fluorescent probes and bioconjugation chemistries for ...€¦ · tronic state.3,4,7 a list of...

JOURNAL OF CHEMICAL PHYSICS VOLUME 117, NUMBER 24 22 DECEMBER 2002

Fluorescent probes and bioconjugation chemistries for single-moleculefluorescence analysis of biomolecules

Achillefs N. Kapanidisa) and Shimon WeissDepartment of Chemistry and Biochemistry, and Department of Physiology, University of Californiaat Los Angeles (UCLA), Los Angeles, California 90095-1569

~Received 7 March 2002; accepted 18 September 2002!

Fluorescence-based detection of single biomolecules in solution and at room temperature hasopened new avenues for understanding biological mechanisms. Single-molecule fluorescencespectroscopy~SMFS! of biomolecules requires careful selection of fluorophores, sites ofincorporation, and labeling chemistries. SMFS-compatible fluorophores should permit extended,uninterrupted observations of fluorescence with high signal-to-noise ratios; more stringentconsiderations apply for specific methodologies, such as fluorescence resonance energy transfer andfluorescence anisotropy. Strategies for site-specificin vitro labeling of small proteins exploit thereactivity of the amino acid cysteine~Cys!, allowing incorporation of one or more fluorophores;labeling of closely spaced Cys residues using bis-functionalized fluorophores allows probing of theorientation of individual protein domains. Forin vitro labeling of large proteins, the options includepeptide ligation, intein-mediated labeling, puromycin-based labeling, unnatural amino acidmutagenesis, and reconstitution from individual subunits or subunit fragments. Forin vivo analysis,one can use proteins that are labeledin vitro and then incorporated in cells; genetic encoding ofspecific protein sequences can also lead toin vivo labeling, either byin vivo targeting byfluorophores or by biosynthesis of protein fusions with natural fluorophores such as the greenfluorescent protein. The existing methods, along with others under development, will bring SMFSto the mainstream and advance significantly our understanding of vital biologicalprocesses. ©2002 American Institute of Physics.@DOI: 10.1063/1.1521158#

iem-bgl-anopmucicns

le

icesmcem

ol-o

S-as-iesk atture

ol-of

ro-lec-,I.ofalmi-

ma

I. INTRODUCTION

The past decade marked one of the most exciting sctific developments born of the interaction of physics, cheistry, and biology: the ability to perform real-time, roomtemperature observations of individual biomoleculesusing optical microscopy techniques described as sinmolecule detection~SMD!.1 SMD eliminates the ensembleand time-averaging present in conventional biochemicalbiophysical assays. SMD can uncover and analyze subplations and distributions hidden in heterogeneous systeprobe conformational dynamics of biomolecules under eqlibrium conditions, an otherwise impossible task due to laof synchronization; and explore complex reaction kinetand biochemical pathways under nonequilibrium conditioSeveral excellent reviews exist on the topic.2–8

A popular method for probing single molecules is singmolecule fluorescence spectroscopy~SMFS!. SMFS moni-tors the structure, dynamics, and interactions of biochemsystems~e.g., intramolecular and intermolecular distancdomain orientations, binding stoichiometry, equilibriubinding constants! through measurements of fluorescenproperties of individual species, such as fluorescence esion intensity, lifetime, polarization, and quantum yield.6,9

SMFS has allowed striking visualizations of single biomecules at work, including the rotation of the rotor subunit

a!Author to whom all correspondence should be addressed. [email protected]

10950021-9606/2002/117(24)/10953/12/$19.00

Downloaded 11 Dec 2002 to 169.232.130.170. Redistribution subject to

n--

ye-

du-s;i-ks.

-

al,

is-

f

the rotary stepper motor protein F1-ATPase,10 the sliding ofkinesin on microtubules,11,12 the translocation of RNA poly-merase~RNAP! molecules on double-stranded DNA,13 theformation of ribozyme intermediates,14 and the real-time in-fection of a cell by single virus particles.15

In this article, we describe the properties of SMFcompatible fluorophores, list considerations for popularsays, provide examples of site-specific labeling strategthat enabled SMFS observations, and conclude with a loofluorophores and labeling strategies that can answer fuchallenges.

II. PROPERTIES OF SMFS-COMPATIBLEFLUOROPHORES

Optical detection of single fluorescently labeled biomecules using SMFS is based on thousands to millionslaser-induced excitation–deexcitation cycles of the fluophore between the ground state and the first excited etronic state.3,4,7 A list of fluorophores often used for SMFSalong with characteristic properties, is provided in TableFluorophores compatible with the demanding natureSMFS fulfill most of the photophysical and photochemiccriteria set for ensemble fluorescence spectroscopy andcroscopy measurements16,17~high extinction coefficient, highquantum yield, large Stokes shift, high photostability!, aswell as additional properties specific to SMFS.18 Desirablefluorophore properties include the following.il:

3 © 2002 American Institute of Physics

AIP license or copyright, see http://ojps.aip.org/jcpo/jcpcr.jsp

10954 J. Chem. Phys., Vol. 117, No. 24, 22 December 2002 A. N. Kapanidis and S. Weiss

Downloaded 11 De

TABLE I. Characteristic properties of selected fluorophores used for SMFS.a

Fluorophore

Absorbancemaximum

~nm!

Extinctioncoefficient

(cm21 M21)

Emissionmaximum

~nm!Quantum

yieldLaser line

~nm! Photostability

GFP 395 30 000 509 0.8 457 Good470 7 000

Fluorescein 490 67 000 520 0.5–0.9 488 PoorAlexa-488 495 80 000 520 0.5–0.9 488 ModerateTetramethyl-rhodamine

554 85 000 585 0.2–0.5 514 532 Good

Cy3 550 150 000 585 0.2 514 532 GoodTexas Red 596 85 000 620 0.5 514 532 GoodCy5 650 250 000 670 0.15 633 Moderate

aValues were taken from Refs. 16 and 17, manufacturers of reactive forms of the fluorophores~MolecularProbes, Amersham Pharmacia Biotech!, or measured in our laboratory~see Ref. 80!.

hth

hemn

datlyremn

ytv

ldo

leuron

iotehe-

thodesomrg

forra-

laybe

ipletthats for

anld

f-urethe

gnal

to-ly

hehe, re-

ntial.ortnses

ionith

A. Brightness

Brightness is a measure of the rate of fluorescence ptons emitted by the fluorophore, and is represented byproduct of the extinction coefficient of the fluorophore at twavelength of excitation,eexc, and the fluorescence quantuyield of the fluorophore at the wavelength of emissioQF ;16 for typical SMFS-compatible fluorophores,eexc

.20 000 cm21 M21 andQF.0.1. Although less bright fluo-rophores can be detected using SMFS, the above-staterameters set limits for the extraction of reliable biologicinformation from SMFS experiments. These limits curreneliminate fluorophores that are useful for ensemble fluocence or phosphorescence, such as the fluorescent aacid tryptophan, the environmental probes dansyl aIAEDANS, and the phosphorescent probes eosin and errosin. Brightness can be affected significantly by local enronment and buffer conditions (pH, viscosity, concentrationof organic solvents!; accurate evaluation of brightness shoube performed with the fluorophore incorporated at the siteinterest and in the appropriate assay buffer.

B. Singlet-state saturation

During the excitation–deexcitation cycle of a singfluorophore, a transition from ground to excited state occdue to matching of the excitation wavelength to a fluophore transition, whereas a transition from excited to groustate occurs due to photon emission~fluorescence! or thermaldeactivation. Since in SMFS the prerequisite for reexcitatof a singlefluorophore is the relaxation to the ground stathe maximum number of emitted photons is limited by trate of fluorescence,kF .3 Therefore, fluorophores with relatively short fluorescence lifetimes~0.5–5 ns! are preferablefor SMFS.

C. Triplet-state saturation

Intersystem crossing from the singlet excited state offluorophore to the triplet state~an excited state lasting up tseveral milliseconds that is characterized by a spin-forbidtransition to the ground state! can lead to saturation that ieven more severe than singlet-state saturation. For sfluorophores, efficient intersystem crossing leads to a la

c 2002 to 169.232.130.170. Redistribution subject to

o-e

,

pa-l

s-inodh-i-

f

rs-d

n,

e

n

ee

decrease in the signal intensity, rendering them unsuitableSMFS. The lifetime of a triplet state can be shortened dmatically ~by a factor of 103– 105) through quenching by theground triplet state of molecular oxygen (3O2);19 however,since the oxygen concentration is often minimized to dephotodestruction of the fluorophore, a compromise has toreached between removing oxygen and quenching the trstate.19 This dilemma can be addressed by using reagentsact as both oxygen scavengers and triplet-state quenchera specific fluorophore.

D. Signal stability and fluorescence intermittency„‘‘blinking’’ …

Depending on their rotational freedom, fluorophores cinteract transiently with their local environment to yiestates associated with variable emission spectra~‘‘spectraljumps’’! or quantum yield. Although such transitions are eficient reporters of the local environment, they can obscmore interesting, dynamic processes. Moreover, some ofstates adopted by the fluorophore can be long~up to seconds!and exhibit low quantum yield~‘‘dark’’ states!, resulting influorescence intermittency that reduces the detectable siand obscures time trajectories of fluorescence.

E. Resistance to photodestruction

Every organic fluorophore is prone to irreversible phodestruction~due to elusive photochemical reactions, mainphoto-oxidation20!, limiting the total number of emittedphotons;3 SMFS-compatible fluorophores emit 105– 106

photons before photodestruction.4 The quantum yield forphotodestruction ~or ‘‘photobleaching’’! varies greatlyamong fluorophores,21 with the series fluorescein,Cy5,Cy3,tetramethylrhodamine being a general rule for tphotostability of common fluorophores. Depending on ttime scales of the observation and the processes studiedsistance to photobleaching ranges from desirable to esseFor example, surface-immobilized fluorophores that repon long time trajectories of complex biochemical reactioshould possess correspondingly long photosurvival timthat allow probing of several time scales along the reactcoordinate. Photosurvival can be extended w


it

sy

-

th

ighterf

reunte,numckbowin

efloc

orcae-bthosts

helthcisevsions-olt

ho-a-

lesed.

e towo

itesrs-

n-rely.fromn-nalady-

nd

e

nergyf

10955J. Chem. Phys., Vol. 117, No. 24, 22 December 2002 Fluorescence probes for biomolecules

various methods that minimize triplet-state oxygen andhighly reactive metabolites~such as singlet oxygen@1O2#,hydroxyl-radical@OH•# and hydrogen peroxide@H2O2#); thisis performed through deoxygenation,19 use of molecular-oxygen scavengers ~such as n-propyl gallate,p-phenylenediamine, or the glucose oxidase/catalasetem!, singlet-oxygen scavengers~such as carotenoids!,oxygen-metabolite scavengers~such as ascorbic acid, cysteine, and imidazole!, and reducing agents~such asb-mercaptoethanol!.4,17

F. Spectral resolution from background signaland noise

Reduction of background and its associated noise iscentral theme of all SMFS methods.3,4,6,7 The signal-to-background ratio~SBR! and the signal-to-noise ratio~SNR,as it refers to background-associated noise! signify the abil-ity of the optical system to detect single molecules with hstatistical accuracy; high SBR and SNR allow reliable demination of several fluorescence parameters, such as fluocence lifetime and anisotropy. To increase SBR and SNRSMFS, one should select excitation sources, fluorophoand optics that discriminate fluorescence from backgroarising from Rayleigh scattering, Raman scattering of waimpurities in reaction buffers and reaction chambers, andthe case of cells, autofluorescence of cellular componeFluorophores emitting at the far-red portion of the spectrcan be distinguished easily from most sources of baground, and are preferable for studies with cells and tursamples. Also, fluorophores with large Stokes shifts alleasy discrimination of fluorescence from Rayleigh scatter

G. Size of fluorophore and linker

Fluorophores with small and short rigid linkers are prerable because they tend to be less perturbative to theirenvironment and to contain less ‘‘fluorophore noise’’~fluc-tuations of the fluorescent properties of a single fluorophdue to unknown and uncontrolled changes of its loenvironment6!. Moreover, use of hydrophilic fluorophores rduces hydrophobicity-driven aggregation and unpredictabinding to hydrophobic protein surfaces, thus reducinguncertainty in the determination of the location of the flurophores and improving the accuracy of fluorescence renance energy transfer~FRET!-based distance measuremen

H. Compatibility with laser excitation sources, opticalcomponents, and detectors

Matching the properties of the optical system to tproperties of the fluorophore~s! used for SFMS is a criticaconsideration. The large variety of existing cw lasers in400–700 nm range provides flexibility in choosing an extation source; recently introduced all-solid-state pulsed laat the popular wavelengths of 532 and 635 nm are connient and powerful light sources for time-resolved analyon single molecules. Customized optics supplement the llist of available microscopy filters and allow efficient dicrimination of fluorescence from samples containing twomore fluorophores; this ability has been extended by mu


s

s-

e

r-es-ors,dr,ints.

-id

g.

-al

el

lee-o-.

e-rse-sg

ri-

channel detectors that allow simultaneous detection of ptons of multiple wavelengths, generating spectral informtion for a single molecule as a function of time.22

III. CHOOSING THE RIGHT FLUOROPHOREFOR THE RIGHT ASSAY

In the case where distances within single biomolecuhave to be monitored, the technique of FRET can be usFRET is a nonradiative physical process that occurs duweak interactions between the transition dipoles of tcomplementary fluorophores, a donor (D) and an acceptor(A). FRET allows measurements of distances between sspaced by 20–100 Å.18,23 The efficiency of energy transfe(E) is inversely proportional to the sixth power of the ditance (R) betweenD andA:

E5@11~R/R0!6#21,

whereR0 is the Forster radius, characteristic of theD –A pairand the conditions of the solution; forR5R0 , E550%. R0

depends, in part, on the relative orientation ofD and A di-poles, compounded in the orientation factork2; since k2

cannot be measured experimentally for a singleD –A pair,extreme values ofk2 ~which range from 0 to 4, with 2/3being the value for an isotropic distribution of dipole orietations! can complicate the distance measurement seveTherefore, FRET-based distance measurements benefitrapid reorientation of the fluorophores since it minimizes ucertainties associated with dipole orientation. The rotatiofreedom of the fluorophores can be evaluated using stestate and time-resolved fluorescence anisotropy,24,25 and un-certainty limits can be set for eachD –A pair.26,27

To maximize the FRET sensitivity in detecting static adynamic heterogeneity, theR0 values for aD –A pair shouldlie in the linear range of theE–R dependence curve, with thtwo extreme-distance end points lying in the range 0.7R0

,R,1.5R0 . This is a consequence of the steep (1/R6)distance-dependence of FRET~Fig. 1!. For example, for aD –A pair with R0540 Å, E changes dramatically forRvalues borderingR0 : E is 85% for R530 Å, 50% atR

FIG. 1. Dependence of the dynamic range of fluorescence resonance etransfer~FRET! on the Fo¨rster radiusR0 . The numbers present at the left oeach curve correspond to theR0 of each curve~in Å!. The dotted linesdelineate the regime of maximum sensitivity for each pair with differentR0 .D –A, donor–acceptor.


s

-

Å

leth

eif

fi-ecc

sie

s

olyit-

-

eteinp.3e-

ba-

ntf

ornccegtolsxpofte

o-

per-rolrmnsfic

of

os-c-of

of

nce

n-gesringen-

ptortheenal

f

el-of

gi-

esofla-

ty,sy,tro-icle,o-cid

of

ive

her

act


540 Å, and 21% atR550 Å; thus, the dynamic range i;28– 60 Å ~note: measurements ofRD –A.60 Å are, in part,hindered due to the tendency of several acceptors~such asthe cyanine fluorophore Cy5! to exist in nonabsorbing, nonfluorescent states28!. Furthermore, aD –A pair of R0570 Åhas no discriminating ability for distances in the 25–45range since for the entire range,E of 100%. Extensive tableswith R0 values forD –A FRET pairs are available.29

If no high-resolution structural information is availabfor evaluation of possible distances, one can considerdiameter of the protein of interest~treating it as a globularprotein, unless it shows dimensional anisotropy! to estimatea range of distances. Moreover, one can prepare sevD –A pairs, with each pair featuring the same donor but dferent acceptor~or vice versa!, thereby exploring a wideranges ofR0 values. Considering the high extinction coefcients and good quantum yields required for efficient dettion of single fluorophores, the range of measurable distanextends to;100 Å; however, due to the special photophycal properties of SMFS-compatible fluorophores, it becomchallenging to measureshort distances (R,30 Å). For ex-ample, theR0 for the D –A pair fluorescein-Cy5~two fluo-rophores with apparently small spectral overlap! is ;44 Å.30

The inability to probe the short-distance regime~1–10 Å!,however, might soon be addressed by use of electron-tranmolecular rulers.8

In contrast to FRET, fluorescence anisotropy~FA! ex-plores conformational dynamics and relative orientationprotein elements by probing the transition dipole of rigidattached fluorophores. To achieve this, a fluorophore wshort fluorescence lifetimes~to minimize fluorescence depolarization during the time scale of fluorescence emission! andconsiderable hydrophobicity~to favor hydrophobic interactions with protein functionalities instead of solvation! is in-troduced at each of various sites on proteins, and thesemble FA is used to determine the site with the grearestriction of fluorophore movement due to noncovalentteractions with the local protein surfaces. Typical anisotrovalues used for single-molecule FA range from 0.25 to 0~with limiting anisotropy for a single-dipole fluorophore bing in the 0.36–0.4 range!. Fluorophore immobilization canalso be achieved in a rational and predictable fashionusing intramolecular crosslinking of bis-functional derivtives of fluorophores.

Other assays also have special requiremeFluorescence-lifetime-based assays, which are useful‘‘gating’’ the short-lived cellular autofluorescence and fseparating mixture components with different fluorescelifetimes, benefit from fluorophores with long fluorescenlifetimes; however, the lifetimes should not be too lonsince this will shift the onset of singlet-state saturationlower excitation intensities. Several fluorophores can aserve as sensors of the local environment, measuring esure to solvent,pH, and ion concentration; a combinationsensors with FRET or FA probes allows multiparameanalysis of complex systems.


e

ral-

-es-s

fer

f

h

n-st-y2

y

s.or

e

,

oo-

r

IV. CHEMISTRIES FOR SITE-SPECIFIC LABELINGWITH FLUORESCENT PROBES

Currently, most SMFS experiments are performedinvitro, using fluorophores introduced extrinsically after bisynthesis and purification.16 It is possible to use SMFS tostudy biomolecules without protein labeling, as has beenformed in enzymatic-turnover studies of cholesteoxidase31 ~where fluorescence arose from the oxidized foof the enzyme cofactor!, and in nucleotide-binding studies othe molecular motor myosin32 ~where fluorescent derivativeof nucleotides were used!. These cases are protein speciand often require complementary and orthogonal labelingadditional components to extract information.In vivo label-ing with genetically encoded fluorescent probes is also psible, and its anticipated use in SMFS will allow unpreedented mechanistic investigations in the natural milieuthe cell.

Although the exact site or the high site-specificitylabeling is not required for some imaging applications~pro-vided that labeling does not affect functionality!, site-specificity becomes an important issue when precise distaor orientation information is sought. Stochastic~or nonspe-cific! labeling is inadequate to extract reliable biological iformation and should be restricted to the preliminary staof SMFS assay development. This was the case duFRET-based analysis of conformational dynamics of thezyme staphylococcal nuclease~SNase!,33 where the donorwas incorporated in a site-specific fashion, and the accewas incorporated nonspecifically on lysine residues ofenzyme. Since SNase has.10 surface-exposed lysines, thexact sites of incorporation are unknown. Moreover, the filabeled product contained several species:D-only SNase~major species!, D –A SNase~with the acceptor in each oseveral sites!, D –A–A SNase~with the acceptors in two ofseveral sites!, and minor species with higher degree of labing. The labeled proteins allowed the developmentgroundbreaking methodology; however, no reliable biolocal information could be extracted from these studies.

Site-specific labeling of biomolecules with fluorophorrequires careful choice of labeling chemistry, optimizationthe labeling reaction and rigorous characterization of thebeled biomolecules for labeling efficiency, site-specificiand retention of functionality. Labeling nucleic acids is eaand several fluorophores or reactive groups can be induced using automated solid-phase synthesis. In this artwe will focus on strategies for site-specific labeling of prteins, multiprotein complexes, and protein–nucleic acomplexes with one or more fluorophores.

The most common method for site-specific labelingproteins with fluorophores is thecysteine-specific labelingwith thiol-reactive reagents. During this reaction, proteinswith surface-exposed cysteine~Cys! residues are covalentlymodified by maleimide, iodoacetamide, or other reactconjugates of fluorophores.16,34,35 This is the method ofchoice for small proteins (,500 residues!, since Cys is arare amino acid, and can be substituted easily with otamino acids using site-directed mutagenesis,36 a standardmolecular biology technique.

If the protein of interest has no Cys residues, the ex


h

ng

mys

ysu

Ccidfo

laglncll

e-

rug

cca

on

otayyfss

lyth

eec

na-o

thoribapaiesof

ofs-e

-the

st-

n

l-s,ulew

sinpri-el

s the

ents.D,-d

toules.


site of incorporation is selected after inspection of a higresolution three-dimensional structure~generated using x-raycrystallography or nuclear magnetic resonance! and consid-eration of the question to be addressed. Labeling shouldperturb the enzymatic activity or the accurate spatial arranment of the protein sequence~also known as the ‘‘proteinfold’’ !. Subsequently, an existing amino acid~preferablyhaving a side chain of charge, size, and hydrophobicity silar to that of Cys! at the site of choice is substituted by a Cusing site-directed mutagenesis.36

If the unmodified protein has a single preexisting Cstructural information, along with measurements of the sface accessibility of Cys side chain,24 will determine whetherthe existing Cys can be used; otherwise, the preexistingcan be converted to the structurally similar amino aserine, and the procedure for Cys-free proteins can belowed.

SMFS experiments using single-surface Cys proteinsbeled using thiol-specific reagents include imaging of sinmyosin molecules using low-background epifluorescemicroscopy;37 imaging of single myosin molecules, as weas individual cycles of adenosino-triphosphate~ATP, whichserves as a biological energy source! binding and hydrolysis~‘‘ATP turnovers’’! by total internal reflection fluorescencmicroscopy~TIRFM!;37 and imaging of single kinesin molecules as they move processively along microtubules.11 In-terestingly, labeling efficiencies as low as 10% were instmental in establishing new biological information usinSMFS.38

Fluorophores incorporated on surface Cys residuesalso become efficient reporters of the orientation of loprotein structure. Tetramethylrhodamine~TMR!-labeledmyosin featuring high anisotropy allowed the differentiatibetween conformational states of myosin.39,40 In an exten-sion of the seminal experiments that demonstrated the rtion of F1-ATPaseg-subunit, Kinosita and co-workers usedsingle fluorophore~instead of a long and extremely bulkfluorescent actin filament! to probe the rotation process bsingle-fluorophore angular imaging.10 In this case, the site ofluorophore incorporation was chosen after several cycletrial and error that resulted in maximization of the fluorecence anisotropy in the labeled protein.

The orientation of a single dipole can be fixed not onthrough noncovalent interactions of the fluorophore withlocal environment, but also byintramolecular cross-linkingof two appropriately spaced Cys residueswith a bis-functional Cys-reactive fluorophore. This approach has bused only for proteins with an existing high-resolution struture. Intramolecular cross-linking restricts the local rotatiomobility of the fluorophore significantly, allowing monitoring of the orientation and dynamics of protein domainsother protein structural elements.41 More importantly, knowl-edge of the protein structure permits precise design ofexactorientation of the fluorophore relative to the proteinto an interacting molecule; such a selection is not possfor immobile fluorophores attached to a single Cys. Thisproach was introduced in studies of the myosin light-chorientation relative to actin filaments by ensemble fluorcence polarization41 and was employed recently in studies


-

ote-

i-

,r-

ys

l-

-ee

-

anl

a-

of-

e

n-l

r

e

le-

n-

kinesin intermediates during translocation42,43~Fig. 2!. Sosaand co-workers used the crystallographic structurekinesin44 @Fig. 2~b!# to design a kinesin derivative with Cyresidues spaced by;16 Å, a distance suitable for crosslinking by the bis-functional reactive fluorophorbis-@(N-iodoacetyl)-piperazinyl#-sulfonerhodamine @BSR;Fig. 2~a!; compare with the mono-functional TMR fluorophore#. Site-directed mutagenesis was used to prepareprotein derivative by substituting eight of the nine preexiing Cys residues of kinesin with alanine residues~keepingonly Cys174! and by introducing a second Cys at positioCys169. The two Cys lie on either side of a shortb-sheet@Fig. 2~b!#, orienting the transition dipole of BSR to be amost perpendicular (75°) to the long axis of microtubuleand tangential to a radius centered on the microtubcylinder.42 The double-Cys kinesin was labeled using a loreactive fluorophore-to-protein molar ratio~1:1; which is

FIG. 2. Strategy for fluorescent labeling and observation of kinemolecules prepared using intramolecular cross-linking of two approately spaced Cys residues.~a! Structures of the fluorophores used to laba single cysteine~Cys174! and a pair of proximal cysteines~Cys169 andCys174!. TMR, tetramethylrhodamine; BSR, bis-@(N-iodoacetyl)-piperazinyl#-sulfonerhodamine.~b! Model of kinesin motor domain show-ing the positions of the selected proximal cysteines; the arrow representorientation of microtubules. ADP, adenosine diphosphate.~c! Schematic ofthe epifluorescence microscope used for the polarization measuremAOM, acousto-optical modulator; EOM, electro-optical modulator; ICCintensified charged-coupled device camera.~d! Schematic showing the random orientation of axonemes on thexy plane of the microscope. The dottearrow lines of 0° and 90° denote the two excitation polarizations usedprobe the orientation of the axonemes and associated kinesin molec~Reprinted from Ref. 43 with permission from the Biophysical Society!.


o-nsnge

cts-kssinthth

as

css

chu

inr tsbl

sc

ctono-

fs:

rewsly

sanstie

io

o-l-

or

tic

nzy-se

rterareab-a-tiden-

hek-in-

onde

low-on

tide

yn-as-

hendot-a

e

di-

asby

tic--ith

-in

ralin-iesan

ng


10–100-fold lower than typical reactive fluorophore-tprotein molar ratios used for labeling of single-Cys protei!to ensure stoichiometric labeling of the site; initial labeliof the first Cys increased the local concentration of the sond reactive group of BSR around the second, unreaCys, and allowed BSR cross-linking to generate BSR-kine~labeling efficiency: 60%–75%!. Mass spectrometry of protein digestion products verified the presence of cross-linalthough it is not clear what was the exact fraction of crolinked kinesin in the labeled kinesin product. The spacand exact orientation of Cys side chains was critical, andmargin for error was small; a similar kinesin derivative withe two Cys spaced by six residues~Cys168 and Cys174! didnot result in intramolecular cross-linking. BSR-kinesin wfunctional and polarization-TIRFM@Fig. 2~c!# shows that itbinds to microtubules with the expected [email protected]~d!#. Analysis of microtubule-bound BSR-kinesin dynamiuncovered a previously unknown disordered state of kinein the presence of adenosine-diphosphate~ADP!, and led tonew structural models that describe the translocation menism of kinesin and the chemical–mechanical coupling ding each step.

Analysis of static or dynamic heterogeneity in protesamples~due to the presence of distinct subpopulations oconformational dynamics! using single-pair FRET requirethe incorporation of two complementary, SMFS-compatifluorophores within a single protein. Initially, this seemeasily attainable by generating a pair of appropriately spasurface-exposed Cys residues (Cys1– Cys2– protein),and statistical labeling of the two sites with two distinfluorophores. However, this is true only for dual-Cys prteins with sites of incorporation of the same enviroment, making SMFS an excellent option for studying hmodimeric proteins~symmetric dimers of two identical subunits or protomers! with one surface-exposed Cysper pro-tomer (Cys1– Cys18– protein). Statistical labeling oCys1– Cys18– protein will generate the following specieCys1

D – Cys18A – protein, the equivalent Cys1

A– Cys18D – protein,

the donor-only species Cys1D – Cys18

D – protein ~which canserve as the perfect donor-only control for FRET measuments, especially after photodestruction of one of the tdonor fluorophores!, and the acceptor-only specieCys1

A– Cys18A – protein ~which can serve as an acceptor-on

control!. SMFS differentiates easily amongD –A, D-only,andA-only species, and can use theD –A species for analy-sis of structure and dynamics~with D-only and A-onlypresent as internal controls!. However, homodimeric proteinconstitute a special case; the local environment for two rdomly chosen Cys in other proteins is different, and statical labeling results in the nonequivalent specCys1

D – Cys2A– protein and Cys1

A– Cys2D – protein, which have

to be resolved, either by purification before data acquisit~e.g., using liquid chromatography25,45! or by ‘‘optical’’ pu-rification during data analysis. Both of the purification prcedures are very difficult or protein specific; to our knowedge, this approach has not been used in SMFS.

An elegant general method to incorporate one or mdistinct fluorophores within a single protein involvespeptideligation, the assembly of full-length proteins from synthe


c-edin

s,-

ge

in

a-r-

o

e

ed

---

-o

-i-s

n

e

or biosynthetic peptide fragments.46–50 Peptide ligation en-compasses various techniques that allow chemical or ematic covalent linking of peptides in aqueous solutions. Uof peptides carrying fluorescent probes or other repogroups and modifications yields protein molecules thatsite-specifically labeled at multiple sites. The most estlished version of peptide ligation is the native chemical ligtion, a two-step reaction that occurs between a first pepcontaining aC-terminal thioester and a second peptide cotaining anN-terminal Cys@Fig. 3~a!#. In the first, reversiblestep, theN-terminal thiol of the second peptide attacks telectrophilic center of the thioester of the first peptide, lining the two peptides. In the second, irreversible step, antramolecular transfer reaction generates a peptide b@R1– (C5O) – NH–R2# between the two fragments. Thstriking property of this system is that only theN-terminalCys participates in the second, spontaneous reaction, aling any number of Cys to be present in virtually any positiof the two peptideswithout interference.

If both peptides are prepared using solid-phase pepsynthesis, small proteins or protein domains~up to ;100residues, due to the 50-residue limit for reliable peptide sthesis! can be assembled. Using the conformationallysisted version of peptide ligation,50,51which allows rapid andefficient linking of peptides under conditions that favor tfolded conformations of the assembled peptides, Deniz aco-workers prepared a doubly labeled version of chymrypsin inhibitor 2~CI2!, a 64-residue protein that serves asmodel for the folding of single protein domains.52 Fragment~1-39!CI2 featuring a C-terminal thioester@functionalityR– (C5O)-SR8] was synthesized and labeled at thN-terminus ~position 1! using TMR-succinimidyl ester~anamine-reactive fluorophore!. Fragment~40-64!CI2 was syn-thesized carrying anN-terminal Cys~Cys40!, a prerequisitefor reaction with theC-terminal thioester of the~1-39!CI2fragment. The peptides were ligated under folding contions, and Cy5~an efficient FRET acceptor for TMR! wasincorporated into the resulting~1-64!CI2 using disulfide ex-change~a typical thiol-specific reaction16! of CI2 with anactivated Cy5-disulfide. Donor–acceptor-labeled CI2 wused to observe folded and unfolded subpopulationssingle-pair FRET.

Larger proteins (.100 residues! can be labeled usingpeptide ligation to link short, fluorescently labeled synthepeptides to larger ‘‘recombinant’’~i.e., prepared by biosynthesis in bacteria! proteins. To achieve this, as in fully synthetic peptide ligation, one should generate proteins wC-terminal thioesters and peptides withN-terminal Cys. Togenerate a protein with aC-terminal thioester, chemists borrowed a few ideas from the biological process of protesplicing, the remarkable self-cleaving activity of sevenaturally occurring proteins. In a process resembling an ‘‘verted’’ peptide ligation, certain proteins go through a serof isomerizations and group-transfer reactions to exciseinternal protein fragment~an ‘‘intein’’ ! that is nested be-tween two protein fragments~‘‘exteins’’ !; ligation of the ex-teins yields the final protein product@Fig. 3~b!#. Modulationof the cleavage reaction through protein engineering53–56hasresulted in inteins that promote only the first step of splici


-

p--o.

es


FIG. 3. Principles of peptide ligationand protein splicing.~a! Example of apeptide ligation reaction that yields along peptide product through the initial reaction of aC-terminal thioester~in peptide 1! with the thiol of anN-terminal cysteine~in peptide 2! andsubsequent isomerization. The petides can carry various internal functional groups, since such groups dnot interfere with the ligation reaction~b! Example of protein splicing thatyields a shorter, processed peptidthrough a multistep reaction that leadto the excision of the central portion~‘‘intein’’ ! of the initial peptide or pro-tein. ~Adapted from Ref. 46 with per-mission from Elsevier Science.!

-te

o

a

h

la

fi-

ng

-ein-thee-he

ns-

yl-

and allow formation ofC-terminal thioesters through intermolecular reactions with excess of thiols. Intein-medialabeling was used for site-specific labeling ofEscherichiacoli RNAP for systematic, FRET-based, structural analysisRNAP and RNAP–DNA complexes.57–59 Each of thea andb8 subunits were labeled afterin vivo assembly of RNAPcore enzymes containing the modifieda or b8 subunits; themodified subunits had an intein module for labeling, andaffinity tag ~CBD: chitin binding domain! for capturing themodified RNAP on a solid support@Fig. 4~a!#. While immo-bilized, a reaction of the transient thioester moiety of tmodified RNAP with Cys-fluorescein@Fig. 4~a!, middlepanel# simultaneously cleaved theC-terminal intein, incor-porated aC-terminal fluorescein group, and eluted thebeled RNAP from the solid support@Fig. 4~a!, left-handpanel#. Fluorescein-RNAP was fully functional, and the ef


d

f

n

e

-

ciency and site-specificity of labeling were [email protected]%; Fig. 4~b!#; the protein yield was relatively low(;0.1 mg/L of cultured cells!, but adequate for several ensemble fluorescence and SMFS experiments. FluorescRNAP was used to show that, contrary to the held belief,initiation factor s70 can remain associated with RNAP byond the transition of the enzyme from the initiation to telongation mode of transcription.

Proteins can also be labeled using cell-free RNA tralation systems that covalently attachfluorescent derivativesof the antibiotic puromycinto the C-terminus of the codedprotein ~Fig. 5!. Puromycin inhibits protein synthesis bbinding to theA site~the site where the incoming aminoacytransfer RNA@tRNA# binds! of the ribosome~the protein-synthesis factory of the cell!; it can also covalently link thetranslated protein and the messenger RNA~mRNA!.60 How-


of


FIG. 4. Intein-mediated labeling of RNA polymerase~RNAP!. ~a! Incorporation of fluorescein in RNAP core. Left-hand side; Intein-mediatedC-terminalincorporation of fluorescein using Cys-fluorescein. Center; synthesis of Cys-fluorescein. Right-hand side site-specific incorporation of fluorescein into RNAPcore atb8 1377, b 643 or b 937, anda II 235. CBD, chitin binding domain; F, fluorescein; H6 , hexahistidine.~b! Labeled RNAP core. Denaturingpolyacrylamide electrophoresis gel of RNAP core derivatives labeled atb8 1377,b 643,b 937, ora II 235. Single bands demonstrate the high specificitylabeling.~Reprinted from Ref. 57 with permission from Elsevier Science.!

oftidon

reth

flela

g-rooronn

tdos

m,

oferiond

i-o-ithreses

ed

ive,as

one

la-

-bac-

o-d to

o-llyb-

ever, for mRNAwithout a stop codon~the triplet of nucle-otides in mRNA that provides the signal for terminationprotein synthesis and release of the complete polypepchain!, the ribosome stalls and allows covalent modificatiof the nascent polypeptide chain by puromycin.61,62 If thepuromycin is supplied as a conjugate with a fluorophothen the targeted protein becomes fluorescent. This mewas used to incorporate fluorescein61 and Cy562 @Fig. 5~a!# inproteins, with efficiencies of up to;90%.62 SMFS assays okinesin movement on microtubules showed that Cy5-labekinesin prepared using puromycin behaves similarly to unbeled kinesin,62 reinforcing the notion of the nonperturbincharacter ofC-terminal labeling. Although the yield of labeled protein is lower compared to the intein-mediated ptein yields, puromycin-mediated labeling is well-suited fSMFS analysis of proteins, since the high sensitivitySMFS allows several assays with a simple, 2 h preparatiolabeled protein~Fig. 5!; however, protein characterizatiousing conventional assays becomes complicated due tolow protein yield. The generality of the method depenpartly on the availability of various fluorescent derivativespuromycin; controlled-pore glass with puromycin groupconvenient for solid-phase synthesis of fluorescent purocin derivatives, is commercially available~Glen ResearchSterling, VA!.

Unnatural amino acid mutagenesisalso usesin vitrotranscription/translation for site-specific labelingproteins.63–65Amino acids with side chains carrying reportor functional groups can be incorporated at any positwithin a protein by using synthetic amino-acylated tRNA a


e

,od

d-

-

fof

hesf,y-

n

complementary sequences in the protein-coding DNA;63 ex-tensions of this method allowed protein modificationinvivo.66,67 Incorporation of unnatural amino acids permits drect or indirect introduction of fluorophores. Direct incorpration is performed by using unnatural amino acids wfluorescent side chains; however, the introduced fluorophoare restricted to derivatives of intrinsic protein fluorophor~such as 7-aza-tryptophan, a tryptophan derivative!, and arenot SMFS-compatible. Indirect incorporation is performby using unnatural amino acids with nonproteinogenic~i.e.,absent in naturally occurring proteins! reactive groups, suchas the ketone carbonyl group; such groups allow selectsite-specific labeling in a postsynthetic fashion. This wdemonstrated by the site-specific labeling of a single-ketprotein with fluorescein hydrazide.65 Despite the power ofthe approach, it has not been used widely for preparingbeled proteins mainly due to the very low yields of theinvitro transcription/translation reaction~e.g., large-scaleinvitro reactions to generate a keto-protein resulted in;10mgprotein,64 .100-fold lower than the typical amount of recombinant proteins that are prepared from a small-scaleterial culture!.

A popular way to label large multiprotein or nucleoprtein complexes is to label components of the complex anassemble the complex byin vitro reconstitution from purifiedcomponents. For example, a hypothetical four-subunit prtein with subunit composition ABCD can be site-specificalabeled at subunits A and B by site-specific labeling of suunit A with fluorophore F1~to prepare AF1); site-specificlabeling of subunit B with fluorophore F2~to prepare BF2);


nla-

theful

nla-al

--m-eran-

ofto

tt

thatP-blee

r-rat-

isforrep-

res

-sheo-singg

ro-ll

aly

an

ofnd.


FIG. 5. Preparation of puromycin-labeled kinesin for single-molecule ansis of motility. ~a! Structure of Cy5-puromycin conjugate.~b! Schematic ofthe procedure. Messenger RNA without a stop codon is prepared usinginvitro transcription system containing the gene for kinesin; addition of aninvitro translation mix and Cy5-puromycin and incubation allow formationC-terminally-labeled kinesin. After purification, the enzyme is diluted aused for motility assays performed on surface-immobilized microtubules~c!Fluorescent kinesin molecules moving along microtubules~top panel! arevisualized using TIRFM. Scale bar, 5mm. @Reprinted from Ref. 62 withpermission from the FEBS society.#


and mixing labeled subunits AF1 and BF2 with purified, un-labeled subunits C and D~to prepare the desired AF1BF2CD).This can also be achieved by subunit exchange of an ubeled component with excess of a labeled component~e.g.,ABCD1AF1→AF1BCD1A). Once more, it is critical to es-tablish that fluorescent labeling does not adversely affectassembly of the complex. This method is especially usefor large proteins, such as myosin39,40 and RNAP.57,58 Formyosin, the regulatory light chain~RLC! was site-specifically labeled and subsequently exchanged with ubeled RLC, allowing probing of myosin conformationstates. For RNAP, subunits70 was labeled with thiol-reactivefluorophores and incubated with unlabeled RNAP core~sub-unit compositionb8ba2v) to form RNAP holoenzyme~fors→DNA FRET experiments within RNAP–DNAcomplexes57,58!, or with fluorescein-labeled RNAP core~forRNAP core→s FRET experiments within RNAP holoenzyme or RNAP–DNA complexes57,58!. The method has revealed the existence of subpopulations of RNAP–DNA coplexes with different behavior relative to promoter escap59

and allowed investigation of asynchronous processes in tscription.

The reconstitution approach is not limited to assemblycomplexes using labeled individual subunits, but extendsassembly of complexes using labeledfragmentsof individualsubunits~‘‘split subunits’’!, thus allowinginternal labelingofvery large protein subunits. In an illustration of this concep~Fig. 5!, the b subunit of RNAP was internally labeled aeach of two positions~643 and 937! by preparingb~1-643!-F/ andb~1-937!-F ~using intein-mediated labeling!, andby reconstituting@b~1-643!-F/b~644-1342!#-RNAP core and@b~1-937!-F/~938-1342!#-RNAP corein vitro.57 This comple-mented a panoply of site-specific labeling methodologiesresulted in incorporation of fluorophores at 4 sites in RNAcore, 20 sites ins70, and 3 sites on DNA, allowing measurement of a large set of interprobe distances by ensemFRET and yielding a detailed picture of RNAP holoenzymand the RNAP–DNA open complex in solution.

V. SITE-SPECIFIC LABELING OF PROTEINSIN LIVING CELLS

In vitro analysis of biological processes has led to fomulation of elaborate models about the mechanisms opeing in the living cell. In vivo analysis of such processesessential for the validation of the proposed models, andthe understanding of additional processes that cannot belicated in vitro due to inability to account for all interactingcomponents. Observation of SMFS-compatible fluorophoin living cells is more complicated thanin vitro, primarilydue to the cellular autofluorescence~fluorescence background in the cell due to naturally occurring fluorophore!,the difficult control of the fluorophore concentration, and tmultitude of interactions that can interfere with the fluorphore detection. There are three major strategies for usite-specifically labeled proteins to perform SMFS in livincells.

In the first and most popular strategy, the labeled pteins are preparedin vitro, and are introduced into the ce

-


catoF

-

ic

uwi

,e,n

inr

ulio

iangy

g

-

e-caellsina

ai

s-

fo

np

cetere

nla

ar

is

rba-

ithess,g

ein

FMthenm

f-ointsrrow-spots

the


through a variety of cell-loading techniques, such as endotosis, permeabilization, or microinjection. This strategy hallowed SMFS observations of the epidermal growth fac~EGF! dimerization by monitoring FRET between Cy3-EGand Cy5-EGF;68 imaging of single b-galactosidase molecules~with ;5 Alexa-488 fluorophores per molecule! in thecell nucleus;69 and detection of diffusing proteins and nucleacids ~as fluorescence bursts! within living cells.70 The invitro-labeled biomolecules can also be large structures, sas virus particles. Labeling of adeno-associated virusesa single far-red fluorophore~Cy5! allowed real-time TIRFMtrajectories of the viral infection pathway in living cellsstarting with the initial contact of virus with the cell surfaccontinuing with its journey through the cytoplasm, and cocluding with invasion of the nucleus.15

In the second strategy, a high-affinity protein–ligandteraction is used forin situ labeling of specific sequences ostructures in the cell. This task is much more difficult thaninvitro labeling, since the site-specificity of the reagent shobe exceptional to avoid nonspecific binding or incorporatto the tens of thousands of different biomolecules foundthe cell; the labeling reagent should also be nontoxiccell-permeant. This strategy was used for labeling of sinion channels on the membrane of Jurkat cells, where Clabeled toxin targeted individual channels.71 A variation ofthis strategy allows covalent labeling of proteins in livincells by targeting a 4-Cysa-helical motif incorporated to thetargeted protein.72 The validity of this strategy has been demonstrated with the fluorescein-related reagent FLASH~fluo-rescein arsenical helix binder!, and has been extended rcently to a second fluorophore. FLASH-based labelingbe used in conjunction with genetically-encoded fluorescproteins for real-time FRET measurements in living ceHowever, this strategy is limited to two fluorophores, and,some cell lines, is not applicable due to nonspecific intertions with other Cys-rich proteins.73 Another variation,termed ‘‘epitope tagging,’’ uses expression of single-chantibodies at specific intracellular locations~as receptor-antibody fusion proteins! and labeling of specific sitethrough high-affinity interactions of the antibody with fluorescent conjugates of its ligand.74 The last two variations ofthe in situ labeling strategy have not been used yetSMFS.

In the third strategy, the fluorophore is a genetically ecoded fluorescent protein, such as the green fluorescenttein ~GFP! from the jellyfishAequorea victoria.75 Accordingto this method, the DNA sequence coding for GFP is plaimmediately adjacent to the sequence coding for the proof interest. During biosynthesis, the protein will be prepaas a ‘‘GFP-fusion’’ protein~i.e., a hybrid protein containingGFP and the protein of interest!, and thus, it will be fluores-cent. After the DNA is introduced to the cells, the cells sythesize the GFP fusion, which then travels to the cellustructures or compartments that it normally occupies. Vants of GFP~blue, cyan, and green/yellow! or other geneti-cally encoded fluorescent proteins can also be used, allowflexibility in terms of excitation and emission wavelengthand generating genetically encodedD –A pairs for in vivoFRET. However, use of GFP has its limitations;72 it can only


y-sr

chth

-

-

dnnd

le5-

nnt.

c-

n

r

-ro-

dind

-ri-

ng,

be introduced at the protein termini and can cause pertutions due to its large size~238 amino acids in length!. Fur-thermore, its limited brightness and its spectral overlap wcellular autofluorescence complicates SMFS. Nonethelthe first observation of single GFP-fusion proteins in livincells was reported recently76 ~Fig. 6!. Using TIRFM @Fig.6~a!#, single copies of a GFP fusion of the adhesion prot

FIG. 6. Single-molecule imaging of GFP-cadherin in living cells.~a! Sche-matic of the chamber-objective assembly used for the objective-type TIRmeasurements. The cell membrane facing the coverslip is illuminated byevanescent wave generated by the total internal reflection of the 488laser excitation beam.~b! Image of membrane showing immobile and difusing molecules exhibiting GFP fluorescence. The single arrowhead pto a possible monomeric GFP-cadherin species, whereas the double ahead points to a possible dimeric GFP-cadherin species. The dimmerare attributed to diffusing species Scale bar, 1mm. ~c! Single-step pho-tobleaching of monomeric GFP-cadherin species. The arrow points toexact timing of photobleaching.~Reprinted from Ref. 76 with permissionfrom the Biophysical Society.!


ofstadralipecef

ehibfchopiio

ifiit-y,o

pr

ninnfo

l-vetioreiofosein

thtyei

er0opa

ceo-s

r

fors ofe inen--il-S-lsoS-iedlife-

nceom-

al--tar-

of

s ofkebyies-y–calon

dyuse

onlye

ise

tic

-that

ds

asss-es,es

s for

fontorialtes


cadherin~E-cad-GFP! were observed on the membranefibroblast cells, and their oligomerization status was invegated. To allow the observation of single copies of E-cGFP, the cell line was optimized to minimize the concenttion of biosynthesized E-cad-GFP, and the coversproximal membrane was exposed to the evanescent wava few seconds to reduce the concentration of the fluoresspecies~by photobleaching!, thus allowing observation oindividual, well-separated fluorescent spots@Fig. 6~b!#. His-tograms of fluorescence intensity for individual spots showdistributions of quantized fluorescence intensity; spots exiting the basic quantized intensity~presumably monomers oE-cad-GFP! also display single-step photobleaching, whiserves as a diagnostic test for observing a single fluorophBased on these results, an oligomerization-induced trapmodel was proposed for the mechanism of cell-adhesstructure formation.

VI. NEW FLUOROPHORES AND NEW CHEMISTRIESFOR IN VITRO AND IN VIVO LABELING

Despite the battery of available strategies, site-speclabeling with SMFS-compatible fluorophores is still a liming step in establishing SMFS assays. Since the popularitSMFS for analysis of biomolecules is growing rapidly1

SMFS is likely to be used extensively for the analysisnumerous human proteins. However, the vast number ofteins found in the human proteome~30 000–40 000!, thelarge number of multiprotein and other multicomponecomplexes, and the intricate network of transient proteprotein interactions involved in the structural, regulatory, acommunication network of the cell, underlines the neednew fluorophores and new labeling strategies.

Fluorescent semiconductor nanocrystals~NC; or quan-tum dots! comprise a novel family of fluorophores for labeing biomolecules.77,78 NC possess several advantages oconventional organic fluorophores, such as broad excitaspectra, narrow and tunable emission spectra, long fluocence lifetimes, and high resistance to photodestructHowever, bioconjugation and cell-targeting chemistriesNC are still immature, and the intermittency of their fluorecence complicates their use. However, since NC definparticularly active area of research, it is certain that excitfuture developments are on the horizon.

Another challenge for both physicists and chemists isexploration of the UV wavelength range for SMFS. Abilito operate in the UV will allow smaller, less perturbativfluorophores to be used, and will extend the available emsion range for SMFS, allowingsimultaneousprobing of alarger set of fluorophores. Currently, SMFS uses only vbright fluorophores with emissions confined in the 450–7nm spectral range. SMFS in the UV requires the develment of brighter UV fluorophores with large Stokes shifts,well as reliable, intense, and inexpensive UV laser sourInnovative ways to amplify and to detect the faint signalintrinsic protein fluorophores~such as tryptophan and its redshifted analogs!, or of DNA-structure-sensitive fluorophore~such as 2-aminopurine@a reporter of DNA double-helicity#or intercalating dyes! will create further opportunities fomultiparametric analysis of complex systems.


i----fornt

d-

re.ngn

c

of

fo-

t–dr

rns-n.r-a

g

e

s-

y0-

ss.f

It is not an exaggeration to say that the best probesSMFS might already be on the shelves, since thousandfluorophores have never been screened for performancSMFS ~although they have been evaluated for use insemble fluorescence assays!. High-throughput characterization of the available libraries of fluorophores for compatibity with SMFS could generate a new category, the ‘‘SMFoptimal’’ probes. Such a comprehensive search could agenerate rules that will allow the rational design of SMFcompatible fluorophores. This effort should be accompanby a quest for reagents that both extend photobleachingtime of fluorophores and quench triplet states.

Site-specific labeling strategies of greater convenieand speed should also be pursued. One way to label recbinant proteinsin vitro or in vivo is to target ‘‘affinity tags’’~short sequences introduced usually at protein termini tolow rapid purification!, similar to the FLASH-based sitespecific labeling strategy. Another sequence that can begeted is the amino acid sequence His6 ~‘‘hexahistidine tag’’!that binds tightly to transition-metal complexes. TargetingHis6 by (Ni21-nitrilotriacetic acid)2-fluorophore conjugateswas reported recently;30 however, the interaction of His6

with the fluorophore conjugates had dissociation constant.300 nM, requiring a large increase in the affinity to mathe approach useful for SMFS. This might be possibleusing the concept outlined in the construction of antibodwith ‘‘infinite affinity.’’ 79 During this remarkable feat of protein engineering, the high-affinity, noncovalent antibodligand interaction was used to increase the effective loconcentrations of complementary reactive groups placedeach component, resulting in covalent linkage of antiboand ligand and preventing dissociation. In general, to‘‘affinity-labeling’’ for labeling of living cells, it is necessaryto devise chemistries that render the reagent fluorescentafter incorporation to the site of interest, thus minimizing thbackground of unincorporated reagent that will otherwoverwhelm the SMFS signal.

We conclude with a note on the potential of the genecode expansion~by unnatural amino acid mutagenesis! torevolutionize in vivo site-specific labeling. Wang and coworkers have recently developed a sophisticated strategycombines novel nucleic acids~tRNA molecules! and engi-neered enzymes~amino-acyl tRNA synthetases! for in vivosite-specific incorporation of two unnatural amino aci~L-3-@2-napthyl#-alanine66 or O-methyl-L-tyrosine67! in pro-teins. If this breakthrough work is extended to encompside chains with SMFS-compatible fluorophores, it will esentially miniaturize the genetically encoded fluorophorby reducing the number of additional amino acid residurequired for in vivo fluorescence from 238~for GFP! to 1~the single unnatural amino acid!. This advance will helpunravel the deep secrets of the cell and open new avenueexciting research.

ACKNOWLEDGMENTS

The authors thank So¨ren Doose for the critical reading othe manuscript, Dr. Richard H. Ebright for helpful discussiand access to unpublished data, and Joanne Tang for ediassistance. This work was funded by the National Institu


.S

T

an

K.

T

T

lag

C

m

e

H

T.

nd

a,

,

cts

tu

, T

dy

Sc

ruct.

s. J.

re

ate

n,cad.

Q.

.

.

nd

ett.

T.

io-

ll,

and

s,

P.

ci.


of Health, Grant No. 1 R01 GM65382:01, and by the UDepartment of Energy, Grant No. DE-FG03-02ER63339.

1A. M. Kelley, X. Michalet, and S. Weiss, Science292, 1671~2001!.2Y. Harada, T. Funatsu, M. Tokunaga, K. Saito, H. Higuchi, Y. Ishii, andYanagida, Methods Cell Biol.55, 117 ~1998!.

3W. P. Ambrose, P. M. Goodwin, J. H. Jett, A. Van Orden, J. H. Werner,R. A. Keller, Chem. Rev.99, 2929~1999!.

4S. Nie and R. N. Zare, Annu. Rev. Biophys. Biomol. Struct.26, 567~1997!.

5S. Weiss, Nat. Struct. Biol.7, 724 ~2000!.6S. Weiss, Science283, 1676~1999!.7W. E. Moerner and M. Orrit, Science283, 1670~1999!.8S. Xie, Single Molecules2, 229 ~2001!.9R. Kuhnemuth and C. A. M. Seidel, Single Molecules2, 251 ~2001!.

10K. Adachi, R. Yasuda, H. Noji, H. Itoh, Y. Harada, M. Yoshida, andKinosita, Proc. Natl. Acad. Sci. U.S.A.97, 7243~2000!.

11R. D. Vale, T. Funatsu, D. W. Pierce, L. Romberg, Y. Harada, andYanagida, Nature~London! 380, 451 ~1996!.

12D. W. Pierce, N. Hom-Booher, and R. D. Vale, Nature~London! 388, 338~1997!.

13Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama, andYanagida, Biophys. J.76, 709 ~1999!.

14X. Zhuang, L. E. Bartley, H. P. Babcock, R. Russell, T. Ha, D. Herschand S. Chu, Science288, 2048~2000!.

15G. Seisenberger, M. U. Ried, T. Endress, H. Buning, M. Hallek, andBrauchle, Science294, 1929~2001!.

16A. Waggoner, Methods Enzymol.246, 362 ~1995!.17R. Tsien and A. Waggoner, inHandbook of Biological Confocal Micros-

copy, edited by J. D. Pawley~Plenum, New York, 1995!, pp. 267–279.18T. Ha, Methods25, 78 ~2001!.19C. G. Hubner, A. Renn, I. Renge, and U. P. Wild, J. Chem. Phys.115,

9619 ~2001!.20F. Wilkinson, D. J. McGarvey, and O. F. Olea, J. Phys. Chem.98, 3762

~1994!.21C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, Anal. Che

70, 2651~1998!.22M. E. Dickinson, G. Bearman, S. Tilie, R. Lansford, and S. E. Fras

BioTechniques31, 1272~2001!; 31, 1274~2001!.23P. R. Selvin, Nat. Struct. Biol.7, 730 ~2000!.24A. N. Kapanidis, Y. W. Ebright, R. D. Ludescher, S. Chan, and R.

Ebright, J. Mol. Biol.312, 453 ~2001!.25M. P. Lillo, J. M. Beechem, B. K. Szpikowska, M. A. Sherman, and M.

Mas, Biochemistry36, 11261~1997!.26R. E. Dale and J. Eisinger, Proc. Natl. Acad. Sci. U.S.A.73, 271 ~1976!.27R. E. Dale, J. Eisinger, and W. E. Blumberg, Biophys. J.26, 161 ~1979!.28T. J. Ha, A. Y. Ting, Liang, A. A. Deniz, D. S. Chemla, P. G. Schultz, a

S. Weiss, Chem. Phys.247, 107 ~1999!.29P. Wu and L. Brand, Anal. Biochem.218, 1 ~1994!.30A. N. Kapanidis, Y. W. Ebright, and R. H. Ebright, J. Am. Chem. Soc.123,

12123~2001!.31H. P. Lu, L. Xun, and X. S. Xie, Science282, 1877~1998!.32A. Ishijima, H. Kojima, T. Funatsu, M. Tokunaga, H. Higuchi, H. Tanak

and T. Yanagida, Cell92, 161 ~1998!.33T. Ha, A. Y. Ting, J. Liang, W. B. Caldwell, A. A. Deniz, D. S. Chemla

P. G. Schultz, and S. Weiss, Proc. Natl. Acad. Sci. U.S.A.96, 893 ~1999!.34R. P. Haugland,Handbook of Fluorescent Probes and Research Produ,

8th ed.~2002!.35P. R. Selvin, Methods Enzymol.246, 300 ~1995!.36T. A. Kunkel, K. Bebenek, and J. McClary, Methods Enzymol.204, 125

~1991!.37T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, Na

~London! 374, 555 ~1995!.38R. Yamasaki, M. Hoshino, T. Wazawa, Y. Ishii, T. Yanagida, Y. Kawata

Higurashi, K. Sakai, J. Nagai, and Y. Goto, J. Mol. Biol.292, 965~1999!.39D. M. Warshaw, E. Hayes, D. Gaffney, A. M. Lauzon, J. Wu, G. Kenne

K. Trybus, S. Lowey, and C. Berger, Proc. Natl. Acad. Sci. U.S.A.95,8034 ~1998!.

40I. Sase, H. Miyata, S. Ishiwata, and K. Kinosita, Jr., Proc. Natl. Acad.U.S.A. 94, 5646~1997!.


.

.

d

.

.

,

.

.

r,

.

re

.

,

i.

41J. E. Corrie, B. D. Brandmeier, R. E. Fergusonet al., Nature~London!400, 425 ~1999!.

42H. Sosa, E. J. Peterman, W. E. Moerner, and L. S. Goldstein, Nat. StBiol. 8, 540 ~2001!.

43E. J. Peterman, H. Sosa, L. S. Goldstein, and W. E. Moerner, Biophy81, 2851~2001!.

44F. J. Kull, E. P. Sablin, R. Lau, R. J. Fletterick, and R. D. Vale, Natu~London! 380, 550 ~1996!.

45M. Sinev, P. Landsmann, E. Sineva, V. Ittah, and E. Haas, BioconjugChem.11, 352 ~2000!.

46G. J. Cotton and T. W. Muir, Chem. Biol.6, R247~1999!.47T. W. Muir, D. Sondhi, and P. A. Cole, Proc. Natl. Acad. Sci. U.S.A.95,

6705 ~1998!.48K. Severinov and T. W. Muir, J. Biol. Chem.273, 16205~1998!.49J. P. Tam, J. Xu, and K. D. Eom, Biopolymers60, 194 ~2001!.50P. E. Dawson and S. B. Kent, Annu. Rev. Biochem.69, 923 ~2000!.51P. E. Dawson, T. W. Muir, I. Clark-Lewis, and S. B. Kent, Science266,

776 ~1994!.52A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Marti

D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, Proc. Natl. ASci. U.S.A.97, 5179~2000!.

53M. Q. Xu and F. B. Perler, EMBO J.15, 5146~1996!.54S. Mathys, T. C. Evans, I. C. Chute, H. Wu, S. Chong, J. Benner, X.

Liu, and M. Q. Xu, Gene231, 1 ~1999!.55T. C. Evans, Jr., J. Benner, and M. Q. Xu, J. Biol. Chem.274, 3923

~1999!.56S. Chong, F. B. Mersha, D. G. Combet al., Gene192, 271 ~1997!.57V. Mekler, E. Kortkhonjia, J. Mukhopadhyay, J. Knight, A. Revyakin, A

Kapanidis, W. Niu, Y. W. Ebright, R. Levy, and R. H. Ebright, Cell108, 1~2002!.

58J. Mukhopadhyay, A. N. Kapanidis, V. Mekler, E. Kortkhonjia, Y. WEbright, and R. H. Ebright, Cell106, 453 ~2001!.

59A. N. Kapanidis, T. Lacoste, V. Mekler, J. Mukhopadhyay, S. Weiss, aR. H. Ebright, Biophys. J.80, 478A ~2001!.

60N. Nemoto, E. Miyamoto-Sato, Y. Husimi, and H. Yanagawa, FEBS L414, 405 ~1997!.

61N. Nemoto, E. Miyamoto-Sato, and H. Yanagawa, FEBS Lett.462, 43~1999!.

62J. Yamaguchi, N. Nemoto, T. Sasaki, A. Tokumasu, Y. Mimori-Kiyosue,Yagi, and T. Funatsu, FEBS Lett.502, 79 ~2001!.

63D. Mendel, V. W. Cornish, and P. G. Schultz, Annu. Rev. Biophys. Bmol. Struct.24, 435 ~1995!.

64V. Cornish, K. M. Hahn, and P. G. Schultz, J. Am. Chem. Soc.118, 8150~1996!.

65V. W. Cornish, D. R. Benson, C. A. Altenbach, K. Hideg, W. L. Hubbeand P. G. Schultz, Proc. Natl. Acad. Sci. U.S.A.91, 2910~1994!.

66L. Wang, A. Brock, and P. G. Schultz, J. Am. Chem. Soc.124, 1836~2002!.

67L. Wang, A. Brock, B. Herberich, and P. G. Schultz, Science292, 498~2001!.

68Y. Sako, S. Minoghchi, and T. Yanagida, Nat. Cell Biol.2, 168 ~2000!.69T. Kues, R. Peters, and U. Kubitscheck, Biophys. J.80, 2954~2001!.70T. A. Byassee, W. C. Chan, and S. Nie, Anal. Chem.72, 5606~2000!.71G. J. Schutz, V. P. Pastushenko, H. J. Gruber, H-G. Knaus, B. Pragl,

H. Schindler, Single Molecules1, 25 ~2000!.72B. A. Griffin, S. R. Adams, and R. Y. Tsien, Science281, 269 ~1998!.73K. Stroffekova, C. Proenza, and K. G. Beam, Pfluegers Arch.442, 859

~2001!.74J. Farinas and A. S. Verkman, J. Biol. Chem.274, 7603~1999!.75R. Y. Tsien, Annu. Rev. Biochem.67, 509 ~1998!.76R. Iino, I. Koyama, and A. Kusumi, Biophys. J.80, 2667~2001!.77M. Bruchez, Jr., M. Moronne, P. Gin, S. Weiss, and A. P. Alivisato

Science281, 2013~1998!.78X. Michalet, F. Pinaud, T. D. Lacoste, M. Dahan, M. P. Bruchez, A.

Alivisatos, and S. Weiss, Single Molecules2, 261 ~2001!.79A. J. Chmura, M. S. Orton, and C. F. Meares, Proc. Natl. Acad. S

U.S.A. 98, 8480~2001!.80A. N. Kapanidis, R. H. Ebright, and S. Weiss~unpublished!.


fluorescent probes and bioconjugation chemistries for ...€¦ · tronic state.3,4,7 a list of...

Documents