Sumida Forsythe Pusey J Cryst Growth 232 2001 308

Journal of Crystal Growth 232 (2001) 308–316

Preparation and preliminary characterization of crystallizingfluorescent derivatives of chicken egg white lysozyme

John P. Sumidaa, Elizabeth L. Forsythea, Marc L. Puseyb,*aUSRA, 4950 Corporate Drive, Suite 100, Huntsville, AL 35806, USA

bBiophysics SD48, NASA/MSFC, Huntsville, AL 35812, USA

Abstract

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins areintrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivativesadded as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have preparedfluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the proteinmolecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to theside chain carboxyl of Asp101 using a carbodiimide coupling procedure. Asp101 lies within the active site cleft, and it isbelieved that the probes are ‘‘buried’’ within that cleft. Lucifer yellow and EDANS probes with iodoacetamide reactivegroups have been bound to His15, located on the ‘‘back side’’ of the molecule relative to the active site. All thederivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused ondetermination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenchingby added solutes. Quenching studies show that, as postulated, the Asp101–bound probes are partially sheltered from thebulk solution by their location within the active site cleft. Probes bound to His15 have quenching constants about equalto those for the free probes, indicating that this site is highly exposed to the bulk solution. # 2001 Elsevier Science B.V.All rights reserved.

Keywords: A1. Biocrystallization; A1. Characterization; A1. Nucleation; B1. Biological macromolecules; B1. Lysozyme; B1. Proteins

1. Introduction

Fluorescence, one of the most powerful techni-ques available for studying proteins and theirinteractions in solution, has only occasionally beenused in protein crystal growth studies [1,2].Intrinsic fluorescing amino acids generally cannotbe used due to the high protein concentrationsemployed in crystallization experiments. Fluores-

cent techniques, relative to absorption methods,use low concentrations of the probe species to keepwithin a linear response region. Protein concentra-tions are typically high in crystal nucleation andgrowth studies. A practical way around theseconflicting requirements is to covalently attach afluorescent probe to a subpopulation of theprotein molecules.

For this strategy to be effective the derivatizedmolecules must behave ‘‘normally’’ in the crystalnucleation and growth process, i.e., the probe mustnot participate in that process. Such an approachhas been shown to work with ribonuclease [1],

*Corresponding author. Tel.: +1-256-544-7823; fax:+1-256-544-6660.

E-mail address: [email protected] (M.L. Pusey).

0022-0248/01/$ - see front matter # 2001 Elsevier Science B.V. All rights reserved.PII: S 0 0 2 2 - 0 2 4 8 ( 0 1 ) 0 1 0 6 1 - 2

where the probe N-[[(iodoacetyl)amino]ethyl]-5-napthylamine-1-sulfonic acid has been attachedsuch that it lies within the active site cleft.Subsequent X-ray crystallographic studies con-firmed the location of the probe [2].

Pan and Berglund [3] carried out fluorescenceanisotropy measurements using lysozyme withpyrene butyric acid bound to the N-terminalamine. Time resolved measurements showed thatthe probe was not rigidly fixed in place and had asegmental motion. Once this motion was takeninto account, the rotational motion of the proteincould be independently analyzed. In the case ofribonuclease the probe was rigidly bound to theprotein and no correction to the data forindependent probe motion was required.

We have used a carbodiimide procedure pre-viously described [4–6] to covalently attach theprobes lucifer yellow (LY) and 5-((2-amino-ethyl)amino)napthalene-1-sulfonic acid (EDANS)to Asp101 of lysozyme. The His15 group, located onthe back of the protein relative to the active site,can be reacted with probes containing an iodo-acetamide reactive group [7–9]. Using this ap-proach, we have bound lucifer yellow iodoaceta-mide (LYI) and N-[[(iodoacetyl)amino]ethyl]-5-napthylamine-1-sulfonic acid (IEDANS) to His15.In all cases the derivatized proteins have beenshown to crystallize. Herein, we report on thepreparation and preliminary fluorescent and crys-tallographic characterization of these derivatives.

2. Materials and methods

Chicken egg white lysozyme, (CEWL, Sigma)was purified by cation exchange chromatographyand recrystallized as previously described [10]. Therecrystallized protein was dialyzed against dH2Oprior to derivatization. Unless otherwise noted allchemicals were of reagent grade or better. Thefluorescent probes lucifer yellow CH lithium salt(LY), 5-((2-aminoethyl)amino)napthalene-1-sulfo-nic acid, (EDANS), lucifer yellow iodoacetamide(LYI), and N-[[(iodoacetyl)amino]ethyl]-5-napthy-lamine-1-sulfonic acid (IEDANS), were fromMolecular Probes. 1-Ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDAC) was from Aldrich

Chemical Co. Sodium iodide and 2-picolylaminewere from Sigma. Potassium iodide and primulinewere from Aldrich. Cobalt chloride, thalliumchloride, cesium chloride, trifluoroacetamide, and2,2,2-trichloroethanol were from Fluka. Acryla-mide was from BioRad, and trypan blue was fromFlow Laboratories.

2.1. CEWL derivatization

Derivatization reactions of Asp101 were carriedout using the method of Yamada et al. [6]. To100ml of 10mg/ml lysozyme solution in dH2Owere added 10–100mg of the probe to be attached.The protein was kept in excess relative to the probeto maximize the amount of probe bound and favorreaction at a single site. All reactions wereperformed with stirring under reduced light levels.After adding the probe, the solution pH wasadjusted to 5.0 with dilute HCl or NaOH asneeded. Solid EDAC was then added at an8 : 1molar ratio, EDAC :CEWL, and the pHmaintained at 5.0� 0.02 by addition of diluteHCl or NaOH as needed. After 1–2 h the reactionmixture was covered with parafilm and stirringcontinued in the dark an additional 12–16 h atroom temperature.

Derivatization of His15 was carried out using themethod previously described for ribonuclease [11].To 0.5 g of CEWL at �10mg/ml in 0.1M Naphosphate buffer, pH 5.2, is added 100mg ofreactive probe (IEDANS or LYI). The additionand subsequent reaction are carried out withstirring in the dark at room temperature for 40 h.Then 10 ml of mercaptoethanol were added and thesolution was stirred for 2 h, to take up anyunreacted probe.

2.2. Purification of the fluorescent labeledlysozyme

The reaction mixture was first dialyzed at roomtemperature in the dark against 0.25M NaCl tofacilitate removal of excess un-reacted or non-covalently bound probe, and then dialyzed againstdH2O. Following dialysis, the protein solution waschromatographed on a BioPrep CM cation ex-change column (2.5� 50 cm2) equilibrated with

J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316 309

0.05M sodium phosphate, pH 7.0 at roomtemperature. After loading the protein onto thecolumn it was briefly washed with equilibratingbuffer, then developed with a gradient of 0.35–0.65M NaCl at a flow rate of 3.0ml/min. with atotal gradient volume of 1200ml. Fractions of24ml size were collected, and the protein andappropriate probe absorptivities determined foreach fraction. Additional purification, if needed,was carried out using a semi-preparative HPLCcation exchange column (HEMA-IEC CM 10 mmcolumn, 22.5� 250mm2). The peaks were col-lected, and those with derivatized protein at anappropriate probe : protein ratio were pooled,dialyzed against dH2O, lyophilized, and stored at�208C until use.

2.3. Steady state absorption

UV/V is absorption spectra were obtained usinga Hewlett Packard 8425A diode array spectro-photometer. All absorption samples were taken inquartz cuvettes. Free probe and derivatizedprotein samples were blanked against the 0.05MTris buffer used to make up the sample.

2.4. Fluorescence characterization

Steady state and frequency domain fluorescencemeasurements were made with an ISS K2 digitalspectrofluorometer. Rhodamine B in conjunctionwith a neutral density filter in the referencechannel was used as a quantum counter for steadystate measurements. For all measurements a 2mmslit width was used in the excitation channel anda width of 1.0 or 0.5mm in the emission channel,depending upon the sample. Quantum yielddeterminations were made using the comparativemethod with quinine sulfate in 0.1M H2SO4 as thereference. Optical densities for fluorescence workwere maintained below 0.1 at the excitation(absorption maximum) wavelength in order toavoid inner filter effects or significant self-absorp-tion of sample fluorescence. All solutions werebubbled with argon prior to measurement.

Lifetime measurements were made with theinstrument in the lifetime acquisition mode, andthe data collected using the fastscan feature. Data

were collected by measuring the modulation andphase difference between the excited and emittedlight as a function of the excitation modulationfrequency over the range of 2–200MHz. Measure-ments were made using the appropriate long passfilter in the emission path. A dilute glycogensolution was used as a reference scatterer for thelifetime determinations. Frequency domain dataanalyses were carried out using the GlobalsUnlimited software package.

2.5. Crystallization

Crystallizations were done using the sitting dropvapor diffusion method. The reservoir solutionwas 0.05M tris-HCl buffer, 7% NaCl, pH 7.5. Wehave previously shown that tetragonal crystals canbe readily grown in this pH range once the proteinhas been purified [10]. Crystallizations were alsocarried out at 0.1M sodium acetate, 5% NaCl,pH 4.6. Lyophilized protein was dissolved intodH2O to a concentration of 25mg/ml, and serialdilutions of this solution were mixed in a 1 : 1 ratiowith reservoir buffer for the crystallization solu-tion. The sitting drop plates were kept in anincubator maintained at 208C.

3. Results

3.1. Preparation and purification of the modifiedlysozyme

All of the probes absorb at 280 nm, and thecalculated concentration and binding ratio dataneed to be corrected for this absorption. The probeabsorption maximum: 280 nm absorption ratioswere determined for the un-reacted probes indH2O and are given in Table 1. The ratios weredetermined on the basis of the probe absorptivitiesgiven in the Molecular Probes catalogue. Themolar absorptivity of CEWL at 280 nm wascalculated to be 37� 103, based upon a massabsorptivity of 26.4 [12].

Upon completion of the probe binding reaction,the solutions were first dialyzed against 0.25MNaCl and then against distilled water. If the

J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316310

solution was cloudy after dialysis, it was subse-quently clarified by filtration through a 0.45 mmmembrane. The first chromatographic purificationwas on a BioPrep CM column, with all of thereaction mixture being loaded onto the column.All chromatography steps were performed at roomtemperature to avoid crystallization of the proteinon the column or in the fractions. Correctedprobe : protein ratios were calculated for eachfraction, and those peaks having the highest ratios41 were separately pooled, dialyzed againstdistilled water, and concentrated by ultrafiltration.The concentrated protein was subsequently re-chromatographed on a HEMA-IEC CM column.The peak derivative-labeled fractions were pooled,again dialyzed against distilled water, concen-trated, lyophilized and stored at �208C.

3.2. Absorption characterization of the derivatives

Steady state absorption spectra were obtainedfor free EDANS, LY, and CEWL, and for theHis15 and Asp101 derivatives. The absorptionspectra were obtained in 0.05M Tris buffer, 7%NaCl, pH 7.5. In the case of the free EDANS threeabsorption peaks are found at 210, 252, and336 nm. For free LY three major absorption peaksare found at 230, 280, and 428 nm.

EDANS bound at Asp101 exhibits strong andbroad absorption peaks with maxima at 212 and282 nm, where both EDANS and lysozymeabsorb. Since Asp101 is within the active site cleftof the protein, interaction between the probe andprotein is expected. Fig. 1 shows the spectrum ofthe derivatized protein and the sum spectrum of

the free probe and protein, representing a linearcombination of their absorption spectra. Both the253 and 336 nm bands of the free probe are redshifted by 10 nm in the derivatized protein. In thecase of LY (not shown), the sum spectra isessentially equivalent to that of the derivatizedprotein, indicating little perturbation of either theprotein or probe absorption by covalent attach-ment within the active site cleft.

The absorption spectra of the His15 derivativesof IEDANS and LYI were also examined withsimilar results. For the IEDANS derivative aredshift is observed, analogous to that for theAsp101 derivative. In this case the shift wassomewhat less, about 2 nm, but broadened. ForLYI no shifts were observed and, as for theAsp101 derivative, the absorption spectrum of

Fig. 1. Absorption spectrum of underivatized lysozyme, freeEDANS, the sum spectrum of lysozyme and free EDANS, andof the Asp101-bound EDANS–CEWL.

Table 1Measured properties of the prepared derivatives of lysozymea

Derivative Location Abs. l maximum(nm)

Abs. ratio 280=lmax(free probe)

Emission maximum l(nm)

Probe quantumyield

Probe lifetime(ns)

EDANS Free Probe 336 } 494 0.152� 0.003 12.34� 0.05LY Free Probe 428 } 534 0.219� 0.001 5.04� 0.07EDANS-lys Asp101 346 0.13 498 0.121� 0.003 12.41� 0.15IEDANS-lys His15 498 0.151� 0.006 12.03� 0.21LY-lys Asp101 425 2.12 528 0.066� 0.001 4.56� 0.05LYI-lys His15 526 0.356� 0.003 7.02� 0.13

aFrom: Handbook of Fluorescent Probes and Research Products, at http://www.probes.com/handbook.


LYI–CEWL was a linear sum of its components,indicating little if any interaction between theprobe and protein.

3.3. Steady state fluorescence characterizationof the derivatives

Basic steady state fluorescence and time resolveddata were collected to characterize the free probeand derivatized probe-CEWL systems. Fig. 2shows the fluorescence emission spectra for freeEDANS and the EDANS and IEDANS–CEWLderivatives. The quantum yields were determinedreferenced to quinine sulfate in 0.1M H2SO4, andwere found to be 0.152, 0.121, and 0.151,respectively. For both derivatives there is a 4 nmredshift in the emission maximum. Similar mea-surements, shown in Fig. 3, were made for free LYand the LY and LYI–CEWL derivatives. In thesecases the quantum yields were determined to be0.219, 0.066, and 0.356, respectively, i.e., the His15derivative has an enhanced fluorescence quantumyield over the free probe. Again, wavelength shiftsin the emission maximum were observed relative tothe free probe, in this case being blue shifted from534 nm for the free probe to 528 and 526 nm forthe LY and LYI derivatives, respectively.

3.4. Fluorescence lifetime characterizationof the derivatives

Table 1 summarizes the frequency domain dataobtained for the free probes and their respectiveAsp101 and His15 derivatives. In all cases a singlelifetime was sufficient to obtain a reasonable fit tothe data. In the case of EDANS the fluorescencelifetime was relatively unaffected by its location onthe surface, being �12 ns and comparable to thatfor the free probe. The steady state quenching andthe reduction in quantum yield observed for theAsp101 derivative is not apparent in the lifetimedata. In the case of LY and its derivatives, a singlelifetime is sufficient to give a good fit to the data.In this case, however, there is a pronouncedchange in the lifetimes that are reflected in thequantum yield measurement. The lifetime datashows quenching of the probe at the Asp101 siteand enhancements at the His15 site.

3.5. Collisional quenching of fluorescent probes

Collisional quenching of a bound fluorescentprobe by an exogenous species can be used to gaininsight into the probes exposure. A number ofcollisional quenchers were investigated to generatequenching data for the derivatized protein sys-tems. None gave strong quenching for either thefree or bound probes. However, sodium iodide,

Fig. 2. Fluorescence emission spectra for free EDANS, theAsp101-bound EDANS–CEWL, and His15-bound IEDANS–CEWL. Emission spectra are obtained in 7% NaCl, 0.05MTris-HCl buffer, pH 7.5 and are corrected to reflect the probequantum yields.

Fig. 3. Fluorescence emission spectra for free LY, the Asp101-bound LY–CEWL, and His15-bound LYI–CEWL. Emissionspectra are obtained in 7% NaCl, 0.05M Tris-HCl buffer, pH7.5 and are corrected to reflect the probe quantum yields.


potassium iodide, and acrylamide were sufficientlystrong quenchers to enable data collection.

Acrylamide gave the best quenching results withEDANS (Fig. 4). Quenching of the free probe wasmeasured as a function of the acrylamide concen-tration and a Stern–Volmer plot of F0=F vs. [Q],the molar concentration of the added quencher,was constructed based uponF0=F ¼ 1þ Ksv½Q�; ð1Þ

where F and F0 are the measured fluorescence withand without quencher, respectively. The slope ofthe plot gives Ksv, the Stern–Volmer quenchingconstant. For unbound EDANS quenchedwith acrylamide, Ksv ¼ 8:78. Assuming efficientdiffusion limited quenching of the probe,Ksv ¼ ðkqÞðt0Þ, where kq is the bimolecular quench-ing constant and t0 is the fluorescence lifetime ofthe probe in the absence of quenching (=12.39 ns),then kq ¼ 7:1� 108 1/s. For the structurally simi-lar IEDANS product with mercaptoethanol, kqwas found to be 1.2� 109 1/s [13]. In the case of theAsp101 EDANS derivative, Ksv ¼ 4:09 was deter-mined, resulting in a kq of 3.3� 108 1/s. As the Ksvvalue is �60% that of the free probe, this showsthat the probe bound at the Asp101 site is not asaccessible to the quencher as the free probe.Together with the steady state and quantum yielddata, this indicates a perturbation of the electronicenvironment of the probe as well as a hinderedapproach from the solution, both of whichcorroborate the binding of the probe within theactive site cleft.

Potassium and sodium iodide were used togenerate quenching data in the case of LY and itsCEWL derivatives. For the unbound LY a valueof Ksv ¼ 15:65 was obtained. For the Asp101

derivative a value of Kapparent ¼ 5:97 was obtained.The positive upward deviation of the curveindicates that the quenching observed is not purelydynamic, but is a combination of both dynamicand static quenching of the probe fluorescence(Fig. 5).

Static and dynamic quenching act in differentways to decrease probe fluorescence. For staticquenching non-fluorescent complexes formed inthe ground state between the fluorescent proteinderivatives and quencher decrease the population

of the excited state species thus decreasing theintensity of the observed fluorescence. In the caseof dynamic quenching, the fluorescence intensityis decreased as a result of the number of non-radiative decay pathways available to the excitedstate species due to collisions with the quencher.As a result, static quenching does not affect thefluorescence lifetime of the probe so an analysis ofthe time lifetime data with quencher concentrationshould produce a linear curve with a slope whichequals the dynamic Stern–Volmer quenching con-stant:t0=t ¼ 1þ Ksv½Q�; ð2Þ

where [Q] is the molar concentration of quencherand t0 and t are the lifetimes in the absence andpresence of quencher, respectively. The inset toFig. 5 shows such a plot. In the case of LY–CEWLthe slope obtained was the dynamic quenchingconstant Ksv ¼ 9:18. The data for EDANS–CEWLshows that the approach to the probe fromsolution is hindered and corroborates that theprobe is bound within the active site cleft.

The total quenching observed is the product ofthe dynamic and static quenching, and after

Fig. 4. Fluorescence quenching of free EDANS (*), theAsp101-bound EDANS–CEWL (n), and His15-bound IE-DANS–CEWL (&) by acrylamide in 7% NaCl, 0.05M Tris-HCl buffer, pH 7.5. The slopes, equal to the Stern–Volmerquenching constant, are 8.78, 4.09, and 5.02, respectively.


rearrangement:Kapp ¼ ððF0=FÞ � 1Þ=½Q�

¼ ðKsv þ KstaticÞ þ KsvKstatic½Q�: ð3Þ

A plot of Kapp vs. Q yields a straight line withKsv þ Kstatic as the intercept and KsvKstatic as theslope. This results in a calculated static quenchingconstant of 0.30 1/M for LY–CEWL. A similaranalysis was performed for the LYI–CEWLderivative, which also showed upward curvaturein the Stern–Volmer quenching curve, and a Ksv ¼15:77 and Kstatic ¼ 0:86 were obtained. The deter-mined Ksv for the LYI–CEWL was considerablycloser to that determined for the free probe,indicating that the probe in this case is somewhatmore exposed to the bulk solution as expected.

3.6. Crystallization of CEWL fluorescentderivatives

Sitting drop crystallizations were set up at 208Cwith reservoir solutions of 7% NaCl in 0.05MTris-HCl, pH 7.5 or 0.1M NaAc buffer, pH 4.6with 5% NaCl. The results are shown in Fig. 6.

Fig. 6A and B show EDANS–CEWL and LYI–CEWL crystals, respectively, grown at pH 7.5under white light illumination. Fig. 6C and Dshow EDANS–CEWL and LY–CEWL crystalsgrown at pH 4.6 under fluorescing illumination.

4. Discussion

The carbodiimide coupling procedure at pH 5.0has been used to covalently attach a number ofdifferent species to the CEWL Asp101 side chaincarboxyl group [6]. The efficacy of the couplingprocedure has been found to vary somewhat,depending upon the ligand being attached. Theoverall yield for formation of the Asp101 EDANSand LY derivatives of CEWL are somewhat lowerthan previously found for other small molecules,typically �5–10%. The reasons for the low yieldsare not clear at this time. Experiments to test if theoverall percentage yield of derivative could beincreased by increasing the probe : protein ratiodid not meet with success (data not shown).Initially the derivatization reactions were carriedout at low protein concentrations, ca. 5mg/ml.No difference in the yield was found when theCEWL concentration was increased to 10mg/mlor higher.

In addition to LY and EDANS, a number ofdifferent probes having free aliphatic amines weretested for binding to the Asp101 site. Not everyprobe tried could be covalently attached using theabove procedure. Success has been had with theprobe cascade blue, but in this case the fluores-cence was very highly quenched, with a quantumyield of �0.01. Attempts at incorporating eitherdansyl ethylenediamine or dansyl cadaverine wereuniformly unsuccessful. Similarly, attempts atincorporating probes such as pyrene methyl amine,amino methyl fluorescein, and 5-(aminoacet-amido)fluorescein were unsuccessful as well. Inmany cases initially promising results turned outto be a strong non-covalent interaction, with theprobe : protein ratio never substantially improvingwith successive purification steps. Ultimately inthese cases tryptic digestion and peptide analysisshowed that the probe was not in fact covalentlyattached to the protein. Due to the range in sizes of

Fig. 5. Fluorescence quenching of free LY (*), the Asp101-bound LY–CEWL (n), and His15-bound LYI–CEWL (&), byI� ion in 7% NaCl, 0.05M Tris-HCl buffer, pH 7.5. The insetshows the corresponding lifetime Stern–Volmer plot for theAsp101-bound LY–CEWL (n) and His15-bound LYI–CEWL(&), which give slopes of 9.18 and 15.77, respectively.


both the successes and failures we tentatively ruleout the ability of the probe to fit within the activesite as a limiting factor. One apparent constant inthe three known successes (LY, EDANS, cascadeblue) is the presence of one or more sulfonategroups attached to the aromatic portions of themolecule. The unsuccessful probes did not havesuch a group, or, in the case of the dansylstructures, it is an aliphatic amino sulfonamide.How or why this would make a difference, ifindeed it does, is not clear at this time.

Earlier work suggested that CEWL has anesterase activity centered on the sole histidine,His15, which is located in a ‘‘pocket’’ on the backside away from the active site cleft [8]. This singlehistidine can be labeled with iodoacetamides [7–9].While the procedure used typically calls for thereaction to be carried out at 408C, CEWL isknown to undergo a tetragonal$ orthorhombicphase transition upon warming above �258C[10,14–17]. Warming of the protein solution intothe orthorhombic region has been shown to have asignificant effect on the subsequent nucleation rate[10], and therefore potentially on the self-associa-

tion reactions leading up to the formation of thecritical nucleus. Reactions were carried out atroom temperature to avoid these complications.As with the Asp101 site, we have found thatapparently only those probes having an aromaticsulfonate group could be readily bound to His15,and that they were not always successful either.Probes such as N-(1-pyrenemethyl)iodoacetamide,2-(40-(iodoacetamido)anilino)naphthalene-6-sulfo-nic acid, and 7-diethylamino-3-((40-(iodoacetyl)amino)phenyl)-4-methylcoumarin, have all beentried with no success to date.

The purpose of making fluorescent derivatives isfor use in protein crystal nucleation and crystalgrowth studies. The fluorescent bound probesmust meet certain criteria for use in these studies.A variety of fluorescent derivatives thus provide atoolbox for use in subsequent crystal nucleationand growth studies. In the case of resonanceenergy transfer, used to measure the distancebetween two different probes, they must havesome overlap in the emission and excitationspectra. For fluorescence quenching to be usefulin nucleation studies the quenching constant

Fig. 6. Crystals of CEWL fluorescent derivatives: All crystals were grown by sitting drop vapor diffusion at 208C. Fig. 6A: EDANS–CEWL crystals, and Fig. 6B, LYI–CEWL crystals, both grown from 0.05M Tris-HCl, 7% NaCl, pH 7.5. The scale bar in panels A andB is 100mm. Fig. 6C: EDANS–CEWL crystals grown from 0.1M sodium acetate, pH 4.6, 5% NaCl, under fluorescing illumination.Fig. 6D: LY–CEWL crystal grown from 0.1M sodium acetate, pH 4.6, 5% NaCl. Here one panel shows the crystals under white lightand the other under fluorescing illumination.


should be as high as possible, minimizing theamount of quencher that must be added to thesolution. In the case of the derivatives presentedabove this is not the case, and thus they are oflimited utility in studies using this approach.However, the quenching studies presented abovedo show that, as expected, the Asp101-boundprobes are at least partially shielded for the bulksolution.

In the case of anisotropy studies it is useful forthe probes to be rigidly bound to the protein, i.e.,not have any motion independent of the under-lying protein, and that they have a decay timecomparable to or longer than the rotationalcorrelation time of the protein. The rotationalcorrelation time y can be estimated byy ¼ ðZM=RTÞðnþ hÞ; ð4Þ

where Z is the viscosity, M the molecular weight, Rthe gas constant, T the temperature, n the specificvolume of the protein and h the hydration. Forlysozyme, assuming 40% hydration, we calculate arotational correlation time of �6.85 ns. Thus theEDANS derivatives should have suitable lifetimesfor use in anisotropy studies of lysozyme nuclea-tion up through the dimer (estimated y � 13:7 ns)stage, and possibly through the tetramer stage(estimated y � 27 ns). However EDANS is notrigidly bound but has an independent motion thatmust be taken into account in the data analysis.

It is apparent from the crystallizations that theprobes can affect the crystal nucleation and growthprocess, particularly those attached to His15.However, the crystals in Fig. 6 were grown from100% derivatized protein, from a �2mM proteinsolution. Fluorescence studies are more typicallyconducted at 410�6M probe concentrations.Further, the least effect (if any) is found withprobes in the Asp101 site. We currently postulatethat the initial stages of CEWL self-associationleading to nucleation and crystal growth unitformation are the formation of 43 helix-like

structures in solution. Asp101-bound probes wouldbe buried within the interior of such a structure.His15-bound probes would lie on the outside, andnot participate in these interactions, but couldinterfere with subsequent helix–helix interactionsduring nucleation or crystal growth.

Acknowledgements

Funding support for this work was providedunder a National Aeronautics and Space Admin-istration grant, UPN number 101-11-32.

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Sumida Forsythe Pusey J Cryst Growth 232 2001 308