1. Journal of Crystal Growth 232 (2001) 308–316
Preparation and preliminary characterization of crystallizing
fluorescent derivatives of chicken egg white lysozyme
John P. Sumidaa, Elizabeth L. Forsythea, Marc L. Puseyb,*
a
USRA, 4950 Corporate Drive, Suite 100, Huntsville, AL 35806, USA
b
Biophysics 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 are
intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives
added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared
fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein
molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the
side chain carboxyl of Asp101 using a carbodiimide coupling procedure. Asp101 lies within the active site cleft, and it is
believed that the probes are ‘‘buried’’ within that cleft. Lucifer yellow and EDANS probes with iodoacetamide reactive
groups have been bound to His15, located on the ‘‘back side’’ of the molecule relative to the active site. All the
derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on
determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching
by added solutes. Quenching studies show that, as postulated, the Asp101–bound probes are partially sheltered from the
bulk solution by their location within the active site cleft. Probes bound to His15 have quenching constants about equal
to 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 cent techniques, relative to absorption methods,
use low concentrations of the probe species to keep
Fluorescence, one of the most powerful techni- within a linear response region. Protein concentra-
ques available for studying proteins and their tions are typically high in crystal nucleation and
interactions in solution, has only occasionally been growth studies. A practical way around these
used in protein crystal growth studies [1,2]. conflicting requirements is to covalently attach a
Intrinsic fluorescing amino acids generally cannot fluorescent probe to a subpopulation of the
be used due to the high protein concentrations protein molecules.
employed in crystallization experiments. Fluores- For this strategy to be effective the derivatized
molecules must behave ‘‘normally’’ in the crystal
nucleation and growth process, i.e., the probe must
*Corresponding author. Tel.: +1-256-544-7823; fax:+1-256-
544-6660. not participate in that process. Such an approach
E-mail address: marc.pusey@msfc.nasa.gov (M.L. Pusey). has been shown to work with ribonuclease [1],
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

2. J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316 309
where the probe N-[[(iodoacetyl)amino]ethyl]- Chemical Co. Sodium iodide and 2-picolylamine
5-napthylamine-1-sulfonic acid has been attached were from Sigma. Potassium iodide and primuline
such that it lies within the active site cleft. were from Aldrich. Cobalt chloride, thallium
Subsequent X-ray crystallographic studies con- chloride, cesium chloride, trifluoroacetamide, and
firmed the location of the probe [2]. 2,2,2-trichloroethanol were from Fluka. Acryla-
Pan and Berglund [3] carried out fluorescence mide was from BioRad, and trypan blue was from
anisotropy measurements using lysozyme with Flow Laboratories.
pyrene butyric acid bound to the N-terminal
amine. Time resolved measurements showed that 2.1. CEWL derivatization
the probe was not rigidly fixed in place and had a
segmental motion. Once this motion was taken Derivatization reactions of Asp101 were carried
into account, the rotational motion of the protein out using the method of Yamada et al. [6]. To
could be independently analyzed. In the case of 100 ml of 10 mg/ml lysozyme solution in dH2O
ribonuclease the probe was rigidly bound to the were added 10–100 mg of the probe to be attached.
protein and no correction to the data for The protein was kept in excess relative to the probe
independent probe motion was required. to maximize the amount of probe bound and favor
We have used a carbodiimide procedure pre- reaction at a single site. All reactions were
viously described [4–6] to covalently attach the performed with stirring under reduced light levels.
probes lucifer yellow (LY) and 5-((2-amino- After adding the probe, the solution pH was
ethyl)amino)napthalene-1-sulfonic acid (EDANS) adjusted to 5.0 with dilute HCl or NaOH as
to Asp101 of lysozyme. The His15 group, located on needed. Solid EDAC was then added at an
the back of the protein relative to the active site, 8 : 1 molar ratio, EDAC : CEWL, and the pH
can be reacted with probes containing an iodo- maintained at 5.0 Æ 0.02 by addition of dilute
acetamide reactive group [7–9]. Using this ap- HCl or NaOH as needed. After 1–2 h the reaction
proach, we have bound lucifer yellow iodoaceta- mixture was covered with parafilm and stirring
mide (LYI) and N-[[(iodoacetyl)amino]ethyl]-5- continued in the dark an additional 12–16 h at
napthylamine-1-sulfonic acid (IEDANS) to His15. room temperature.
In all cases the derivatized proteins have been Derivatization of His15 was carried out using the
shown to crystallize. Herein, we report on the method previously described for ribonuclease [11].
preparation and preliminary fluorescent and crys- To 0.5 g of CEWL at $10 mg/ml in 0.1 M Na
tallographic characterization of these derivatives. phosphate buffer, pH 5.2, is added 100 mg of
reactive probe (IEDANS or LYI). The addition
and subsequent reaction are carried out with
2. Materials and methods stirring in the dark at room temperature for 40 h.
Then 10 ml of mercaptoethanol were added and the
Chicken egg white lysozyme, (CEWL, Sigma) solution was stirred for 2 h, to take up any
was purified by cation exchange chromatography unreacted probe.
and recrystallized as previously described [10]. The
recrystallized protein was dialyzed against dH2O 2.2. Purification of the fluorescent labeled
prior to derivatization. Unless otherwise noted all lysozyme
chemicals were of reagent grade or better. The
fluorescent probes lucifer yellow CH lithium salt The reaction mixture was first dialyzed at room
(LY), 5-((2-aminoethyl)amino)napthalene-1-sulfo- temperature in the dark against 0.25 M NaCl to
nic acid, (EDANS), lucifer yellow iodoacetamide facilitate removal of excess un-reacted or non-
(LYI), and N-[[(iodoacetyl)amino]ethyl]-5-napthy- covalently bound probe, and then dialyzed against
lamine-1-sulfonic acid (IEDANS), were from dH2O. Following dialysis, the protein solution was
Molecular Probes. 1-Ethyl-3-(3-dimethylamino- chromatographed on a BioPrep CM cation ex-
propyl)carbodiimide (EDAC) was from Aldrich change column (2.5 Â 50 cm2) equilibrated with

3. 310 J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316
0.05 M sodium phosphate, pH 7.0 at room were collected by measuring the modulation and
temperature. After loading the protein onto the phase difference between the excited and emitted
column it was briefly washed with equilibrating light as a function of the excitation modulation
buffer, then developed with a gradient of 0.35– frequency over the range of 2–200 MHz. Measure-
0.65 M NaCl at a flow rate of 3.0 ml/min. with a ments were made using the appropriate long pass
total gradient volume of 1200 ml. Fractions of filter in the emission path. A dilute glycogen
24 ml size were collected, and the protein and solution was used as a reference scatterer for the
appropriate probe absorptivities determined for lifetime determinations. Frequency domain data
each fraction. Additional purification, if needed, analyses were carried out using the Globals
was carried out using a semi-preparative HPLC Unlimited software package.
cation exchange column (HEMA-IEC CM 10 mm
column, 22.5 Â 250 mm2). The peaks were col-
2.5. Crystallization
lected, and those with derivatized protein at an
appropriate probe : protein ratio were pooled,
Crystallizations were done using the sitting drop
dialyzed against dH2O, lyophilized, and stored at
vapor diffusion method. The reservoir solution
À208C until use.
was 0.05 M tris-HCl buffer, 7% NaCl, pH 7.5. We
have previously shown that tetragonal crystals can
2.3. Steady state absorption
be readily grown in this pH range once the protein
has been purified [10]. Crystallizations were also
UV/V is absorption spectra were obtained using
carried out at 0.1 M sodium acetate, 5% NaCl,
a Hewlett Packard 8425A diode array spectro-
pH 4.6. Lyophilized protein was dissolved into
photometer. All absorption samples were taken in
dH2O to a concentration of 25 mg/ml, and serial
quartz cuvettes. Free probe and derivatized
dilutions of this solution were mixed in a 1 : 1 ratio
protein samples were blanked against the 0.05 M
with reservoir buffer for the crystallization solu-
Tris buffer used to make up the sample.
tion. The sitting drop plates were kept in an
incubator maintained at 208C.
2.4. Fluorescence characterization
Steady state and frequency domain fluorescence
measurements were made with an ISS K2 digital 3. Results
spectrofluorometer. Rhodamine B in conjunction
with a neutral density filter in the reference 3.1. Preparation and purification of the modified
channel was used as a quantum counter for steady lysozyme
state measurements. For all measurements a 2 mm
slit width was used in the excitation channel and All of the probes absorb at 280 nm, and the
a width of 1.0 or 0.5 mm in the emission channel, calculated concentration and binding ratio data
depending upon the sample. Quantum yield need to be corrected for this absorption. The probe
determinations were made using the comparative absorption maximum: 280 nm absorption ratios
method with quinine sulfate in 0.1 M H2SO4 as the were determined for the un-reacted probes in
reference. Optical densities for fluorescence work dH2O and are given in Table 1. The ratios were
were maintained below 0.1 at the excitation determined on the basis of the probe absorptivities
(absorption maximum) wavelength in order to given in the Molecular Probes catalogue. The
avoid inner filter effects or significant self-absorp- molar absorptivity of CEWL at 280 nm was
tion of sample fluorescence. All solutions were calculated to be 37 Â 103, based upon a mass
bubbled with argon prior to measurement. absorptivity of 26.4 [12].
Lifetime measurements were made with the Upon completion of the probe binding reaction,
instrument in the lifetime acquisition mode, and the solutions were first dialyzed against 0.25 M
the data collected using the fastscan feature. Data NaCl and then against distilled water. If the

4. J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316 311
Table 1
Measured properties of the prepared derivatives of lysozymea
Derivative Location Abs. l maximum Abs. ratio 280=lmax Emission maximum l Probe quantum Probe lifetime
(nm) (free probe) (nm) yield (ns)
EDANS Free Probe 336 } 494 0.152 Æ 0.003 12.34 Æ 0.05
LY Free Probe 428 } 534 0.219 Æ 0.001 5.04 Æ 0.07
EDANS-lys Asp101 346 0.13 498 0.121 Æ 0.003 12.41 Æ 0.15
IEDANS-lys His15 498 0.151 Æ 0.006 12.03 Æ 0.21
LY-lys Asp101 425 2.12 528 0.066 Æ 0.001 4.56 Æ 0.05
LYI-lys His15 526 0.356 Æ 0.003 7.02 Æ 0.13
a
From: Handbook of Fluorescent Probes and Research Products, at http://www.probes.com/handbook.
solution was cloudy after dialysis, it was subse-
quently clarified by filtration through a 0.45 mm
membrane. The first chromatographic purification
was on a BioPrep CM column, with all of the
reaction mixture being loaded onto the column.
All chromatography steps were performed at room
temperature to avoid crystallization of the protein
on the column or in the fractions. Corrected
probe : protein ratios were calculated for each
fraction, and those peaks having the highest ratios
41 were separately pooled, dialyzed against
distilled 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, Fig. 1. Absorption spectrum of underivatized lysozyme, free
again dialyzed against distilled water, concen- EDANS, the sum spectrum of lysozyme and free EDANS, and
trated, lyophilized and stored at À208C. of the Asp101-bound EDANS–CEWL.
3.2. Absorption characterization of the derivatives the free probe and protein, representing a linear
combination of their absorption spectra. Both the
Steady state absorption spectra were obtained 253 and 336 nm bands of the free probe are red
for free EDANS, LY, and CEWL, and for the shifted by 10 nm in the derivatized protein. In the
His15 and Asp101 derivatives. The absorption case of LY (not shown), the sum spectra is
spectra were obtained in 0.05 M Tris buffer, 7% essentially equivalent to that of the derivatized
NaCl, pH 7.5. In the case of the free EDANS three protein, indicating little perturbation of either the
absorption peaks are found at 210, 252, and protein or probe absorption by covalent attach-
336 nm. For free LY three major absorption peaks ment within the active site cleft.
are found at 230, 280, and 428 nm. The absorption spectra of the His15 derivatives
EDANS bound at Asp101 exhibits strong and of IEDANS and LYI were also examined with
broad absorption peaks with maxima at 212 and similar results. For the IEDANS derivative a
282 nm, where both EDANS and lysozyme redshift is observed, analogous to that for the
absorb. Since Asp101 is within the active site cleft Asp101 derivative. In this case the shift was
of the protein, interaction between the probe and somewhat less, about 2 nm, but broadened. For
protein is expected. Fig. 1 shows the spectrum of LYI no shifts were observed and, as for the
the derivatized protein and the sum spectrum of Asp101 derivative, the absorption spectrum of

5. 312 J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316
LYI–CEWL was a linear sum of its components,
indicating little if any interaction between the
probe and protein.
3.3. Steady state fluorescence characterization
of the derivatives
Basic steady state fluorescence and time resolved
data were collected to characterize the free probe
and derivatized probe-CEWL systems. Fig. 2
shows the fluorescence emission spectra for free
EDANS and the EDANS and IEDANS–CEWL
derivatives. The quantum yields were determined Fig. 2. Fluorescence emission spectra for free EDANS, the
referenced to quinine sulfate in 0.1 M H2SO4, and Asp101-bound EDANS–CEWL, and His15-bound IEDANS–
were found to be 0.152, 0.121, and 0.151, CEWL. Emission spectra are obtained in 7% NaCl, 0.05 M
respectively. For both derivatives there is a 4 nm Tris-HCl buffer, pH 7.5 and are corrected to reflect the probe
redshift in the emission maximum. Similar mea- quantum yields.
surements, shown in Fig. 3, were made for free LY
and the LY and LYI–CEWL derivatives. In these
cases the quantum yields were determined to be
0.219, 0.066, and 0.356, respectively, i.e., the His15
derivative has an enhanced fluorescence quantum
yield over the free probe. Again, wavelength shifts
in the emission maximum were observed relative to
the free probe, in this case being blue shifted from
534 nm for the free probe to 528 and 526 nm for
the LY and LYI derivatives, respectively.
3.4. Fluorescence lifetime characterization
of the derivatives
Table 1 summarizes the frequency domain data
obtained for the free probes and their respective
Fig. 3. Fluorescence emission spectra for free LY, the Asp101-
Asp101 and His15 derivatives. In all cases a single bound LY–CEWL, and His15-bound LYI–CEWL. Emission
lifetime was sufficient to obtain a reasonable fit to spectra are obtained in 7% NaCl, 0.05 M Tris-HCl buffer, pH
the data. In the case of EDANS the fluorescence 7.5 and are corrected to reflect the probe quantum yields.
lifetime was relatively unaffected by its location on
the surface, being $12 ns and comparable to that
for the free probe. The steady state quenching and
the reduction in quantum yield observed for the 3.5. Collisional quenching of fluorescent probes
Asp101 derivative is not apparent in the lifetime
data. In the case of LY and its derivatives, a single Collisional quenching of a bound fluorescent
lifetime is sufficient to give a good fit to the data. probe by an exogenous species can be used to gain
In this case, however, there is a pronounced insight into the probes exposure. A number of
change in the lifetimes that are reflected in the collisional quenchers were investigated to generate
quantum yield measurement. The lifetime data quenching data for the derivatized protein sys-
shows quenching of the probe at the Asp101 site tems. None gave strong quenching for either the
and enhancements at the His15 site. free or bound probes. However, sodium iodide,

6. J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316 313
potassium iodide, and acrylamide were sufficiently
strong quenchers to enable data collection.
Acrylamide gave the best quenching results with
EDANS (Fig. 4). Quenching of the free probe was
measured 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 upon
F0 =F ¼ 1 þ Ksv ½QŠ; ð1Þ
where F and F0 are the measured fluorescence with
and without quencher, respectively. The slope of
the plot gives Ksv , the Stern–Volmer quenching
constant. For unbound EDANS quenched
with acrylamide, Ksv ¼ 8:78. Assuming efficient
diffusion limited quenching of the probe,
Ksv ¼ ðkq Þðt0 Þ, where kq is the bimolecular quench-
ing constant and t0 is the fluorescence lifetime of Fig. 4. Fluorescence quenching of free EDANS (*), the
Asp101-bound EDANS–CEWL (n), and His15-bound IE-
the probe in the absence of quenching (=12.39 ns), DANS–CEWL (&) by acrylamide in 7% NaCl, 0.05 M Tris-
then kq ¼ 7:1 Â 108 1/s. For the structurally simi- HCl buffer, pH 7.5. The slopes, equal to the Stern–Volmer
lar IEDANS product with mercaptoethanol, kq quenching constant, are 8.78, 4.09, and 5.02, respectively.
was found to be 1.2 Â 109 1/s [13]. In the case of the
Asp101 EDANS derivative, Ksv ¼ 4:09 was deter-
mined, resulting in a kq of 3.3 Â 108 1/s. As the Ksv of the excited state species thus decreasing the
value is $60% that of the free probe, this shows intensity of the observed fluorescence. In the case
that the probe bound at the Asp101 site is not as of dynamic quenching, the fluorescence intensity
accessible to the quencher as the free probe. is decreased as a result of the number of non-
Together with the steady state and quantum yield radiative decay pathways available to the excited
data, this indicates a perturbation of the electronic state species due to collisions with the quencher.
environment of the probe as well as a hindered As a result, static quenching does not affect the
approach from the solution, both of which fluorescence lifetime of the probe so an analysis of
corroborate the binding of the probe within the the time lifetime data with quencher concentration
active site cleft. should produce a linear curve with a slope which
Potassium and sodium iodide were used to equals the dynamic Stern–Volmer quenching con-
generate quenching data in the case of LY and its stant:
CEWL derivatives. For the unbound LY a value t0 =t ¼ 1 þ Ksv ½QŠ; ð2Þ
of Ksv ¼ 15:65 was obtained. For the Asp101
derivative a value of Kapparent ¼ 5:97 was obtained. where [Q] is the molar concentration of quencher
The positive upward deviation of the curve and t0 and t are the lifetimes in the absence and
indicates that the quenching observed is not purely presence of quencher, respectively. The inset to
dynamic, but is a combination of both dynamic Fig. 5 shows such a plot. In the case of LY–CEWL
and static quenching of the probe fluorescence the slope obtained was the dynamic quenching
(Fig. 5). constant Ksv ¼ 9:18. The data for EDANS–CEWL
Static and dynamic quenching act in different shows that the approach to the probe from
ways to decrease probe fluorescence. For static solution is hindered and corroborates that the
quenching non-fluorescent complexes formed in probe is bound within the active site cleft.
the ground state between the fluorescent protein The total quenching observed is the product of
derivatives and quencher decrease the population the dynamic and static quenching, and after

7. 314 J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316
Fig. 6A and B show EDANS–CEWL and LYI–
CEWL crystals, respectively, grown at pH 7.5
under white light illumination. Fig. 6C and D
show EDANS–CEWL and LY–CEWL crystals
grown at pH 4.6 under fluorescing illumination.
4. Discussion
The carbodiimide coupling procedure at pH 5.0
has been used to covalently attach a number of
different species to the CEWL Asp101 side chain
carboxyl group [6]. The efficacy of the coupling
procedure has been found to vary somewhat,
depending upon the ligand being attached. The
overall yield for formation of the Asp101 EDANS
and LY derivatives of CEWL are somewhat lower
than previously found for other small molecules,
Fig. 5. Fluorescence quenching of free LY (*), the Asp101-
typically $5–10%. The reasons for the low yields
bound LY–CEWL (n), and His15-bound LYI–CEWL (&), by
IÀ ion in 7% NaCl, 0.05 M Tris-HCl buffer, pH 7.5. The inset are not clear at this time. Experiments to test if the
shows the corresponding lifetime Stern–Volmer plot for the overall percentage yield of derivative could be
Asp101-bound LY–CEWL (n) and His15-bound LYI–CEWL increased by increasing the probe : protein ratio
(&), which give slopes of 9.18 and 15.77, respectively. did not meet with success (data not shown).
Initially the derivatization reactions were carried
rearrangement: out at low protein concentrations, ca. 5 mg/ml.
Kapp ¼ ððF0 =FÞ À 1Þ=½QŠ No difference in the yield was found when the
CEWL concentration was increased to 10 mg/ml
¼ ðKsv þ Kstatic Þ þ Ksv Kstatic ½QŠ: ð3Þ
or higher.
A plot of Kapp vs. Q yields a straight line with In addition to LY and EDANS, a number of
Ksv þ Kstatic as the intercept and Ksv Kstatic as the different probes having free aliphatic amines were
slope. This results in a calculated static quenching tested for binding to the Asp101 site. Not every
constant of 0.30 1/M for LY–CEWL. A similar probe tried could be covalently attached using the
analysis was performed for the LYI–CEWL above procedure. Success has been had with the
derivative, which also showed upward curvature probe cascade blue, but in this case the fluores-
in the Stern–Volmer quenching curve, and a Ksv ¼ cence was very highly quenched, with a quantum
15:77 and Kstatic ¼ 0:86 were obtained. The deter- yield of $0.01. Attempts at incorporating either
mined Ksv for the LYI–CEWL was considerably dansyl ethylenediamine or dansyl cadaverine were
closer to that determined for the free probe, uniformly unsuccessful. Similarly, attempts at
indicating that the probe in this case is somewhat incorporating probes such as pyrene methyl amine,
more exposed to the bulk solution as expected. amino methyl fluorescein, and 5-(aminoacet-
amido)fluorescein were unsuccessful as well. In
3.6. Crystallization of CEWL fluorescent many cases initially promising results turned out
derivatives to be a strong non-covalent interaction, with the
probe : protein ratio never substantially improving
Sitting drop crystallizations were set up at 208C with successive purification steps. Ultimately in
with reservoir solutions of 7% NaCl in 0.05 M these cases tryptic digestion and peptide analysis
Tris-HCl, pH 7.5 or 0.1 M NaAc buffer, pH 4.6 showed that the probe was not in fact covalently
with 5% NaCl. The results are shown in Fig. 6. attached to the protein. Due to the range in sizes of

8. J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316 315
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 0.05 M Tris-HCl, 7% NaCl, pH 7.5. The scale bar in panels A and
B is 100 mm. Fig. 6C: EDANS–CEWL crystals grown from 0.1 M sodium acetate, pH 4.6, 5% NaCl, under fluorescing illumination.
Fig. 6D: LY–CEWL crystal grown from 0.1 M sodium acetate, pH 4.6, 5% NaCl. Here one panel shows the crystals under white light
and the other under fluorescing illumination.
both the successes and failures we tentatively rule tion reactions leading up to the formation of the
out the ability of the probe to fit within the active critical nucleus. Reactions were carried out at
site as a limiting factor. One apparent constant in room temperature to avoid these complications.
the three known successes (LY, EDANS, cascade As with the Asp101 site, we have found that
blue) is the presence of one or more sulfonate apparently only those probes having an aromatic
groups attached to the aromatic portions of the sulfonate group could be readily bound to His15,
molecule. The unsuccessful probes did not have and that they were not always successful either.
such a group, or, in the case of the dansyl Probes such as N-(1-pyrenemethyl)iodoacetamide,
structures, it is an aliphatic amino sulfonamide. 2-(40 -(iodoacetamido)anilino)naphthalene-6-sulfo-
How or why this would make a difference, if nic acid, and 7-diethylamino-3-((40 -(iodoacetyl)
indeed it does, is not clear at this time. amino)phenyl)-4-methylcoumarin, have all been
Earlier work suggested that CEWL has an tried with no success to date.
esterase activity centered on the sole histidine, The purpose of making fluorescent derivatives is
His15, which is located in a ‘‘pocket’’ on the back for use in protein crystal nucleation and crystal
side away from the active site cleft [8]. This single growth studies. The fluorescent bound probes
histidine can be labeled with iodoacetamides [7–9]. must meet certain criteria for use in these studies.
While the procedure used typically calls for the A variety of fluorescent derivatives thus provide a
reaction to be carried out at 408C, CEWL is toolbox for use in subsequent crystal nucleation
known to undergo a tetragonal $ orthorhombic and growth studies. In the case of resonance
phase transition upon warming above $258C energy transfer, used to measure the distance
[10,14–17]. Warming of the protein solution into between two different probes, they must have
the orthorhombic region has been shown to have a some overlap in the emission and excitation
significant effect on the subsequent nucleation rate spectra. For fluorescence quenching to be useful
[10], and therefore potentially on the self-associa- in nucleation studies the quenching constant

9. 316 J.P. Sumida et al. / Journal of Crystal Growth 232 (2001) 308–316
should be as high as possible, minimizing the structures in solution. Asp101-bound probes would
amount of quencher that must be added to the be buried within the interior of such a structure.
solution. In the case of the derivatives presented His15-bound probes would lie on the outside, and
above this is not the case, and thus they are of not participate in these interactions, but could
limited utility in studies using this approach. interfere with subsequent helix–helix interactions
However, the quenching studies presented above during nucleation or crystal growth.
do show that, as expected, the Asp101-bound
probes are at least partially shielded for the bulk
solution. Acknowledgements
In the case of anisotropy studies it is useful for
the probes to be rigidly bound to the protein, i.e., Funding support for this work was provided
not have any motion independent of the under- under a National Aeronautics and Space Admin-
lying protein, and that they have a decay time istration grant, UPN number 101-11-32.
comparable to or longer than the rotational
correlation time of the protein. The rotational
correlation time y can be estimated by
y ¼ ðZM=RTÞðn þ hÞ; ð4Þ References
where Z is the viscosity, M the molecular weight, R
[1] M. Jullien, FEBS Lett. 253 (1989) 38.
the gas constant, T the temperature, n the specific [2] S. Baudet-Nessler, M. Jullien, M. Crosio, J. Janin,
volume of the protein and h the hydration. For Biochemistry 32 (1993) 8457.
lysozyme, assuming 40% hydration, we calculate a [3] B. Pan, K.A. Berglund, J. Crystal Growth 171 (1997) 226.
rotational correlation time of $6.85 ns. Thus the [4] T. Ueda, H. Yamada, N. Sakamoto, Y. Abe, K. Kawano,
EDANS derivatives should have suitable lifetimes Y. Terada, T. Imoto, J. Biochem. (Tokyo) 110 (1991) 719.
[5] T.-Y. Lin, D.E. Koshland, Science 244 (1969) 505.
for use in anisotropy studies of lysozyme nuclea- [6] H. Yamada, T. Imoto, K. Fujita, K. Okazaki,
tion up through the dimer (estimated y $ 13:7 ns) M. Motomura, Biochemistry 20 (1981) 4836.
stage, and possibly through the tetramer stage [7] F.J. Hartdegen, J.A. Rupley, Biochim. Biophys. Acta 92
(estimated y $ 27 ns). However EDANS is not (1964) 625.
[8] D. Piszkiewicz, T.C. Bruice, Biochem. 7 (1968) 3037.
rigidly bound but has an independent motion that
[9] R.I. Artyukh, G.S. Kachalova, B.A. Samaryanov,
must be taken into account in the data analysis. V.P. Timofeev, J. Mol. Biol. 29 (1995) 87.
It is apparent from the crystallizations that the [10] F.L. Ewing, E.L. Forsythe, M. van der Woerd,
probes can affect the crystal nucleation and growth M.L. Pusey, J. Crystal Growth 160 (1996) 389.
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[13] M.R. Eftink, Fluorescence quenching: theory and applica-
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