Surface recognition of a protein using designed transition metal complexes

Article abstract of DOI:10.1021/ja003193z

Each protein has a unique pattern of histidine residues on the surface. This paper describes the design, synthesis, and binding studies of transition metal complexes to target the surface histidine pattern of carbonic anhydrase (bovine erythrocyte). When

Full text of DOI:10.1021/ja003193z

J. Am. Chem. Soc. 2001, 123, 6283-6290
6283
Surface Recognition of a Protein Using Designed Transition Metal
Complexes
Md. Abul Fazal, Bidhan C. Roy, Shuguang Sun, Sanku Mallik,* and Kenton R. Rodgers
Contribution from the Department of Chemistry, North Dakota State UniVersity,
Fargo, North Dakota 58105
ReceiVed August 28, 2000
Abstract: Each protein has a unique pattern of histidine residues on the surface. This paper describes the
design, synthesis, and binding studies of transition metal complexes to target the surface histidine pattern of
carbonic anhydrase (bovine erythrocyte). When the pattern of cupric ions on a complex matches the surface
pattern of histidines of the protein, strong and selective binding can be achieved in aqueous buffer (pH ) 7.0).
The described method of protein recognition is applicable to proteins of known structures. With rapidly increasing
number of solved protein structures, the method has wide applicability in purification, targeting, and sensing
of proteins.
Strong and selective binding to a complex biomolecule in
aqueous solution by synthetic receptors is an area of active
research.1 Molecules that bind to a particular protein with a high
affinity have many potential applications including design of
new and selective enzyme inhibitors,2 design of stationary phases
for chromatographic purification of proteins3 and construction
of protein sensors.4 Recognition of an enzyme can be achieved
by exploiting a structurally well-defined enzyme active site or
a unique section of the protein’s surface topology.
strated.9 Linking two or more of these receptors improves the
binding affinity and the selectivity by large increments.10
Another approach for recognizing the surface pattern of amino
acids of a protein involves template polymerization.11 The
methodology has been performed on a surface12 or using a gel.13
In this approach, a pattern complementary to that of the protein
template is created on the polymer.
We are interested in the recognition of peptides14 and
proteins15 employing metal-ligand interactions. Various transi-
tion metal ions (e.g., Cu2+, Ni2+, Zn2+ etc.) bind to the imidazole
side chains of surface exposed histidines of proteins.16 This
coordination interaction (M2+-His) has been used for protein
purification by immobilized metal affinity chromatography
(IMAC),17 protein targeting to a surface18 and two-dimensional
Each protein has a unique pattern of amino acid residues on
the surface. It is this pattern that a surface receptor recognizes
for binding and selectivity. Protein surface patterns can be
recognized by polypeptides,5 non-peptide charged moieties,6
sugars,7 etc. If the receptor binds on the surface of an enzyme
close to the active site, this method can lead to efficient
inhibitors8a,b or disruptors for protein-protein interactions.8c,d
Combinatorial approaches to generate peptides and small
molecules capable of binding to a protein have been demon-
(8) (a) Park, H. S.; Lin, Q.; Hamilton, A. D. J. Am. Chem. Soc. 1999,
121, 8-13. (b) Hamuro, Y.; Calama, C.; Park, H. S.; Hamilton, A. D.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2680-2683. (c) Jain, R. K.;
Hamilton, A. D. Org. Lett. 2000, 2, 1721-1723. (d) Peczuh, M. D.;
Hamilton, A. D. Chem. ReV. 2000, 100, 2479-2494.
(1) (a) Baine, R. E.; Bender S. L. Chem. ReV. 1997, 97, 1359-1372. (b)
Bohm, H. J.; Klebe, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 2588-
2614. (c) Schneider, S. E.; Anslyn, E. V. AdVances in Supramolecular
Chemistry; Gokel, G. W., Ed.; JAI Press: Stanford, CT, 1999; Vol. 5, pp
55-120. (d) Zutshi, R.; Brickner, M.; Chmielewski, J. Curr. Opin. Chem.
Biol. 1998, 2, 62-66.
(2) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H. Chem.
ReV. 1999, 99, 2735-2776. (b) Keinar, S.; Avnir, D. J. Am. Chem. Soc.
2000, 122, 4378-4384. (c) Petrassi, H. M.; Klabunde, T.; Sacchettini, J.;
Kelly, J. W. J. Am. Chem. Soc. 2000, 122, 2178-2192. (d) Danishefsky,
S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 112, 882-912.
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R. E. Inorg. Chem. 2000, 39, 1180-1186. (b) Zhang, C. M.; Reslewic, S.
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Santarelli, X. J. Chromatogr., B 2000, 737, 143-150.
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10.1021/ja003193z CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/05/2001

6284 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
Fazal et al.
protein crystallization.19 For these applications, the proteins are
distinguished on the basis of their surface histidine contents.
The goal of this study is to use metal-ligand interactions to
selectively bind to peptides and proteins by recognizing unique
patterns of surface-exposed histidines. Herein, we report our
results on the recognition of the enzyme, carbonic anhydrase
from bovine erythrocytes (25 mM HEPES buffer, pH ) 7.0,
25 °C). Recognition has been achieved with ligands that bind
three cupric ions such that the metal centers can coordinate to
multiple surface histidie residues simultaneously. The reported
method of protein recognition is applicable to proteins of known
structures. With rapidly increasing number of solved protein
structures available from the data bank,20 the method has wide
applicability.
Metal-ligand (M-L) interactions offer several advantages
in recognition compared to hydrogen bonding, ion-pair or other
interactions. The M-L interactions are stronger in water21
compared to other interactions mentioned; thus, a fewer number
of simultaneous and complementary interactions can lead to tight
and selective binding. The spectroscopic properties of the metal
ions can be used to monitor the binding process and to obtain
structural information about the resultant complex.22 A variety
of transition metal ions are available and are used by nature in
biological systems for molecular recognition and catalysis.23 This
and 93 are separated from the targeted histidine triad by a
peptide loop. Calculations (using insight II and Discover)
suggest that these two histidines are less exposed to solvent
compared to histidines 1, 7, 14 (or 12, Supporting Information).
Three other proteins were used as controls (chicken egg
albumin, horse skeletal muscle myoglobin, and chicken egg
lysozyme). Chicken egg albumin (CEA) has six solvent-exposed
histidines (22, 23, 329, 332, 363, 371). CEA has the same
number of surface histidines as CA, but the pattern is different.
Myoglobin (Mb) has seven histidines on the surface (36, 48,
81, 97, 113, 116, 119). Thus, Mb has a greater number of surface
histidines than the target protein (CA) but with a different
distribution. Lysozyme has only one histidine (15), and it is
exposed on the surface. The histidine distributions of the control
proteins are also depicted in Figure 1.
All of these proteins (target and the controls) are known to
interact with transition metal ions and complexes through the
surface-exposed histidines.26 If recognition is based solely on
the number of surface histidine residues (e.g., IMAC), then Mb
is expected to have the highest affinity for the metal complexes;
the target protein CA and CEA should exhibit similar binding
strengths.
To bind to CA selectively, the tris-Cu2+ complex 2 (Figure
2) was designed. The presence of the (1,3,5-triphenyl)phenyl
core makes the complex rigid. Iminodiacetic acid (IDA) was
chosen as the ligand to chelate the cupric ions. IDA has strong
affinity for Cu2+ (K ≈ 1012 M-1),27 and the resultant complex
has been widely used in protein purification by IMAC.17 This
ensures that the complex will not be demetalated even at high
protein concentrations. It is reported in the literature that IDA-
Cu2+ complexes bind to proteins (pH ) 7.0) primarily through
the histidine residues on the protein surface.28 Complexes 1, 3,
4, 5, and 6 (Figure 2) were designed as the controls for these
studies.
The inter-copper distances were estimated by molecular
modeling employing the software Spartan (version 5.2, Wave
function Inc., Irvine, CA). The complexes were energy-
minimized in the gas phase (employing Merck molecular
mechanics force field) followed by systematic conformational
searches to locate the lowest energy conformations. The
distances between the Cu2+ ions were estimated to be ∼12 Å
for 1, ∼16 Å for 2, ∼12-16 Å for 3, ∼14-18 Å for 4, and
∼8-12 Å for 5.
allows fine-tuning of each of the interactions and opens the
24
possibility of catalysis following recognition
strated in this study).
(not demon-
Results and Discussion
Design and Syntheses of the Metal Complexes. To dem-
onstrate the proof-of-concept, carbonic anhydrase (CA, bovine
erythrocyte) was taken as the target protein. CA has six
histidines on the surface (1, 7, 12, 14, 61, and 93 determined
from the X-ray structure25 using the modeling software insight
II and Discover, version 98.0, Molecular Stimulations Inc.,
Burlington, MA). The distances separating the histidines 1, 7,
14 (or 12) are 13 ( 2, 16 ( 2, and 17 ( 2 Å respectively. In
the studies reported here, either His-14 or His-12 can participate
in binding. In this study, no attempts were made to dissect these
two binding modes as either one is consistent with the
conclusions reached.
The reported inter-histidine distances were measured using
the ꢀ-N atom of the imadazole moiety (Figure 1). Histidines 61
Complex 1 was designed to position three cupric ions at a
distance that does not match the inter-histidine distances of CA.
Complexes 3 and 4 were designed to probe the role of rigidity
versus flexibility in the recognition process. Complex 5 has five
cupric ions, but the pattern is not complementary to histidine
pattern of the target protein (CA). Complex 6 has one Cu2+
and was used as another control for these recognition studies.
The syntheses of 1, 2, 3, 4, and 5 are indicated in Scheme 1.
Elemental analyses indicated that the complexes had the correct
number of cupric ions. Determination of Cu2+ by UV-vis
spectrometry (employing EDTA) also indicated the proper
number of Cu2+ ions. The complexes were found to be stable
in aqueous buffer solution (25 mM HEPES buffer, pH ) 7.0,
25 °C) in air for more than a month.
(17) (a) Changa, G.; Bochkariov, D. E.; Jokhadze, G. G.; Hopp, J.;
Nelson, P. J. Chromatogr., A 1999, 864, 247-256. (b) Sideneus, U.; Farver,
O.; Jons, O.; Gamelgaard, B. J. Chromatogr., B 1999, 735, 85-91. (c)
Wang, M. Y.; Kau, Y. Y.; Lee, M. S.; Doong, S. R.; Ho, J. Y.; Lee, L. H.
Biotechnol. Bioeng. 2000, 67, 104-111. (d) Wu, H. P.; Bruley, D. F.
Biotechnol. Prog. 1999, 15, 928-931.
(18) Ng, K.; Pack, D. W.; Sasaki, D. Y.; Arndd, F. H. Langmuir 1995,
11, 4048-4055. (b) Dietrich, C.; Boscheinen, O.; Scharf, K. D.; Schmitt,
L.; Tampe, R. Biochemstry 1996, 35, 1100-1105. (c) Pack, D. W.; Arnold,
F. H. Chem. Phys. Lipids 1997, 86, 135-152. (d) Sigal, G. B.; Bamdad,
C.; Barberies, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68,
490-497. (e) Kroger, D.; Liley, M.; Schiweck, W.; Skerra, A.; Vogel. H.
Biosens. Bioelectr. 1999, 14, 155-161.
(19) Pack, D. W.; Chen, G.; Maloney, K. M.; Chen, C. T.; Arnold. F.
H. J. Am. Chem. Soc. 1997, 119, 2479-2487.
(20) The URL for the protein data bank on the internet is: http://
www.rcsb.org/pdb/.
(21) Frey, P. A.; Cleland, W. W. Bioorg. Chem. 1998, 26, 175-192.
(22) Kendrick, M. J.; May, M. T.; Plishka, M. J.; Robinson, K. D. Metal
in Biological Systems; Ellis Horwood: New York, 1992; pp 17-56.
(23) Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry;
University Science Books: Mill Valley, CA, 1994; pp 349-376.
(24) Dugas, H. Bioorganic Chemistry: A Chemical Approach to Enzyme
action; Springer, New York, 1996; pp 388-460.
(25) The structure coordinates were obtained from the pdb server. The
files used for modeling are: 1g6v.pdb (for CA), 1ova.pdb (for CEA),
1azf.pdb (for lysozyme), 1myt.pdb (for myoglobin).
(26) (a) Sadler, P. J.; Viles, J. H. Inorg. Chem.1996, 35, 4490-4496.
(b) Singh, A. Markowitz, M.; Tsao, L. I.; Deschamps, J. ACS Symposium
Series 556; American Chemical Society: Washington, DC, 1993; pp 252-
263.
(27) Martell, A. E.; Smith, P. M. Critical Stability Constants; Plenum
Press: New York, 1975; Vol. 2.
(28) Jiang, W.; Graham, B.; Spiccia, L.; Hearn, M. T. W. Anal. Biochem.
1998, 255, 47-58.

Surface Recognition of a Protein
J. Am. Chem. Soc., Vol. 123, No. 26, 2001 6285
Figure 1. Histidine distributions of the target protein CA (1A) and the controls, lysozyme (1B), CEA (1C) and Mb (1D) used in this study. The
solvent accessibility of the histidine residues are included in Supporting Information.
in the ITC cell was titrated with a solution of the metal complex.
The heat of dilution of the metal complex was then separately
determined by injection of a solution of the complex in buffer.
The software provided by the manufacturer (Bindworks, v 3.0,
Calorimetry Sciences Corporation, Provo, UT) was used to
correct for the heat of dilution and subsequent data processing
by nonlinear regression of the titration data. A multiple
independent binding site model fits the data best. Other binding
models (two sets of independent binding sites and cooperative
binding) did not fit the titration data well. A typical titration
curve (raw and processed data) is shown in Figure 3 (titration
of CA with 2). Results of ITC titration experiments are shown
in Table 1. The data processing gave the stoichiometry (n),
affinity (K), and the enthalpy (∆H) of the interactions. The
entropy values were calculated using the experimentally deter-
mined parameters (K, ∆H).
The control metal complex 6 (containing one cupric ion)
showed weak affinity for the proteins tested (K < 7500 M-1).
Myoglobin bound more than more equivalents of 6; with CEA,
the affinity was weak, and reliable values were difficult to
obtain. The control protein lysozyme (with one histidine on the
surface) was found to have low affinity (K < 5000 M-1) for all
of the complexes used in this study.
Figure 2. Structures of the metal complexes.
Strong binding to the target protein (CA) was achieved by
the distance-matched tris-Cu2+ complex 2 (K ) 299 000 (
Binding Studies by Isothermal Titration Microcalorim-
etry. Binding studies were performed in HEPES buffer (25 mM,
30 000 M-1). When other metal complexes were used (distances
pH ) 7.0, 25 °C) and were studied by isothermal titration
(29) Ladbury, J. E.; Chowdhry, B. Z. Biocalorimetry Applications of
microcalorimetry (ITC)29 or by UV-vis spectrometry. In a
Calorimetry in the Biological Sciences; John Wiley & Sons: New York,
typical ITC experiment, a solution of the protein (∼100 µM)
1998.

6286 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
Fazal et al.
Scheme 1a
Figure 3. Raw titration curve (3A) and processed data (3B) for the
titration of CA with the complex 2 (25 mM HEPES buffer, pH ) 7.0,
25 °C, [CA] ) 0.12 mM, [2] ) 1.0 mM, 42 × 5 µL injections).
binding constants with CA were also similar. The flexible
complex 3 had low entropic loss upon binding to CEA (we do
not have any explanations yet for this observation), leading to
a moderate affinity for the protein (K ) 70 600 M-1). If the
organic ligands corresponding to the metal complexes were used,
no binding was detected by ITC. This demonstrated the role of
the metal ions in the binding process.
a Reagents and conditions: a: HN(CH2CO2Et)2 (3 equiv), K2CO3
(10 equiv), CH3CN. Sonication, 24 h. b: LiOH (9 equiv), MeOH-
THF, 25 °C, 15 h then H3O+ (pH ) 3.0). c: CuCl2‚2H2O (3 equiv),
MeOH-H2O, 25 °C, 3 h. d: H2NCH2CH2N(CH2CO2Et)2 (11), Et3N,
BOP, CH3CN.
To demonstrate selective binding of 2 to the target protein, a
mixture of CA and complex 6 ([CA] )100 µM; [6] )100 µM)
was titrated with 2 ([2] ) 0-500 µM). No change in the binding
constant was observed for 2. On the other hand, when a mixture
of CA and 2 ([CA] ) 100 µM; [2] ) 100 µM) was titrated
with 6 ([6]) 0-1 mM), no binding was detected. A mixture of
the tested proteins (100 µM each) was titrated with complex 2
to obtain an unchanged affinity of 2 (280 000 M-1). If CA was
omitted from the protein mixture, very weak binding (K < 1000
M-1) was detected. The selectivity reported here for complex
2 is distinctly different when compared to a random distribution
of cupric ions (on chelating Sepharose fast flow with IDA-
Cu2+ at pH ) 7.0, 22 °C) either in equilibrium binding
experiments or in chromatography).30
Effect of pH. The protein-ligand interactions are very
sensitive to pH of the buffer as pH controls the protonation-
deprotonation equilibria of both the protein and ligand mol-
ecules. The interactions between carbonic anhydrase and
complex 2 were studied over the pH range 6.0-8.0. The lower
affinity at pH ) 6.0 (K ) 199 000 ( 35 000 M-1) is due to the
partial protonation of the imidazole nitrogen (pKa ) 6.8). This
protonation prevents the nitrogen atom of the imidazole groups
not matched), lower affinities were observed. Similarly if the
pattern of cupric ions was kept constant (i.e., complex 2) and
the histidine pattern on the protein surface was varied (i.e.,
proteins other than CA), binding constants were lower. A metal
complex containing more number of cupric ions (5, inter-Cu2+
distances not matched with the inter-histidine distances of the
targeted histidine triad) demonstrated weak affinities for all of
the proteins tested. The binding constants reported in Table 1
are graphically illustrated in Figure 4.
Comparison of the binding constants indicated that complex
2 is very selective for CA compared to CEA (300:1); with
myoglobin, the selectivity is moderate (20:1). For myoglobin,
two of the cupric ions of 2 may simultaneously bind to two
histidine residues on the protein surface (His 113 and 116 or
119, see the inter-histidine distances in Figure 1). This will lead
to decreased selectivity of 2 in distinguishing CA from
myoglobin. Lysozyme precipitated in the presence of the
complex 2; this prevented us from determining the binding
constant using ITC. With the complex 1, possibly the binding
is more strained with CA (∆H ) -12.7 kcal/mol for 1; -15.1
kcal/mol for 2). The flexible complexes 3, 4, and 5 showed
less strain of binding compared to 1 and 2 but considerably
more loss of entropy upon binding to CA. The three flexible
complexes have similar distances among the cupric ions. The
(30) Hutchers, T. W.; Yip, T.-P.; Porath, J. Anal. Biochem. 1998, 170,
168-182.

Surface Recognition of a Protein
J. Am. Chem. Soc., Vol. 123, No. 26, 2001 6287
Table 1. Stoichiometry (n), Binding Constant (K, M-1), Enthalpy (∆H, kcal mol-1), Entropy (T∆S, kcal mol-1) and Free Energy (∆G, kcal
mol-1) for the Interactions of the Metal Complexes with the Proteins
proteins
control 6
complex 1
<103
complex 2
complex 3
complex 4
complex 5
< 103
lysozyme
n
K
1.2 ( 0.3
PPT
0.71 ( 0.04
(4.8 ( 0.72) × 103
15.81 ( 1.36
5.01
PPT
(1.6 ( 0.2) × 103
-∆H 7.0 ( 0.5
-∆G 4.36
-T∆S 2.64
10.8
0.98 ( 0.03
carbonic anhydrase
chicken egg albumin
myoglobin
n
K
0.90 ( 0.03
1.3 ( 0.01
0.94 ( 0.006
(299 ( 30) × 103
15.1 ( 0.3
7.46
0.83 ( 0.03
1.22 ( 0.03
(3.5 ( 0.7) × 103 (75 ( 6) × 103
(36.7 ( 7.3) × 103 (33.3 ( 6.7) × 103 (33.6 ( 4.5) × 103
-∆H 39.2 ( 2.0
12.7 ( 0.2
6.64
6.06
1.6 ( 0.07
(16 ( 2.5) × 103
20.9 ( 1.5
5.73
23.62 ( 1.48
6.22
23.40 ( 2.0
6.16
17.24
20.88 (1.2
6.17
14.71
-∆G 4.83
-T∆S 34.37
7.65
17.4
1.55 ( 0.01
n
K
-∆H
-∆G
-T∆S
n
<103
<103
1.70 ( 0.02
0.90 ( 0.06
(76.5 (7.0) × 103 (32.0 ( 3.4) × 103 (18.4 ( 3.3) × 103
14.03 ( 0.26
6.65
6.97 ( 0.14
6.14
0.83
0.56 ( 0.02
(3.5 ( 0.1) × 103
81.19 ( 14.5
4.83
15.54 ( 1.8
5.81
9.73
0.53 ( 0.02
(12.3 ( 1.7) × 103
34.22 ( 5.8
5.57
15.17
0.74 ( 0.03
7.38
1.00 ( 0.04
1.6 ( 0.02
1.62 ( 0.03
K
(7.2 ( 0.6) × 103 (49 ( 6.2) × 103 (20.4 ( 5.2) × 103 (8.2 ( 0.7) × 103
-∆H 4.2 ( 0.4
21.6 ( 1.3
6.39
15.21
16.62 ( 2.4
5.87
10.75
28.09 ( 2.0
5.33
22.76
-∆G 5.25
-T∆S 1.95
76.36
28.65
Table 2. Binding Parameters of CA with 2 as a Function of Salt
Concentration
[NaCl]
M
Ka
-∆H
-∆G
-Th∆S
n
(M-1
)
kcal mol-1 kcal mol-1 kcal mol-1
0.00 0.94 ( 0.006 299,400 ( 30,200 15.1 ( 0.32
0.10 1.06 ( 0.02 232,600 ( 12,200 16.36 ( 0.72
0.20 0.98 ( 0.003 215,800 ( 14,300 16.99 ( 1.03
0.30 0.96 ( 0.002 125,400 ( 8,500 18.34 ( 0.82
0.60 1.21 ( 0.001 78,600 ( 10,200 20.26 ( 0.43
7.46
7.31
7.27
6.95
6.67
7.65
9.05
9.72
11.39
13.59
pH ) 7.0 (25 mM HEPES buffer). The binding constant was
found to decrease with increasing salt concentrations (Table 2).
When the salt concentration was increased above 0.6 M,
complex 2 was found to precipitate. This prevented us from
studying the salt effect beyond the reported concentration range.
The salt effects on immobilized Cu(II) with histidine and
proteins have been studied extensively by various authors.31,35
The interactions between solutes and immobilized Cu(II) consist
of the formation of a coordination compound, the hydrophobic
or van der Waals force, and electrostatic interactions.31 In cases
where donor-acceptor interaction is the primary force of
binding mode, addition of NaCl at a high concentration would
cause the free coordination sites to be occupied by anions. As
a result the binding constant should be decreasing with
increasing NaCl concentration. In addition, ionic strength also
affects the protein surface through accumulation of anions and
cations on it and results in a high repulsive force between the
ligand and protein.
In our study, the change in binding constant as a function of
salt concentration behaved well. In the studied concentration
range of salt, the binding constant decreased steadily. The
binding stoichiometry (n), enthalpy change (∆H), and free
energy change (∆G) suggested that the same type of binding
mechanism was in effect.
Binding Studies by UV-Vis Spectrometry. The binding
of CA with the metal complexes can be followed by UV-vis
spectrometry. Upon addition of CA to a solution of 2, the
absorption maximum of the Cu2+ ions progressively shifted from
727 to 660 nm ([2] ) 0.5 mM; [CA] ) 0.8 mM; 25 mM HEPES
buffer, 25 °C, pH ) 7.0; 10 × 100 µL additions). Further
addition of the protein did not change to the absorption
maximum. This shift indicates the coordination of one imidazole
Figure 4. Binding constants of the metal complexes with the proteins.
to donate the lone pair of electrons to the Cu2+ ions.31 On the
other hand, at high pH (8.0), the IDA-Cu2+ complexes start
forming insoluble precipitates and the binding affinity drops
(K ) 97 000 ( 16 000 M-1). In addition, at pH ) 8.0 the net
charge of IDA-Cu2+ complex is -1 which also reduces the
affinity of the complex to accept the lone pair from imidazole
group.32
Effect of Buffer Structure and Concentration. Three
buffers (HEPES, MOPS, CHES) with different concentrations
(25-100 mM) were used to study carbonic anhydrase-2
interactions. Neither the buffer type nor the buffer concentration
showed any significant effect on the binding affinity. Although
protein-ligand interactions have been reported to be influenced
by buffer types in some cases,33 the reverse is also reported.34
Effect of Ionic Strength. The effects of ionic strength on
the affinity of carbonic anhydrase and Cu2+ complex 2 were
studied by varying NaCl concentration from 0.0 to 0.6 M at
(31) Chen, W. Y.; Wu, C. F.; Liu, C. C. J. Colloid Interfac. Sci. 1996,
180, 135-143.
(32) Zachariou, M.; Hearn, M. T. W. Biochemistry 1996, 35, 202-
211.
(33) (a) Beschiaschvili, G.; Seelig, J. Biochemistry 1992, 31, 10044-
10053. (b) Doyle, M. L.; Louie, G.; Monte, P. R. D.; Sokoloski, T. D.
Methods Enzymol. 1995, 259, 183-193.
(34) Srivastava, D. K.; Wang, S.; Peterson, K. L. Biochemistry 1997,
36, 6359-6366.
(35) Lin, F. Y.; Chen, W. Y.; Sang, L. C. J. Colloid Interfac. Sci. 1999,
214, 373-379.

6288 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
Fazal et al.
from the solutions. In presence of 2, the CD spectrum of CA
was found to change in the region 220-200 nm. The spectra
(normalized to residue molar elipticity) were analyzed with the
software DICROPROT43 by self-consistent method to get
structural information from the spectral changes.
Carbonic anhydrase, in the native form, consists of 7.7%
R-helices and 25.8% â-sheets (calculated by the Structure
Explorer module of the Protein Data Bank20). Analysis of the
CD spectra of CA by DICROPROT predicted that the enzyme
has 7.2% R-helices and 26.5% â-sheets. Analysis of the CD
spectra of CA.2 indicated that the content of R-helices was 8.4%
and the amount of â-sheet was 31.8%.
The histidine triad targeted by complex 2 is located near the
N-terminus of the protein. The N-terminal region of the protein
has no ordered secondary structure; starting from residue 16, a
short R-helical region starts (analyzed by insight II). It is possible
that the random coil containing the histidines (1 and 7) may
organize into a helical structure upon binding to complex 2.
The binding energy of 2 with CA is sufficient to compensate
for the energy requirement of this structural change. We do not
have any hypothesis yet on the increase of â-sheet contents of
CA upon binding to 2. The enzyme was found to retain its full
activity in the presence of two equivalents of complex 2.
Figure 5. EPR spectra of complex 2 in absence and presence of the
protein CA.
group (of histidine) to each cupric ions of the complex 2.36 The
coordination of a second imidazole to the cupric ions is
characterized by a further large blue-shift of the absorption
maximum (628 nm).36 Analysis of the resultant titration curves37
(data not shown) corroborated the ITC results (for stoichiometry
and binding constants).
Electrophoresis Studies. Many transition metal (e.g., Co3+
,
Zn2+, Ni2+, Cu2+) complexes have been reported to cleave
amide bonds in peptides44 and proteins45 under mild conditions
(pH ) 7.0) through hydrolysis. In the literature, SDS-PAGE
has been used to monitor the hydrolysis of protein in the
presence of macrocyclic Cu2+ complexes.45 Results from SDS-
PAGE (data not shown) indicated no cleavage products for the
protein (CA) in the presence of excess 2 (CA:2 ) 1:2, pH )
7.0).
For these titration experiments, an isobestic point was
observed in the UV-vis spectra at 590 nm (Supporting
Information). Another isobestic point started to form at 400 nm;
however, when the molar ratio (complex 2:CA) was close to 2,
this isobestic point was lost. This suggests that at higher
concentrations of 2, CA may not be forming 1:1 complex with
2.
Binding Studies by EPR Spectroscopy. The role of Cu2+
-
Conclusions
histidine interactions in these studies was also demonstrated by
EPR spectroscopy in room-temperature solutions (9.44 GHz,
[CA] ) 1.2 mM; [2] ) 600 µM, 25 °C). Upon addition of the
solution of CA to a solution of the metal complex 2, the apparent
g value of Cu2+ ions decreased from 2.29 to 2.26 (Figure 5).
These values are consistent with the coordination of an imidazole
moiety to the cupric ions and displacement of a bound water
In conclusion, we have demonstrated that a protein can be
targeted with good selectivity by exploiting its surface histidine
pattern using complementary transition metal complexes. The
cupric ions coordinate to histidines exposed on the surface of
the protein being targeted. The protein preserved its native
structure in the presence of the metal complexes. Targeting of
other proteins using this methodology is being carried out and
the results will be reported in future.
molecule from the coordination sphere of IDA-Cu2+ 38
.
Circular Dichroism (CD) Studies. Circular dichroism
spectroscopy has been applied to monitor the secondary and
tertiary structural changes in many protein-ligand,39 protein-
lipid,40 peptide-ligand41 interactions. The far-UV circular
dichroism spectrum (the peptide region) of a globular protein
primarily reflects secondary structure while near-UV circular
dichroism spectra (the aromatic region) primarily reflects its
tertiary structure.42
Experimental Section
1
Syntheses of the Complexes. The H and 13C NMR spectra were
recorded using 300, 400, and 500 MHz spectrometer with TMS as the
internal standard. Elemental analyses were carried out by the in-house
material characterization laboratory. Commercially available reagents
were used as supplied unless stated otherwise. The organic extracts
were dried over anhydrous Na2SO4 and concentrated in vacuo. TLC
was performed with Absorsil Plus 1P, 20 × 20 cm plates (Alltech
Associates, Inc.). Chromatography plates were visualized either with
UV light or in an iodine chamber.
Circular dichroism studies (0.3 mM CA, 0.6 mM metal
complex 2, 25 mM phosphate buffer, pH ) 7.0, 25 °C) were
conducted to detect any structural changes to CA upon binding
to the metal complex (Supporting Information). Extensive
purging with nitrogen was used to remove dissolved oxygen
(43) Deleage, G. DICROPROT, version 2.5; Institute of Biological
Chemistry of Proteins: Lyon, France. The software is available freely from
http://www.ibcp.fr. For references on self-consistent method for analyzing
CD spectra, see: (a) Sreerama, N.; Woody, R. W. Anal. Biochem. 1993,
209, 32-44. (b) Sreerema, N.; Woody, R. W. Biochemistry 1994, 33,
10022-10025.
(44) (a) Kaminskaia, N. V.; Johnson, T. W.; Kostic, N. M. J. Am. Chem.
Soc. 1999, 121, 8663-8664. (b) Parac, T.; Ullman, G. M.; Kostic, N. M.
J. Am. Chem. Soc. 1999, 121, 3127-3135. (c) Allen, G.; Campbell, R. O.
Int. J. Peptide Protein Res. 1996, 48, 265-273.
(36) Dhal, P. K.; Arnold, F. H. Macromolecules 1992, 25, 7051-7060.
(37) Connors, K. A. Binding Constants; Wiley-Interscience, New York,
1987.
(38) Schnek, D. R.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir
1994, 10, 2382-2388.
(39) Dubins, D. N.; Filfil, R.; Macgregor, R. B., Jr.; Chalikian, T. V. J.
Phys. Chem. B 2000, 104, 390-401.
(40) Rakin, S. E.; Wais, A.; Pinheiro, T. T. Biochemistry 1998, 37,
12588-12595.
(41) Peczuh, M. W.; Hamilton, A. D.; Quesada, J. S.; Mendoza, J. D.;
Haack, T.; Giralt, E. J. Am. Chem. Soc. 1997, 119, 9327-9328
(42) Spectroscopic Methods for Determining Protein Structure in Solu-
tion; Havel, H. A., Ed.; VCH Publishers: New York, 1995; pp 5-25.
(45) (a) Puranaprapuk, A.; Leach, S. P.; Kumar, C. V.; Bocarsly, J. R.
Biochem. Biophys. Acta 1998, 1387, 309-316. (b) Allen, G.; Campbell,
R. O. Int. J. Peptide Protein Res. 1996, 48, 265-273. (c) Hegg, E. L.;
Burstyn, J. N. J. Am. Chem. Soc. 1995, 117, 7015-7016.

Surface Recognition of a Protein
J. Am. Chem. Soc., Vol. 123, No. 26, 2001 6289
Complex 1. 1,3,5-Tri-(bromomethyl)-benzene (3.0 g, 8.40 mmol)
and diethyliminodiacetate (4.76 g, 25.18 mmol) were dissolved in dry
CH3CN, and anhydrous K2CO3 (14.11 g, 100.00 mmol) was added.
The resulting reaction mixture was sonicated (125 W bath sonicator)
for 12 h at room temperature. The solids were filtered off, and solvent
was removed under vacuo. The crude yellowish oily liquid was purified
by silica gel column chromatography with 1% methanol in CHCl3 (Rf
) 0.2) to afford the pure ester. Yield: 5.22 g (91%). H NMR (400
MHz, CDCl3) δ 1.15-1.24 (m, 18H), 3.45 (s, 12H), 3.84 (s, 6H), 4.01-
4.18 (m, 12H), 7.24 (s, 3H).
(Rf ) 0.2) to afford the pure product as an oily viscous liquid. Yield:
1
0. 60 g (34%). H NMR (400 MHz, CDCl3) δ 1.23 (t, 18H, J ) 7.1
Hz), 2.94-3.00 (m, 6H), 3.46-3.54 (m, 6H), 3.55 (s, 12H), 4.14-
4.19 (m, 12H), 8.05 (bs, 1H), 8.13 (bs, 2H), 8.65 (s, 3H).
This ester was hydrolyzed (0.54 g, 0.63 mmol) using LiOH‚H2O
(0.32 g, 7.6 mmol) in a mixture of MeOH-THF (10/5 mL). The workup
procedure was the same as the hydrolysis reactions described before.
1
1
Yield: 0.32 g (69%). H NMR (400 MHz, D2O-NaOD) δ 2.79 (t,
6H, J ) 6.6 Hz), 3.21 (s, 12H), 3.44 (t, 6H, J ) 6.6 Hz), 8.28 (s, 3H).
13C NMR (125 MHz, D2O) δ 38.1, 58.6, 59.3, 132.3, 137.0, 172.4,
172.8.
The ester (2.00 g, 2.94 mmol) was dissolved in 30 mL of MeOH-
THF (2:1), and the solution of LiOH‚H2O (1.11 g, 26.43 mmol) in
MeOH (10 mL) was added. The mixture was stirred at room temperature
for 12 h, after which it was acidified with concentrated HCl (pH )
3.0). The white solid was filtered and washed with THF-MeOH
mixture (4:1). This solid was again dissolved in 3 mL of water, THF
was then added until two layers partitioned. The upper layer was
removed carefully, and the bottom layer was dried under vacuo to afford
The acid obtained in the previous step (0.18 g, 0.26 mmol) was
dissolved in water (5 mL) by adding 4 drops of 1 N NaOH solution.
The solution of CuCl2‚2H2O (0.14 g, 0.82 mmol) in MeOH (7 mL)
was added dropwise, and the reaction mixture was stirred for 4 h at
room temperature. The blue solid was filtered and washed with MeOH.
Yield: 0.2 g (77%). Anal. Calcd. for C27H30Cu3N6O15‚3H2O: C, 35.12;
H, 3.93; N, 9.10. Found: C, 35.23, H, 3.81; N, 9.35.
1
the acid as a white solid. Yield: 1.39 g (92%). H NMR (500 MHz,
Tris-bromide 9. A mixture of the 1,3,5-tris(bromomethyl)benzene
(2.78 g, 7.79 mmol), 4-hydroxylbenzyl alcohol (2.90 g, 23.37 mmol),
potassium carbonate (4.14 g, 30 mmol), and 18-Crown-6 (0.53 g, 2
mmol) in dry acetonitrile (100 mL) was refluxed under nitrogen for 4
days with vigorous stirring. The mixture was then cooled and evaporated
to dryness under reduced pressure. Water (150 mL) was added to
dissolve inorganic salts, and the white solid, after being filtered, was
washed with 5% Na2CO3, water, and ether, respectively. The product
was taken to the next step without further purification. Yield: 3.5 g
D2O) δ 3.84 (s, 12H), 4.55 (s, 6H), 7.76 (s, 3H). 13C NMR (125 MHz,
D2O) δ 58.86, 60.70, 133.84, 138.49, 172.54.
To a solution of the acid (0.65, 1.26 mmol) in water (10 mL) was
added 0.65 g (3.80 mmol) of CuCl2‚2H2O in MeOH (15 mL). The
stirring was continued at room temperature for 4 h. The blue solid was
filtered and washed with MeOH. Yield: 85%. Anal. Calcd. for C21H21-
Cu3N3O12‚3H2O: C, 33.54; H, 3.62; N, 5.59. Found: C, 33.53; H, 3.59;
N, 5.54.
Complex 2. A mixture of 746 (1.40 g, 2.07 mmol), diethyliminodi-
caetate (1.17 g, 6.21 mmol), and anhydrous K2CO3 (2.5 g, 17.86 mmol)
in MeCN was refluxed for 12 h and then sonicated for 12 h in a bath
sonicator (125 W). The solids were filtered off, and solvent was
removed under vacuo. A viscous oily yellowish liquid was obtained;
this was purified by column chromatography (silica gel, 2% MeOH in
CHCl3, Rf ) 0.4) to afford the pure ester as a viscous oil. Yield: 1.70
1
(92%); mp 42-44 °C. H NMR (300 MHz, CDCl3) δ 7.48 (s, 3H),
7.23 (d, 6H, J ) 8.1 Hz), 6.98 (d, 6H, J ) 8.1 Hz), 5.12 (s, 6H), 5.07
(t, 3H, J ) 6.1 Hz, OH), 4.43 (d, 6H, J ) 6.1 Hz); 13C NMR (75
MHz, CDCl3) δ 157.2, 137.7, 134.9, 127.9, 126.1, 114.4, 69.0, 62.5.
A suspension of the above compound (1.0 g, 2.05 mmol) in ether
(75 mL, dried by LiAlH4) was cooled to 0 °C, and a solution of PBr3
(0.86 g, 3.2 mmol) in anhydrous ether (25 mL) was added slowly with
stirring; the ice bath was then removed, and it was refluxed for 14 h.
The mixture was cooled down, and the pale yellow solid was collected,
successively washed with 2% HCl, water, NaHCO3, and water, dried
1
g (82%). H NMR (400 MHz, CDCl3) δ 1.27 (t, 18H, J ) 7.0 Hz),
3.58 (s, 12H), 3.78 (s, 6H), 4.18 (q, 12H, J ) 7.0 Hz), 7.48 (d, 6H, J
) 8.0 Hz), 7.63 (d, 6H, J ) 8.0 Hz), 7.75 (s, 3H).
1
The ester (0.94 g, 0.94 mmol) was hydrolyzed with monohydrated
LiOH (0.35 g, 8.33 mmol) in MeOH-THF (10/5 mL). The mixture
was stirred at room temperature for 12 h, after which it was acidified
with concentrated HCl (pH ) 3.0). The white solid was filtered and
washed with THF-MeOH mixture (4:1). This solid was again dissolved
in 3 mL of water, THF was then added until two layers partitioned.
Upper layer was removed carefully, and bottom layer was dried under
in air, and afforded 1.0 g (72%) of tris-bromide: mp 80-82 °C. H
NMR (CDCl3) δ 7.44 (s, 3H), 7.32 (d, 6H, J ) 8.5 Hz), 6.92 (d, 6H,
J ) 8.5 Hz), 5.09 (s, 6H), 4.50 (s, 6H); 13C NMR (CDCl3) δ 158.7,
137.8, 130.6, 130.5, 126.0, 115.2, 69.8, 33.9.
Complex 4. A mixture of 9 (0.3 g, 0.44 mmol), diethyl iminodiac-
etate (0.25 g, 1.23 mmol), and K2CO3 was taken in CH3CN (25 mL)
and sonicated in a bath sonicator (125 W) for 40 h. The workup
procedure was the same as that described before. The resulting crude
oily yellowish liquid was purified by silica gel column chromatography
with 2% MeOH in CHCl3 (Rf ) 0.7). Yield: 58%. 1H NMR (270 MHz,
CDCl3) δ 1.25 (t, 18H, J ) 7.1 Hz), 3.51 (s, 12H), 3.83 (s, 6H), 4.15
(q, 12H, J ) 7.1 Hz), 5.06 (s, 6H), 6.91 (d, 6H, J ) 8.5 Hz), 7.28 (d,
6H, J ) 8.5 Hz), 7.46 (s, 3H).
The ester (0.24 g, 0.24 mmol) was hydrolyzed with LiOH‚H2O (30
mg, 2.14 mmol) in MeOH-THF (5/5 mL) as described for other
compounds. The acid was obtained as a yellowish solid (0.15 g, 87%).
1H NMR (400 MHz, D2O) δ 3.46 (s, 12H), 4.07 (s, 6H), 4.99 (s, 6H),
6.93 (d, 6H, J ) 8.1 Hz), 7.28 (d, 8H, J ) 8.1 Hz), 7.38 (s, 3H). 13C
NMR (125 MHz, D2O) δ 58.1, 60.2, 70.5, 118.0, 124.3, 128.8, 135.2,
139.8, 161.8, 172.5.
1
vacuo to afford the acid as a white solid. Yield: 0.68 g (87%). H
NMR (270 MHz, D2O) δ 3.13 (s, 12H), 3.76 (s, 6H), 7.45 (d, 6H, J )
8.1 Hz), 7.71 (d, 6H, J ) 8.0 Hz), 7.84 (s, 3H). 13C NMR (125 MHz,
D2O-NaOD) δ 59.8, 60.4, 126.9, 129.6, 133.1, 139.3, 141.8, 144.1,
182.1.
To a suspension of the acid (0.50 g, 0.60 mmol) in water/MeOH
(6/10 mL) was added 1 N solution of NaOH solution until the solid
completely dissolved (pH ≈ 7.0). A solution of CuCl2‚2H2O (0.30 g,
1.80 mmol) in MeOH was then added and stirred for 4 h at room
temperature. The blue solid was filtered and washed with MeOH.
Yield: 0.56 g (80%). Anal. Calcd. for C39H33Cu3N3O12‚3H2O: C, 49.60;
H, 3.74; N, 4.45: Found: C, 49.68, H, 3.90, N, 4.52.
Complex 3. To a solution of 1,3,5-benzenetricarboxylic acid (0.44
g, 2.09 mmol) in CH3CN (40 mL), was added Et3N (4 mL, 29.33 mmol)
followed by the addition of BOP reagent (2.8 g, 6.32 mmol) and 11‚
2TFA14 (2.88 g, 6.26 mmol) in CH3CN (20 mL). Stirring was continued
at room temperature for 24 h. The reaction was quenched with saturated
aqueous solution of NaCl. The mixture was then extracted with ethyl
acetate, and the extract was successively washed with 4% aqueous citric
acid, water, 4% aqueous NaHCO3, and water. The organic layer was
dried over anhydrous Na2SO4, and solvent was evaporated under vacuo
to provide the crude amide ester. Purification was achieved by silica
gel column chromatography with 4% MeOH in CHCl3 as the eluant
A solution of CuCl2‚2H2O (0.07 g, 0.42 mmol) in MeOH (5 mL)
was added to the solution of above acid (0.12 g, 0.14 mmol) in water
(5 mL) at room temperature. The workup procedure was the same as
that for complex 1. Yield: 0.12 g (79%). Anal. Calcd. for C42H39-
Cu3N3O15‚3H2O: C, 47.12; H, 4.24; N,3.93. Found: C, 47.34; H, 4.41;
N, 3.73.
Complex 5. Tetraacid 1047 (0.54 g, 1.34 mmol) was dissolved in
DMF-CHCl3 (40 mL, 1:1) mixture in the presence of Et3N (1.5 mL,
10.78 mmol) followed by the addition of 11 (1.68 g, 5.35 mmol) and
BOP reagent (2.30 g, 5.35 mmol). The resultant mixture was stirred
for 30 h at room temperature. The workup procedure was the same as
that described complex 3. The crude product was purified by silica gel
column chromatography (Rf ) 0.4 with 10% MeOH in CHCl3). Yield:
(46) Kim, Y. H.; Beckerbauer, R. Macromolecules 1994, 27, 1968-
1971.
(47) Hamachi, I.; Matsugi, T.; Shinkai, S. Tetrahedron Lett. 1996, 51,
9233-9236.
1
1.06 g (63%), mp 38-40 °C. H NMR (500 MHz, CDCl3) δ 1.27 (t,

6290 J. Am. Chem. Soc., Vol. 123, No. 26, 2001
Fazal et al.
24H, J ) 7.0 Hz), 2.31-2.49 (m, 8H), 2.81-2.88 (m, 8H), 3.15-3.26
(m, 16H), 3.53 (bs, 4H), 3.71-3.76 (m, 4H), 4.15 (q, 16H, J ) 7.0
Hz), 7.40 (bs, 2H), 8.08 (s, 2H).
into the reaction cell containing only the sample buffer. For every
experiment the heat of dilution of the ligand was measured and
subtracted from the sample titration values before data processing.
The temperature-dependent and ionic strength-dependent binding
studies were conducted after the instrument had been equilibrated
overnight at the required temperature or salt concentration. For all the
experiments, the quantity c ) K × [M], where [M] is the initial
macromolecule concentration and K is association constant, was in the
range of 2 < c < 200 as required for isothermal microcalorimetry.
UV-Visible Spectroscopy. Solutions of CA (0.8 mM) and Cu2+
complexes (0.5 mM) were prepared in 25 mM HEPES buffer (pH )
7.0). For routine analysis at room temperature (23 °C), the absorption
spectra were recorded in the 200-800 nm range against the blank
(HEPES buffer). Titration studies were conducted by adding aliquots
of CA to the cell containing a known initial concentration of 2. To
make solutions with a fixed mole ratio (2:CA), appropriate volumes
of prepared solutions were mixed. All measurements were made after
sample equilibration for at least 15 min.
The ester (0.28 g, 0.22 mmol) was hydrolyzed with LiOH‚H2O (0.14
g, 0.33 mmol) in MeOH-THF solvent (5/5 mL) as described
1
previously. Yield: 0.18 g, 59%. H NMR (500 MHz, 60 °C, D2O) δ
3.42-3.60 (m, 4H), 3.64-3.75 (m, 4H), 3.76-3.90 (m, 16H), 3.92-
4.12 (m, 16H), 4.16-4.22 (s, 16H). 13C NMR (125 MHz, D2O) δ 37.4,
39.9, 50.9, 54.8, 57.2, 57.4, 57.8, 58.0, 58.2, 59.5, 59.6, 59.7, 60.7,-
172.72, 172.74, 172.56, 171.78, 172.80.
The acid (0.30 g, 0.28 mmol) was dissolved in 10 mL of H2O (the
insoluble part was filtered off). The solution of CuCl2‚2H2O (0.05 g,
0.29 mmol) in MeOH (7 mL) was added dropwise and stirred at room
temperature for 3 h. The solvent was removed in vacuo. MeOH-H2O
(10 mL, 5:2) was added and stirred another 0.5 h. The solid was filtered
and washed with MeOH. Yield: 0.32 g (86%). Anal. Calcd. for C40H68-
Cu5N12O20‚2Cl-‚5H2O: C, 31.86; H, 4.68; N, 11.14. Found: 31.96;
H, 4.31; N, 10.90.
Determination of Binding Constants. Calorimetric studies were
performed with Isothermal Titration Calorimeter (ITC-4200, Calorim-
etry Sciences Corporation, Provo, UT) linked to a personal computer
for data acquisition and analysis. An external water bath (NESLAB,
RTE-111) was used to maintain the desired temperature ((0.01 °C).
A continuous stream of dry nitrogen was used to prevent moisture
condensation in the unit. Hamilton syringes were used to load the
sample solution (cell volume ) 1.32 mL). A CENTRA-CL2 unit was
used for centrifugation. Absorption spectra were recorded using a
Ultroscope-4000 (Pharmacia Biotech) UV/visible spectrophotometer.
Quartz cuvettes of 10 mm path length were used for all routine studies.
The temperature effect on UV-visible spectra was studied using a
Beckmann-60 unit fitted with a constant temperature accessory. DSC
experiments were carried out with a Perkin-Elmer (DSC 7) unit and
the sample cells provided by the manufacturer (KIT No. 0219-0041)
were used. For EPR study a Varian model E-4 EPR spectrometer was
used. Spectropolarimeter from Japan Spectroscopic Co. Ltd. (JASCO,
J-810) was employed for CD measurements. Mini PROTEAN II from
Bio-Rad was used for electrophoresis studies. Analytical balance (Ohaus
AP 250D) was used for weighing samples (accuracy ) 0.1 mg).
Buffers. Buffers were prepared by adjusting the pH (7.0 ( 0.01) at
25 °C with 1 N NaOH. Sodium azide (1%, W/V) was added as the
preservative. After passing through a 0.22 µm cellulose membrane
(Stericup, Millipore), the final solution was stored at 4 °C. Dilute HCl
and NaOH solutions were used for further adjustment in pH (6.0-
8.0). No consideration was made to take the temperature effect into
account.
Isothermal Titration Calorimetry (ITC). Calorimetric measure-
ments were conducted in aqueous solution (25-100 mM, HEPES,
MOPS, CHES; pH ) 6.0-8.0). Protein samples (0.075-1.5 mM) were
routinely centrifuged prior to the titration and were examined for
precipitates, if any, after the titration. No precipitate was observed even
at the highest working temperature and ionic strength conditions. The
final concentrations of the protein solutions were determined by UV-
visible spectrophotometry at 280 nm. Similarly Cu2+ complexes were
followed at 720-727 nm by UV-vis spectrophotometry. All solutions
were thoroughly degassed by stirring under reduced pressure before
use. The protein solution was placed in the ITC cell for protein-metal
complex study. The metal complex of choice was loaded into the
syringe. For all experiments the freshly prepared protein and Cu2+
complex solutions were used. An equilibration period of 30-45 min
was used before starting the experiment.
Electron Paramagnetic Resonance (EPR). Solutions of CA and
Cu2+ complex were made separately in HEPES buffer (25 mM, pH )
7.0). The solutions were checked for any precipitation and subjected
to centrifugation if any precipitation was observed. The final concentra-
tions of the solutions ([CA] ) 1 mM; [2] ) 0.5 mM) were determined
by UV-vis spectrophotometry. The CA‚2 complex was studied by
mixing the respective solutions in required proportion to get the protein
in excess (CA:2 ) 2:1). All data were under the following conditions:
frequency ) 9.442 GHz; window width ) 2000 G; amplifier gain )
104; modulation amplitude ) 20 G; rate ) 8.3 G/s; T ) 25 °C. The
reported spectra were obtained by averaging four individual scans.
Circular Dichroism (CD). Far-UV (190-260 nm) circular dichro-
ism spectra were acquired for CA and complex 2 separately and in
mixture (phosphate buffer, 25 mm, pH ) 7.0, 25 °C) under continuous
nitrogen purging. The protein and Cu2+ complex (2) concentrations
were 0.3 and 0.6 mM, respectively. The protein-Cu complex mixture
had a molar ratio of 1:2 (CA:2). All spectra were recorded using quartz
cells with 1 mm path length. All spectra were corrected for the
appropriate background. The final spectrum of each sample was an
average of eight scans. The sample temperature was controlled at 23
( 0.2 °C throughout the experiments.
Electrophoresis (SDS-PAGE). CA (0.1 mM) and CA‚2 complex
(CA:2 ) 1:2) solutions were prepared in HEPES buffer (25 mM, pH
) 7.0). The samples (10 µL/well) were then analyzed by electrophoresis
through a 15% polyacrylamide gel with a running buffer (25 mM TRIS,
92 mM glycine, 0.1% SDS) of pH 8.3. The operating voltage was kept
constant at 200 V. At the completion of the run (30-40 min), the gel
was stained with a dye (0.1% Commassie Blue R-250, 45% CH3OH,
45% H2O, and 10% glacial acetic acid) for 20-25 min. Destaining
was achieved by washing the gels repeatedly with freshly prepared
destain solution (10% CH3OH, 10% glacial acetic acid, 80% H2O).
After drying overnight, the gels were scanned to collect the final images.
Acknowledgment. This work was supported by a NSF-
CAREER award (CHE-9896083) and an NIGMS-AREA Grant
(1R15 59594-01, NIH) to S.M.; USDA-NRI (97-35305-5158)
and the Herman Frasch Foundation award (446-HF97) to K.R.R.
S.M. would like to acknowledge Richard Nameth of Jasco for
his help in recording the CD spectra.
Supporting Information Available: Solvent accessibility
surfaces of the histidines shown in Figure 1, ITC titration plots
(raw and processed data) for the results reported in Table 1,
UV-vis titration curve for CA with complex 2 and CD spectra
of CA.2 (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
A typical titration consisted of injecting 5 µL of ligand solution (42
aliquots) into the cell with an interval of 5.0 min to ensure that the
titration peak returned to the baseline prior to the next injection. To
achieve a homogeneous mixing in the cell the stirrer speed was kept
constant at 300 rpm. The heats of dilution were determined under
identical conditions by injecting the appropriate metal complex solution
JA003193Z