN EXTRACTION FORCE FROM VIABLE CELL SURFACE $IA
MONDS REPRESENT THE MOST PROBABLE VALUE IN EACH SET OF DATA AND CIRCLES THE SECOND MOSTPROBABLE CITED FROM REF  WITH PERMISSION *3!0 )NTERNATIONAL .O *ULY NANOMETERS THUS REDUCING THE SLOPE OF THE
PLOT TO AS LOW AS A FEW PICONEWTONS 3INCE
UNDER A SIMILAR LOADING RATE CONDITION THE
FORCE OF UPROOTING INTRINSIC MEMBRANE PROTEIN MUST BE AT LEAST FIVE TIMES LARGER THAN
THE UNBINDING FORCE OF LIGAND RECEPTOR INTERACTION INVOLVING INTEGRINS AND FIBRONECTIN
4HUS IT IS SAFE TO CONDUCT RECEPTOR MAPPINGEXPERIMENTS BASED ON THE FORCE SPECTROSCOPIC
METHOD
4HE EXTRACTION OF A WELL STUDIED MEM
BRANE PROTEIN BACTERIORHODOPSIN HAS BEEN
REPORTED TO SHOW SEVERAL FORCE PEAKS CORRE
SPONDING TO THE SEPARATION OF HELICAL LOOPSFROM EACH OTHER 3INCE MEMBRANE PROTEINSARE CRUCIALLY IMPORTANT IN THE ORIGIN OF CELLULAR
LIFE DETAILED STUDIES ON THEIR ANCHORING FORCE
TO THE LIPID BILAYER WILL SHED LIGHT ON PRINCIPLE
OF COMPARTMENTALIZATION OF BIOCHEMICAL MOL
ECULES(OW THE MEMBRANE PROTEINS ARE EXTRACTED FROM THE CELL MEMBRANE IS NOT A SIMPLE
MATTER BECAUSE THEY INTERACT WITH MEMBRANE
LIPIDS RATHER STRONGLY )T HAS BEEN SUGGESTED
AND BROADLY ACCEPTED THAT MEMBRANE PROTEINS ARE GENERALLY SURROUNDED BY PHOSPHOLIP
ID MOLECULES WITH A HIGHER AFFINITY THAN THATINVOLVED PROTEIN PROTEIN INTERACTION SO THATPROTEINS WOULD NOT FORM LARGE AGGREGATES IN
THE MEMBRANE )N FACT MEMBRANE PROTEINSMAY FORM SPECIFIC DIMERS OR OLIGOMERS BUT
NOT LARGE AGGREGATES EXCEPT FOR A VERY SPECIAL CASE OF BACTERIORHODOPSIN WHERE PROTEIN
MOLECULES FORM PATCHES OF $ CRYSTALS 3UCH
OBSERVATION SUGGESTS THAT PROTEIN LIPID INTERACTION IS GENERALLY SUFFICIENTLY STRONG SO THAT
WHEN PROTEIN MOLECULES ARE EXTRACTED SOME
LIPID MOLECULES ARE LIKELY TO BE EXTRACTED WITH
THE PROTEIN MOLECULES )N ADDITION IT IS ALSOPOSSIBLE THAT THE ASSOCIATED LIPIDS ARE DE
TACHED FROM THE CELL SURFACE AS A SMALL VESICLERATHER THAN AS A FLAT PATCH !LTHOUGH THE ESTI
MATE OF THE MAGNITUDE OF THE FORCE REQUIREDIN EACH POSSIBLE PROCESS IS NOT YET ESTABLISHED"ELL GAVE A SIMPLE METHOD OF ESTIMATION FOR
THE FIRST TWO CASES BY PROVIDING HYDROPHOBIC FREE ENERGY FOR THE TRANSFER OF CONSTITU
ENT AMINO ACID SIDE CHAINS ON THE MEMBRANESPANNING SEGMENT OF GLYCOPHORIN ! A TYPICALMEMBRANE PROTEIN (IS ESTIMATES OF THE FORCEREQUIRED FOR THE EXTRACTION OF A BARE PROTEIN)NVITED 2EVIEW 0APER

SEGMENT STRIPPED OF ALL THE BOUNDARY PHOS
PHOLIPIDS WAS ABOUT P. AND WAS  P.
IF EXTRACTED WITH A MONOLAYER OF SUCH LIPIDS
4O ESTIMATE THE FORCE IN THE CASE OF VES
ICLE DETACHMENT WE NEED TO DETERMINE THE
SIZE OF THE LIPID VESICLE THAT IS CLEAVED OFF
FROM THE CELL SURFACE WITH THE TARGET PROTEIN
MOLECULE )T IS ALSO NECESSARY TO DETERMINETHE SHAPE OF THE CELL MEMBRANE IMMEDIATELYBEFORE VESICLE FORMATION 4HE NEWLY CREATEDVESICLE HAS COMPLETELY DIFFERENT PRINCIPAL AND'AUSSIAN CURVATURES FROM THE ORIGINAL RATHERFLAT CELL SURFACE 3INCE BOTH OF THE CURVATURES
ARE LARGER THAN THOSE OF ORIGINAL SURFACE THE
FREE ENERGY INCREASE DUE TO VESICLE FORMATIONIS POSITIVE $URING THE DEFORMATION OF THE
CELL MEMBRANE BEFORE THE DETACHMENT OF THE
VESICLE THE BENDING FREE ENERGY INCREASES BUT
THAT DUE TO THE 'AUSSIAN CURVATURE REMAINSCONSTANT 7HEN THE POSITIVE BENDING ENERGYIS DIVIDED BY THE ACTIVATION DISTANCE IN THE UP
ROOTING PROCESS ONE GETS THE ESTIMATE OF THE
FORCE REQUIRED FOR THE DETACHMENT OF MEM
BRANE PROTEIN FROM THE CELL SURFACE BY CREAT
ING A SMALL NEW VESICLE -UCH OF THE DETAILEDINFORMATION NECESSARY FOR OBTAINING THE NUMERICAL ESTIMATE IS STILL LACKING IN THIS AREA

 %XTRACTION OF M2.! FROM LIVECELLS(ARVESTING M2.! FROM THE CYTOPLASM
OF LIVE CELLS AND GENOMIC $.! FROM ISOLATED  

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&IG 3%- IMAGES OF !&- TIPS USED FOR EXTRACTION OF CYTOPLASMIC COMPONENTS ,EFT UNUSEDTIP WITH A SCALE BAR OF «M -IDDLE USED TIP WITH CYTOPLASMIC COMPONENTS ADSORBED ON THE SUR
FACE 2IGHT USED TIP THAT WAS BROUGHT CLOSE TO THE CELL BUT NOT INTO THE CELL 4HE BOTTOM lGURESARE MAGNIlED IMAGED OF THE CORRESPONDING TOP lGURES CITED FROM REF  WITH PERMISSION CHROMOSOMES HAS BEEN SUCCESSFUL BUT ARE
STILL BEING DEVELOPED IN OUR GROUP n )N
THE CASE OF M2.! EXTRACTION AN UNMODIFIED!&- TIP WAS FORCIBLY INSERTED INTO THE CELL INTERIOR AND AFTER A SPECIFIED TIME PULLED OUT
AND RECOVERED IN A TEST TUBE 4HE TIP SURFACEWAS UNDOUBTEDLY COVERED WITH VARIOUS COM
PONENTS OF THE CYTOPLASM ESPECIALLY PROTEINSAND M2.!S AMONG OTHERS AS THE 3%- IM
AGE OF ONE OF SUCH TIPS GIVEN IN &IG  SUG
GESTED 4HE TIP RECOVERED IN THE TEST TUBEWAS THEN SUBJECTED TO 0#2 FOR POLYMERASECHAIN REACTION FOR THE AMPLIFICATION OF SPE  

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CIFIC M2.!S BY ADDING PRIMER $.!S WITHKNOWN TARGETED SEQUENCES FOR EXAMPLE A
PARTIAL BASE SEQUENCE MATCHING THAT OF ` ACTIN
M2.! !FTER 0#2 THE AMPLIFIED M2.! WAS
IDENTIFIED BY ELECTROPHORESIS FOLLOWED BY HYBRIDIZATION WITH FLUORESCENCE LABELED $.!
FRAGMENTS WITH A PARTIAL ` ACTIN SEQUENCE)F DUE PRECAUTION IS TAKEN TO ELIMINATE THE
CONTAMINATION OF 2.!S FROM THE CULTURE ME
DIUM IT IS POSSIBLE TO IDENTIFY THE PRESENCE OF
SEVERAL TYPES OF M2.! IN VARIOUS PARTS OF ASINGLE VIABLE CELL &IGURE  SHOWS THE RESULTOF ONE OF SUCH EXPERIMENTS THAT SHOWED THE

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&IG $ETECTION OF ` ACTIN M2.! IN SEVERAL DIFFERENT LOCALS OF VIABLE CELL BY NEW METHOD USING !&TIP A AND B  %XTRACTED FROM NON STIMULATED CELLS AT
INDICATED POSITIONS OF ! " # AND $ WITH A SCALE BAR
OF «M C AND D  %XTRACTED FROM ACTIVATED CELL WITHFETAL CALF SERUM AT POSITIONS ! " # AND $ CITED FROM
REF  WITH PERMISSION *3!0 )NTERNATIONAL .O *ULY 

LOCALIZATION OF ` ACTIN M2.! IN THE REGIONSCLOSE TO THE NUCLEUS IN A RESTING CELL AND ITSSPREADING TO PERINUCLEAR ZONES UPON STIMULA
TION WITH FETAL CALF SERUM
8U AND )KAI SUCCEEDED IN EXTRACTING $.!
FRAGMENTS FROM A SINGLE PIECE OF MOUSE CHROMOSOME USING A MODIFIED !&- TIP COVEREDWITH AMINO GROUPS SO THAT AT P(  THE TIP
WAS UNCHARGED AND USED TO IMAGE A TARGETED
CHROMOSOME !FTER IMAGING THE P( OF THE
SOLUTION WAS CHANGED TO  SO THAT THE TIP
BECAME POSITIVELY CHARGED THEREBY GENERATINGSTRONG INTERACTIONS WITH NEGATIVELY CHARGED$.! 4HE TIPS THAT RECORDED FORCE CURVES IN
DICATING PROLONGED PULLOUT OF $.! FROM THE
CHROMOSOME WERE INDIVIDUALLY RECOVERED IN
TEST TUBES AND ADSORBED $.! WAS 0#2 AM
PLIFIED 4HE AMPLIFIED $.! WAS FLUORESCENCELABELED AND USED FOR FLUORESCENCE IN SITU HYBRIDIZATION &)3(  4HE RESULT SHOWN IN &IG 
SHOWS THE IMAGE OF THE CHROMOSOME WITH
A MARK ON THE SPOT WHERE THE !&- TIP WAS
PUSHED IN TOGETHER WITH THE RESULT OF &)3(
WHICH CONFIRMED THAT THE 0#2 AMPLIFIED $.!
HAD INDEED COME FROM THE CORRESPONDING
SPOT MARKED ON THE TARGETED CHROMOSOME
4HIS LINE OF RESEARCH HAS POTENTIAL FOR APPLICA
TION TO THE IDENTIFICATION OF INDIVIDUAL GENES
AND THEIR GENETIC ABERRATIONS ON SPECIFIC LOCIOF THE CHROMOSOME

&IG % X T R A C T I O N O F $ . ! F R O M A
SPECIlED LOCUS OF MOUSE CHROMOSOME,EFT !N !&- IMAGE OF CHROMOSOME
AT P(  WHERE TIP SAMPLE INTERACTIONIS MINIMAL !FTER CHANGING THE SOLUTION
P( TO  THE TIP WAS PUSHED INTO THE
CHROMOSOME AT THE POSITION INDICATEDBY A SMALL SQUARE 2IGHT 2ESULT OF IN
SITU HYBRIDIZATION SHOWING THAT 0#2
AMPLIlED $.! HYBRIDIZED WITH CHROMOSOMAL $.! AT ESTIMATED POSITION OF
ORIGINAL EXTRACTION CITED FROM REF 
WITH PERMISSION *3!0 )NTERNATIONAL .O *ULY 4HE EXTENDED USE OF PROBE MICROSCOPY
FOR THE ACTUAL HARVEST OF A SMALL NUMBER OF
MACROMOLECULES FROM A SINGLE VIABLE CELL WILLOPEN A NEW FIELD IN BIOCHEMISTRY AND WILLCONTRIBUTE TO THE UNDERSTANDING OF CELLSg IN
DIVIDUAL BEHAVIORS AND BASIC MEDICAL RESEARCHINCLUDING DRUG TESTS $ISCUSSION
4HE MEASUREMENT OF THE FORCE REQUIREDTO DISRUPT BIOSTRUCTURES BASED ON PROTEIN
PROTEIN AND PROTEIN LIPID INTERACTIONS IS THE
FOCUS OF THIS ARTICLE 4HE RESULT OF RECENT MEA
SUREMENT IS EXPECTED TO REVEAL THE MECHANICAL PRINCIPLES OF THE INTERNAL ARCHITECTURE OF
THESE STRUCTURES WHICH CANNOT BE EXPLICITLY
MEASURED USING CONVENTIONAL TECHNOLOGY AND
THEREFORE HAS NOT BEEN INVESTIGATED IN THE
PAST
4HE & n % CURVES OBTAINED DURING THE
FORCED UNFOLDING OF SEVERAL GLOBULAR PROTEINSGAVE A RARE INSIGHT INTO THE PRESENCE OF RIG
IDLY FOLDED LOCAL SUBSTRUCTURES INSIDE THE NA
TIVE CONFORMATION OF SUCH PROTEINS 7HETHERTHIS OBSERVATION REPRESENTS A GENERAL FEATURE
OF PROTEIN MOLECULES IS A QUESTION TO BE AN
SWERED AFTER STUDYING MANY MORE CASES )TIS STILL NOTEWORTHY THAT THE CLASSICAL QUESTIONOF HOW PROTEINS CAN BE SOFT AND YET SUSTAIN
THE ACTIVATED STATE OF COVALENTLY BONDED MOL

ECULES HAS BEEN PARTIALLY ANSWERED BY NEWEVIDENCE OF THE PRESENCE OF LOCALLY RIGID SUB
STRUCTURES $UE TO THE PRESENCE OF SUCH LOCALLY RIGID SUBSTRUCTURES THE INTERNAL MECHANICS
OF PROTEIN IS RATHER MORE COMPLEX THAN HITHERTO IMAGINED )T IS ALSO TRUE AS HAS BEEN SUF
FICIENTLY DEMONSTRATED THAT PROTEINS CANNOTONLY BE RIGID 4HE RIGID PARTS OF A PROTEIN MOLECULE MUST ALSO COORDINATE WITH MORE FLEXIBLEAND SOFT PARTS OF THE SAME MOLECULE FOR THE
WHOLE MOLECULE TO PERFORM ITS BIOLOGICALLYSIGNIFICANT ROLES 7E ARE NOW ENCOUNTEREDWITH SEVERAL NEW QUESTIONS AS FOLLOWS (OW CAN RIGID AND SOFT PARTS BE CREATEDON THE BASIS OF A SMALL NUMBER OF NONCOVALENT INTERACTIONS !RE THE COMPOSITE STRUCTURE OF SOFT AND
HARD PARTS OF PROTEIN MOLECULES REALLY
NECESSARY FOR THESE MOLECULES TO FULFILLTHEIR BIOLOGICALLY SIGNIFICANT ROLES SUCH AS
AN ENZYME
 (OW DO THE TWO MECHANICALLY DIFFERENTPARTS OF A PROTEIN MOLECULE WORK IN ENZY
MATIC CATALYSIS (OW DOES THE FORCE OF ANCHORING OFMEMBRANE PROTEINS TO THE LIPID BILAYERSCOMPETE WITH THE FORCE EXERTED BY CELLULAR MOVEMENT LIKE LOCOMOTION
 (OW EFFECTIVELY CAN WE HARVEST MEM
BRANE PROTEINS ON AN !&- TIP TO PERFORM
PRECISE IN SITU ANALYSIS OF THEIR IDENTITY
DISTRIBUTION AND RELATIVE ABUNDANCE)NVITED 2EVIEW 0APER!NSWERS TO THESE QUESTIONS WILL BE OBTAINEDWHEN TECHNOLOGY FOR MECHANICAL MEASURE
MENTS ON NANO BIOSTRUCTURES HAS MADE CON
SIDERABLE IMPROVEMENTS IN TERMS OF ACCURACY
AND $ RESOLUTION

 #ONCLUSIONS
7E HAVE ESTABLISHED A METHOD OF INVESTIGATING HITHERTO UNEXPLORED PHYSICAL PROPERTIESOF GLOBULAR PROTEINS USING FORCE SPECTROSCOPYBASED ON THE FORCE CURVE MODE OF THE !&-
'LOBULAR PROTEINS HAVE BEEN SHOWN TO POSSESS MECHANICALLY RIGID SUB STRUCTURES WHICH
WHEN UNDER TENSILE STRESS SHOWED FRACTUREMECHANICS SIMILAR TO THAT OF BRITTLE MATERIALS-EMBRANE PROTEINS CAN BE EXTRACTED FROMTHE CELL SURFACE BY A FORCE LESS THAN THAT RE
QUIRED TO BREAK A COVALENT BOND BUT SIGNIFICANTLY LARGER THAN THAT REQUIRED TO SEPARATELIGAND RECEPTOR PAIRS ON THE CELL SURFACE 2E
CEPTOR MAPPING AND CELL MEMBRANE HARVESTCAN THEREFORE BE PERFORMED IN DISTINCT FORCE
RANGES

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!CKNOWLEDGMENTS
4HE AUTHOR EXPRESSES HIS DEEPEST GRATITUDE TO ALL THE PEOPLE WHO WORKED IN THE,ABORATORY OF "IODYNAMICS AND CONTRIBUTEDIN ONE WAY OR ANOTHER TO THE RESULT THAT CULMINATED IN THIS REVIEW ARTICLE (E ALSO THANKSHIS COLLABORATORS IN THIS WORK ESPECIALLY 0RO
FESSORS 5NO #ARLSSON 3HOHEI +OIDE +IN 0ING
7ONG 3USUMU /KAZAKI AND -ASAHIKO (ARA
FOR THEIR KIND HELP IN THIS WORK (E EXPRESSESHIS SINCERE GRATITUDE TO 0ROFESSORS /SAMU .I
SHIKAWA AND 3EIZO -ORITA FOR THEIR CONTINUOUS SUPPORT AND ENCOURAGEMENT IN THE AP
PLICATION OF SCANNING PROBE MICROSCOPY TO
BIOLOGICAL RESEARCH (E ALSO SINCERELY THANKS$R 2EHANA !FRIN FOR HER CRITICAL READING OF
THE MANUSCRIPT 4HIS WORK WAS SUPPORTED BY
GRANTS IN AID FROM THE *303 2ESEARCH FOR THE&UTURE 0ROGRAM 2 AND 3CIENTIFIC 2E
SEARCH ON 0RIORITY !REAS "  ! DONATION FROM
9OKA KAI $IRECTOR $R 4OSHIORO &UJIMOTO IS
ALSO ACKNOWLEDGED

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3CHIMANSKY 'EIER AND 9 - 2OMANOVSKY 7ORLD
3CIENTIlC .EW *ERSEY  #HAP^% %VANS AND & ,UDWIG * 0HYS #ONDENS -ATTER  !
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AND ( % 'AUB 3CIENCE   
4 7ANG AND ! )KAI *PN * !PPL 0HYS  
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% %VANS AND + 2ITCHIE "IOPHYS *   
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
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)CHIKAWA AND ! )KAI #HEMICAL 3ENSES   ( +IM 3 4SURUTA ( !RAKAWA 4 /SADA AND !
)KAI 5LTRAMICROSCOPY   2 !FRIN ( !RAKAWA 4 /SADA AND ! )KAI #ELL
"IOCHEM "IOPHYS   2 !FRIN AND ! )KAI 5LTRAMICROSCOPY  
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+OIDE ( !RAKAWA AND ! )KAI * -OL "IOL  2 (ERTADI AND ! )KAI 0ROTEIN 3CI   

 ! )DIRIS - 4 !LAM AND ! )KAI 0ROTEIN %NG   - 'AO $ #RAIG 6 6OGEL AND + 3CHULTEN *-OL "IOL    3 )ZRAILEV 3 3TEPANIANTS - "ALSERA 9 /ONO AND+ 3CHULTEN "IOPHYS *   
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 ! % %RICKSON AND ! ,ILJAS 0ROTEINS    2 3AITO 4 3ATO ! )KAI AND . 4ANAKA !CTA #RYSTALLOGRAPHICA $    4 7ANG ( !RAKAWA AND ! )KAI "IOCHEM "IO
PHYS 2ES #OMMUN    + "OREN ( 'RANKVIST 0 (AMMARSTROM AND 5
#ARLSSON &%"3 ,ETT    + -ITSUI + .AKAJIMA ( !RAKAWA - (ARA AND! )KAI "IOCHEM "IOPHYS 2ES #OMMUN    4 /KAJIMA ( 3EKIGUCHI AND ! )KAI "IOPHYS#HEM   
 ! ! 'RIFlTH 0HYL 4RANS 2OY 3OC ,ONDON !
   3 3URESH &ATIGUE OF -ATERIALS #AMBRIDGE 5NI
VERSITY 0RESS #AMBRIDGE  ND ED P^ $ * "ROCKWELL % 0ACI 2 # :INOBER ' 3 "ED
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AND 3 % 2ADFORD .ATURE 3TRUCT "IOL  
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EFENAUER AND ! 0LàCKTHUN 0ROC .ATL 3CI !CAD
53!   
 9 -AKINO + !MADA ( 4AGUCHI AND - 9OSHIDA* "IOL #HEM    ( 3EKIGUCHI ( !RAKAWA ( 4AGUCHI 4 )TOH 2
+OKAWA AND ! )KAI "IOPHYS *    - 'RANDBOIS - "EYER - 2IEF ( #LAUSEN
3CHAUMANN AND ( % 'AUB 3CIENCE   & /ESTERHELT $ /ESTERHELT - 0FEIFFER ! %NGEL (% 'AUB AND $ * -àLLER 3CIENCE    4 /SADA ( 5EHARA ( +IM AND ! )KAI * .ANO
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FROM THE 5NIVERSITY OF 4OKYO BOTH IN "IOPHYSICS AND "IOCHEMISTRY AND 0H$  FROM
$UKE 5NIVERSITY !FTER SERVING AS ASSISTANT
LECTURE AND ASSOCIATE PROFESSOR AT THE 5NIVER
SITY OF 4OKYO HE WAS APPOINTED AS 0ROFESSOR
OF "IODYNAMICS AT 4OKYO )NSTITUTE OF 4ECHNOL
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