Inhibition of Ribonuclease A by polyphenols present in green tea
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proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS
Inhibition of Ribonuclease A by polyphenolspresent in green teaKalyan S. Ghosh, Tushar K. Maiti, Joy Debnath, and Swagata Dasgupta*
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
INTRODUCTION
Mammalian ribonucleases are noncytosolic endonucleases, which cleave ribonucleic
acid (RNA) in a mechanism that involves a transphosphorylation step followed by
hydrolysis.1,2 Ribonucleases may be cytotoxic when they adsorb specifically to certain
cells, enter the cytosol, degrade RNA, and consequently inhibit protein syntheses that
results in cell death. Ribonuclease inhibitors, either synthetic or natural have been
intensively sought after for therapeutic purposes. This is largely because a growing
number of ribonuclease type enzymes such as angiogenin,3 eosinophil-derived neuro-
toxin (EDN),4 and bovine seminal RNase A5 exhibit biological activities that have been
shown to play important roles in human disease processes.6
The use of natural compounds as possible functional inhibitors of proteins is cur-
rently gaining importance and the focus on ribonucleases with unusual biological prop-
erties has generated renewed interest. The usual toxicity associated with synthesized
compounds is eliminated to a great extent by the consideration of natural inhibitors.
Epidemiological studies show a lower incidence of cancer among regular drinkers of
green tea.7,8 Green tea extracts, which have a marked antioxidant effect,9 contain the
polyphenols (�)-epicatechin (EC), (�)-epicatechin gallate (ECG), (�)-epigallocatechin
(EGC), and (�)-epigallocatechin gallate (EGCG) (Fig. 1).10
The ribonucleolytic center of RNase A is constituted of multiple subsites (P1, B1, and
B2) (Fig. 2) that bind to the phosphate, nucleobase, and sugar components of the RNA
molecule.11 The most important binding site is the P1 binding site (comprised of His
12, Lys 41, His 119) where cleavage of the phosphodiester bond occurs. Thus inhibition
of the ribonucleolytic activity of RNase A may be possible by directly blocking the
active site12 that would lead to a disruption of enzymatic activity. Apart from
the amino acid residues directly involved in the catalytic process, residues present in
the various subsites are known to play an indirect role in the catalytic mechanism.11
In a previous report we have shown that EGCG acts as a noncompetitive inhibitor of
RNase A, indicating that it does not interact directly with the ribonucleolytic site.13
Apart from an agarose gel based assay and enzyme kinetics conducted for EGCG earlier,
this study has been conducted with the other major components present in green tea
with the inclusion of further spectroscopic and theoretical studies for EGCG. The inter-
actions of the polyphenols with RNase A have been studied by UV–Vis spectroscopy,
Fourier transformed infrared (FTIR), and circular dichroism (CD). Our studies using
similar techniques to investigate the interaction of EGCG with human serum albumin
(HSA) and of catechin and EC with human and bovine serum albumins have been
reported.14,15 Binding parameters obtained from these experiments have been calcu-
Grant sponsor: Council of Scientific and Industrial Research (CSIR), India; Grant number: 37(1188)/04/EMR-II
Tushar K. Maiti’s current address is Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel 76100.
*Correspondence to: Dr. Swagata Dasgupta, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India.
E-mail: swagata@chem.iitkgp.ernet.in
Received 7 December 2006; Revised 8 February 2007; Accepted 16 February 2007
Published online 10 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21484
ABSTRACT
We report the effect of the
natural polyphenolic com-
pounds from green tea on the
catalytic activity of Ribonucle-
ase A (RNase A). The com-
pounds behave as non-
competitive inhibitors of the
protein with inhibition con-
stants ranging from 80–1300
lM. The dissociation con-
stants range from 50–150 lM
for the RNase A–polyphenol
complexes as determined by
ultraviolet (UV) and circular
dichroism (CD) studies. We
have also investigated the
changes in the secondary
structure of RNase A on com-
plex formation by CD and
Fourier transformed infrared
(FTIR) spectroscopy. The pres-
ence of the gallate moiety has
been shown to be important
for the inhibition of enzymatic
activity. Docking studies for
these compounds indicate that
the preferred site of binding is
the region encompassing resi-
dues 34–39 with possible
hydrogen bonding with Lys 7
and Arg 10. Finally we have
also looked at changes in the
accessible surface area of the
interacting residues on com-
plex formation for an insight
into the residues involved in
the interaction.
Proteins 2007; 69:566–580.VVC 2007 Wiley-Liss, Inc.
Key words: green tea poly-
phenols; Ribonuclease A; non-
competitive inhibition; FTIR;
circular dichroism; FlexX sin-
gle molecule docking.
566 PROTEINS VVC 2007 WILEY-LISS, INC.
lated to determine the types of interactions involved. To
substantiate our experimental studies we have also per-
formed docking studies to obtain a clearer insight into
the residues involved in the interaction. This study is
pertinent because RNase A is a convenient model system
of the ribonuclease superfamily. The focus on the ribonu-
cleolytic site in the development of small molecule inhib-
itors is because of its key role in the biological function
of these proteins.16 This study shows that apart from the
ribonucleolytic site, it is reasonable to target other sites
in the rational development of inhibitors of ribonuclease
type proteins.
EXPERIMENTAL
Materials
Bovine pancreatic RNase A, yeast tRNA, 20,30-cCMP,
EC, ECG, EGC, and EGCG were from Sigma Aldrich.
D2O was from Acros Organics and all other reagents
were from SRL India. UV measurements were made
using a Perkin Elmer UV–Vis spectrophotometer (Model
Lambda 25). Concentrations of the solutions were deter-
mined spectrophotometrically using the following data:
for RNase A e278.5 ¼ 9800 M�1 cm�1,17 for 20,30-cCMP
e268 ¼ 8500 M�1 cm�1,18 and e270 values for EC, ECG,
EGC, and EGCG are 4290, 12443, 2088, 11,920 M�1
cm�1 19 respectively.
Methods
Agarose gel-based assay
Inhibition of RNase A by the four polyphenols was
checked qualitatively by the degradation of tRNA in an
agarose gel. In this method, 20 lL of RNase A (1.5 lM)
was mixed with 20 lL each of 0.30 mM EGCG, EGC,
ECG, EC to a final volume of 50 lL and the resulting
solutions were incubated for 6 h at 378C. Twenty micro-
liter aliquots of the incubated mixtures were then mixed
with 20 lL of tRNA solution (5.0 mg/mL tRNA, freshly
dissolved in RNase free water) and incubated for another
30 min. Ten microliter of sample buffer which contains
10% glycerol and 0.025% bromophenol blue was added
to the mixture. Fifteen microliter from each solution was
extracted and loaded onto a 1.1% agarose gel. The gel
was run using 0.04M Tris-Acetic acid-EDTA (TAE) buffer
(pH 8.0). The undegraded tRNA was visualized by ethi-
dium bromide staining under UV light.
Precipitation assay
Inhibition of the ribonucleolytic activity of RNase A
was assayed by the precipitation assay as described by
Bond.20 In this method 10 lL of RNase A (5.6 lM) was
mixed with varying concentrations (0–0.12 mM) of EC,
ECG, EGC, and EGCG to a final volume of 100 lL and
incubated for 2 h at 378C. Twenty microliter of the
Figure 1Structures of the four major polyphenols present in green tea.
Figure 2Schematic representation of the active center cleft in the RNase A substrate
complex. B1, R1, and P1 correspond to the base, ribose and phosphate
recognition sites that comprise the main binding site where the catalytic reaction
takes place.
Inhibition of RNase A by Green Tea Polyphenols
DOI 10.1002/prot PROTEINS 567
resulting solutions from the incubated mixtures were
then mixed with 40 lL of tRNA (5 mg/mL tRNA freshly
dissolved in RNase A free water), 40 lL of phosphate
buffer of pH 6.5 containing 5 mM EDTA and 0.5 mg/
mL HSA. After incubation of the reaction mixture at
258C for 30 min, 200 lL of ice-cold 1.14N perchloric
acid containing 6 mM uranyl acetate was added to
quench the reaction. The solution was then kept in ice
for another 30 min and centrifuged at 48C at 12,000
rpm for 5 min. Hundred microliter of the supernatant
was taken and diluted to 1 mL. The change in absorb-
ance at 260 nm was measured and compared with a
control set.
Inhibition kinetics
The inhibition of RNase A by the polyphenols was
assessed individually by a spectrophotometric method as
described by Anderson et al.18 The assay was performed
in oligo vinylsulphonic acid free (using the method as
described by Smith et al.21) 0.1M Mes-NaOH buffer,
pH 6.0 containing 0.1M NaCl using 20,30-cCMP as the
substrate. The inhibition kinetics for EGCG has been
previously reported from this laboratory.13 For EC,
ECG, and EGC the substrate concentrations ranged
from 0.10 to 0.30 mM and the inhibitor concentration
ranges for EC, ECG, and EGC were from 0 to �0.25
mM. The RNase A concentration used was 12 lM. The
inhibition constants were determined from initial veloc-
ity data. The reciprocal of initial velocity was plotted
against the reciprocal of substrate concentration (Line-
weaver–Burk plot) at a constant inhibitor concentration
according to the equation:
1
V¼ Km
Vmax
1 þ I½ �Ki
� �1
S½ � þ1
Vmax
1 þ I½ �Ki
� �
where V is the initial velocity, [S] the substrate concen-
tration, [I] the inhibitor concentration, Km the Michaelis
constant, Ki the inhibition constant, and Vmax the maxi-
mum velocity. A rearranged form of this equation leading
to the Eadie–Hofstee plot has been used to confirm the
Km, Ki, and Vmax values. In this plot, usually used for
low substrate concentrations in enzyme kinetics, V vs. V/
[S] is plotted to obtain the kinetic constants.
Determination of association constants ofRNase A–Polyphenol complexes
The RNase A–polyphenol binding constants were
determined by spectrophotometric methods according to
published methods.22–24 By assuming that there is only
one type of interaction between the polyphenols and
RNase A in aqueous solution, the overall binding con-
stant can be written as follows considering the equilib-
rium.
RNase A þ Polyphenol , RNase A � Polyphenol
K ¼ ½RNase A � Polyphenol�½RNase A�½Polyphenol�
The double reciprocal plot of 1/(A � A0) versus 1/[L]
is linear, where A0 is the initial absorbance of the free
protein and A is the absorbance at different ligand con-
centrations at 280 nm. The binding constant K can be
estimated from the ratio of the intercept to the slope.
For EC, ECG, EGC, and EGCG the polyphenol concen-
trations were varied from 3.5 to 50 lM. The RNase A
concentration was 4.4 lM for EC, ECG, and EGC. For
EGCG, the protein concentration was kept at 3.5 lM.
CD measurements
CD measurements were made on a Jasco-810 auto-
matic recording spectrophotometer, using a path length
of 10 mm at 258C. The spectra were recorded in the
range of 190–240 nm with a scan rate of 50 nm/min and
a response time of 4 s. For baseline correction, CD spec-
tra of buffer (20 mM phosphate buffer of pH 7.0)
containing variable concentrations of polyphenols were
collected and were subtracted from each sample spectra.
The polyphenol concentrations were varied from 0.05 to
0.55 mM with the RNase A concentration at 11.2 lM.
For EGCG, the RNase A concentration was 14.8 lM. For
comparison with the results from FTIR studies, the CD
spectra of RNase A ligand complexes were collected at
protein to ligand molar ratios of 1:0.5 and 1:1.5. Second-
ary structures were determined using DICHROWEB,25
an online server for protein secondary structure analyses
from CD spectroscopic data.
Calculation of binding constant from CD data
We used the ellipticity ratio (Y222/Y208)26 as a con-
venient parameter (response) to follow changes in the
secondary structure of RNase A upon addition of the
four ligands using the following relationship as described
by Zhai et al.27
R ¼ R0 þ DRsat
½L�Kd þ ½L� ; DRsat ¼ Rsat � R0
where R is the observed response at a given polyphenol
concentration, R0 is the response at zero polyphenol con-
centration, and Rsat is the response at saturation. Kd is
the effective site dissociation constant, and [L] is the free
ligand concentration, which, under the conditions of the
experiment, is approximately equal to the total polyphe-
nol concentration, added to the cuvette. We have also
used this expression to determine the dissociation con-
K.S. Ghosh et al.
568 PROTEINS DOI 10.1002/prot
stants of the complexes formed between EC and catechin
with human and bovine serum albumins.15 It should be
noted that Kd is not the actual site dissociation constant,
but an effective site dissociation constant that reflects the
shape of the binding curve and not the number of sites
present on the macromolecule. If n equivalent and inde-
pendent sites exist on a macromolecule, the number of
bound ligands can be equated to n[L]/(Kd þ[L]). Data
were fit to the above equation using Microcal Origin 6.0.
Fourier transformed infrared studies
For the FTIR studies 15–18 mg/mL RNase A was dis-
solved in 20 mM phosphate buffer of pD 7.2 in 99.9%
D2O. RNase A–polyphenol complex solutions were pre-
pared by mixing the ligands and RNase A maintaining
final ligand-protein molar ratios of 0.5:1 and 1.5:1. FTIR
measurements were carried out at 258C on a Nexus-870
FTIR spectrometer (Thermo Nicolet Corporation)
equipped with a germanium-attenuated total reflection
accessory, a DTGS KBr detector and a KBr beam splitter.
All spectra were taken via the attenuated total reflection
(ATR) method with resolution of 4 cm�1, with 256 scans
each. A control buffer spectra (20 mM phosphate buffer
of pD 7.2) was also recorded under identical conditions.
The background was corrected before every sample.
IR spectra processing procedure
The relative amounts of secondary structural elements
of RNase A and the RNase A complexes with four cate-
chins were determined following the method of Byler and
Susi.28 The spectra obtained after buffer subtraction were
smoothed by an 11 point Savitsky–Golay smooth function
to reduce noise. The protein secondary structure content
was determined from the amide I band located between
1700 and 1600 cm�1. Fourier self-deconvolution and sec-
ondary derivative calculations were applied to estimate the
number and position of the component bands. Based on
these parameters a multiple Gaussian curve-fitting process
was carried out in the region 1700–1600 cm�1 of the am-
ide I band to quantify the area of each component. The
secondary structural elements were obtained from the area
under the Gaussian curve. The resultant fit is analyzed as
follows: each Gaussian band is assigned to a secondary
structure according to the frequency of its maximum: a-
helix (1650–1662 cm�1), b-sheet (1610–1632 cm�1), ran-
dom coil (1636–1644 cm�1), turn (1665–1680 cm�1), and
b-antiparallel (1680–1692 cm�1).29,30 The areas of all the
component bands assigned to a given conformation are
then summed and divided by the total area. The number
obtained is taken as the proportion of the polypeptide
chain in that particular conformation.28
Docking studies
The crystal structure of RNase A (PDB entry 1FS331)
was downloaded from the Protein Data Bank.32 We have
chosen 1FS3 for the docking studies since this is the
structure of the wild type bovine pancreatic RNase A. This
should be able to appropriately complement our experi-
mental results that have been conducted with the same pro-
tein. The 3D structures of green tea polyphenols were gen-
erated by Sybyl6.92 (Tripos, St. Louis) and their energy-
minimized conformations were obtained with the help of
the TRIPOS force field using Gasteiger–Huckel charges
with a gradient of 0.005 kcal/mole. The FlexX software as
part of the Sybyl suite was used for docking of the polyphe-
nols to RNase A. The ranking of the generated solutions is
performed using a scoring function that estimates the free
binding energy DG of the protein-ligand complex.33
PyMol34 was used for visualization of the docked confor-
mations. The theoretical value of the dissociation constants
(Kd) of the docked structures of the protein ligand com-
plexes was computed using PEARLS, which computes inter-
action energies for receptor ligand systems.35
Accessible surface area calculations
The accessible surface area (ASA) of RNase A (uncom-
plexed) and their docked complexes with EC, ECG, EGC,
EGCG were calculated using the program NACCESS.36
The structures corresponding to the minimum score as
obtained from the FlexX analysis of the protein-ligand
docked structures were chosen in each case. Composite
coordinates of the polyphenols and RNase A were gener-
ated to form the docked complex. The change in ASA
for residue, i was calculated using: DASAi ¼ ASAiRNase A�
ASAiRNase A�polyphenol. If a residue lost more than 10 A2
ASA when going from the uncomplexed to the com-
plexed state it was considered as being involved in the
interaction.
RESULTS AND DISCUSSION
The use of natural product based compounds for the
inhibition of RNase A that may subsequently be used for
the rational design of more potent inhibitors is the basis
of this study. This approach to understand the inhibition
of ribonucleases has been investigated by an initial look
at the effect of the green tea polyphenols on RNase A.
The notion being that the inhibition of the ribonucleo-
lytic activity of RNase A, information on which is largely
available, would allow us to be able to rationally design
inhibitors for proteins belonging to the ribonuclease
superfamily e.g. angiogenin, that are known to possess
unusual biological properties.3
The inhibition of the ribonucleolytic activity of RNase
A was initially checked by an agarose gel based assay,
where the degradation of tRNA by RNase A was moni-
tored [Fig. 3(A)]. The most intense band observed in
lane 1 is due to the presence of the control, tRNA.
The faint intensity of the band in lane 2 is due to the
degradation of tRNA by RNase A. The differential inten-
Inhibition of RNase A by Green Tea Polyphenols
DOI 10.1002/prot PROTEINS 569
sity of bands in lanes 3–6 indicates the degree of RNase
A inhibition by EGCG, EGC, ECG and EC respectively.
These results qualitatively show that the compounds are
capable of effectively inhibiting the ribonucleolytic activ-
ity of RNase A. The concentrations of the polyphenols
being the same in each lane, the EGCG lane appeared
more intense than the other three components. This
indicates that the major polyphenolic constituent of
green tea, EGCG acts as the most potent inhibitor
of RNase A. The inhibition of the ribonucleolytic activity
of RNase A by the polyphenols was further confirmed
quantitatively by a precipitation assay. The plots of rela-
tive ribonucleolytic activity versus inhibitor concentration
[Fig 3(B)] give us a comparative idea about the inhibi-
tory powers of the individual polyphenols. Considering a
0.04 mM concentration of EC, ECG, EGC and EGCG the
percent inhibition is found to be 4.4, 12.7, 6.9, and 18.4,
respectively. It is interesting to note that polyphenols
containing the gallate moiety are more effective inhibi-
tors. This feature is also apparent in the other studies
discussed later.
To determine the nature of inhibition and the inhibi-
tion constants, kinetic experiments were conducted. The
reciprocal of reaction velocity was plotted against the
reciprocal of substrate concentration for three different
inhibitor concentrations for each polyphenol. The inhibi-
tion constant values obtained for EC, ECG and EGC are
1336 � 2.5, 392 � 23, and 671 � 18 lM respectively
have been calculated from both the Lineweaver–Burk and
Eadie–Hofstee plots. The unchanged slopes of the lines
in Eadie–Hofstee plots (Fig. 4) are indicative of a non-
competitive type of inhibition. The inhibition constant in
case of EGCG for RNase A is 81 lM as obtained from a
previous study in this laboratory.13 We find that all the
inhibitors behave in a noncompetitive fashion. The order
of values obtained for the inhibition constants correlate
well with those obtained from the precipitation assay.
Our results indicate that the ribonucleolytic inhibitory
power of the catechins follows the order EGCG > ECG
> EGC > EC. Following this study the overall binding
constants for the RNase A–polyphenol complexes were
determined spectrophotometrically. From Figure 5 the
association constants obtained for the EC, ECG, EGC,
EGCG complexes with RNase A are 6.66 � 0.01 3 103,
1.26 � 0.03 3 104, 7.89 � 0.01 3 103, and 2.04 � 0.02
3 104 M�1 respectively. The association constant value
for EGCG is highest, which implies that it forms the
most stable complex with RNase A. This is in good
agreement with the results of the inhibition studies (aga-
rose gel, precipitation assay, and inhibition kinetics),
where EGCG was also found to be the most potent in-
hibitor. It may also be noted that the order of values for
the association constants is EGCG > ECG > EGC > EC.
In both studies, we observe that polyphenols with a gal-
late moiety exhibit a greater potency for inhibition.
The results from the kinetic experiments that indicate
a noncompetitive mode of inhibition prompted us to
further probe the effects of these compounds on the sec-
ondary structure of RNase A by CD and FTIR experi-
ments. CD spectra of RNase A exhibit two negative
bands in the ultraviolet region at 208 and 222 nm that
are characteristic of a-helices in proteins (Fig. 6). The
CD spectroscopic studies indicate that polyphenol bind-
ing to RNase A results in perturbations of the protein
secondary structure upon complexation (Table I). The
CD spectra of free RNase and its complexes with EC,
ECG, EGC, and EGCG are shown in Figure 6. The CD
spectra of RNase A at various concentrations of EC,
ECG, EGC and EGCG were analyzed in terms of the
ratio of ellipticity at 222 to 208 nm. The variations of
Y222/Y208 with the addition of ligands are shown in Fig-
ure 7 for the four compounds. The ellipticity ratio
increased hyperbolically with increasing concentration of
the ligand, yielding dissociation constants (Kd) of 141 �9, 80 � 3, 112 � 12, and 40 � 1 lM for EC, ECG, EGC,
and EGCG, respectively. The binding stoichiometries for
EC, ECG, EGC, and EGCG were found 1.31, 1.16, 1.13,
Figure 3A. Agarose gel based assay for the inhibition of RNase A by the green tea
polyphenols. Lane1: tRNA; Lane 2: RNase A and tRNA; Lane 3: EGCG, RNase
A and tRNA; Lane 4: EGC, RNase A and tRNA; Lane 5: ECG, RNase A and
tRNA; Lane 6: EC, RNase A and tRNA. B. Relative ribonucleolytic activity plots
of inhibition of RNase A by EC (-~-), ECG (-^-), EGC (-n-) and EGCG
(-3-).
K.S. Ghosh et al.
570 PROTEINS DOI 10.1002/prot
and 1.09 as obtained from fitting results. The results
show that the dissociation constants for protein ligand
complexes are consistent with the inhibition constants of
the polyphenols. The reciprocal of the association con-
stants obtained from UV–Vis experiments are found to
be 150 � 0.32, 79 � 2.7, 127 � 0.28, 49 � 0.68 lM for
RNase A-EC, RNase A-ECG, RNase A-EGC, and RNase
A-EGCG, respectively. These correlate well with the
values obtained from CD experiments—the order of
potency remaining the same.
The difference between the Kd and Ki values for these
polyphenols can be explained when we consider the fol-
lowing scheme of simple noncompetitive inhibitors:
E þ S $ ES ! P
þ þI I
l lEI þ S $ ESI
where E, S, and I represent enzyme, substrate and inhibi-
tor respectively. In considering Ki, the inhibitor, I inter-
acts not only with E but is also in equilibrium with two
other species ES and ESI. However, for the Kd calcula-
Figure 4Eadie-Hofstee plots for inhibition of RNase A by (A) EC: concentrations are
0.25 mM (~), 0.13 mM (n), 0 mM (l); (B) ECG: concentrations are 0.19
mM (~), 0.09 mM (n), 0 mM (l); (C) EGC: concentrations are 0.19 mM
(~), 0.09 mM (n), 0 mM (l). RNase A concentration: 12.0 lM.
Figure 5The plot of 1/(A � A0) versus (1/L) for RNase A–Polyphenol complexes at 280
nm. EC (-~-), ECG (-^-), EGC (-n-), and EGCG (-3-). RNase A
concentration: 4.4 lM for EC, ECG and EGC and 3.5 lM for EGCG.
Inhibition of RNase A by Green Tea Polyphenols
DOI 10.1002/prot PROTEINS 571
Figure 6CD spectra of RNase A and its complexes with (A) EC, (B) ECG, (C) EGC and (D) EGCG. RNase A (free) (__) and RNase A with 0.0556 mM (---); 0.074 mM (-~-)
ligand. RNase A concentration: 11.2 lM for EC, ECG and EGC and 14.8 lM for EGCG.
572 PROTEINS DOI 10.1002/prot
K.S. Ghosh et al.
tions by UV and CD methods, in the absence of any sub-
strate, we consider the equilibrium: E þ I $EI where I is
the ligand in these cases.
Secondary structure analyses for CD data was per-
formed using the SELCON method in DICHROWEB for
the RNase A–polyphenol complexes to directly compare
with the FTIR results (Table I). The trends are similar
though the content of secondary structural elements dif-
fers as discussed below. Apart from differences in sample
preparation for the two methods, where a hydrated film
was used for FTIR and an aqueous solution was used for
CD measurements, a major difference lies in the spectro-
scopic signals themselves. FTIR signals arise from the
vibrational modes whereas CD spectra are obtained from
electronic transitions that may be the cause for the differ-
ence in content of their structural information. Similar
differences between FTIR and CD spectroscopic results
for free RNase A have been reported elsewhere.24,37
The percent change in secondary structure content is,
however, well correlated for both studies. The results
indicate that there is a certain degree of order brought
into the structure of the protein on ligand binding,
which is most apparent in ECG where the largest per-
centage increase in a-helicity is observed. This is fol-
lowed by EC and EGCG. For EGC on the other hand,
there is a substantial increase in the b-sheet content on
binding. The difference in the binding pattern of EGC is
also highlighted in the FTIR difference spectra and dock-
ing studies (discussed later).
Hydrogen bonding and the coupling between transi-
tion dipoles are key factors that play a crucial role in
governing the conformational sensitivity of the amide
bands of proteins. The protein amide I and amide II
bands at 1645–1650 cm�1 (mainly C¼¼O stretching) and
1548–1560 cm�1 (C��N stretching coupled with N��H
bending) respectively, are correlated with structural
changes in proteins.28 The difference spectra [(enzyme
solution þ polyphenol solution)—(enzyme solution)]
provide information about the conformational changes
that arise upon interaction between the protein and
ligand. The results are shown in Figure 8. The protein
C��H stretching vibrations exhibited no spectral change
upon ligand interaction and therefore cancel out on spec-
tral subtraction. We observe that the peak positions of
the amide I and II bands in the two difference spectra of
each polyphenol shift, with a simultaneous change in the
relative intensity. This is suggestive of a change in the
secondary structure of the protein. In general, the spec-
tral ranges from 1610–1632 cm�1, 1636–1644 cm�1,
1650–1662 cm�1, and 1665–1680 cm�1 in the amide I
region are attributed to b-sheet, random coil, a-helix,
and turn structures, respectively.29,30
In the difference spectra of EC at lower concentrations,
the observed peak at 1632 cm�1 with two shoulders at
1646 and 1658 cm�1 indicate that the b-sheet, random
structure, and a-helix content of the protein are affected.
At a higher concentration of EC, the peak maximum
shifts to 1649 cm�1 with two shoulders at 1627 and 1678
cm�1, which implies that the a-helical region is further
stabilized. This observation is similar to what is found in
the CD studies. In case of ECG, at low concentrations,
the peak at 1640 cm�1 with a shoulder at 1657 cm�1 is
associated with a change in random coil and a-helix
components respectively. At higher concentrations, the
changes in the peak and the shoulder positions become
more prominent reflecting the changes in content of b-
sheet (1625 cm�1), a-helix (1653 cm�1), and turn (1676
cm�1). The maximum change observed at 1653 cm�1
can be attributed to the stabilization of a-helix of RNase
A. Changes observed in the secondary structural compo-
nents of RNase A on interaction with EGC are distinctly
different from the other polyphenols as is evident from
the difference spectra. The maximum of the amide I
band in the EGC complex at a lower concentration is at
1645 cm�1, which is indicative of a change in the unor-
dered region of the protein. With an increase in concen-
tration the peak maximum shifted to 1632 cm�1. The
difference spectra for EGC are also characterized by an
overall lower absorbance change compared with the other
polyphenols. The maximum change for EGC is observed
in the b-sheet region indicating the stabilization of this
structural element. For EGCG, at higher concentration,
the major changes in absorbance are at 1638 and
1627 cm�1 with two shoulders at 1651 and 1671 cm�1
Table ISecondary Structure Analyses of Free RNase A (in italics) and RNase A Complexes With Four Polyphenols With Protein:Ligand Ratios at 1:1.5 Determined by FTIR and
CD Techniques
a-helix (%) b-sheet (%) Random coil (%) Turn (%)
FTIR CD FTIR CD FTIR CD FTIR CD
RNase A 23.2 15.0 41.8 42.0 23.7 26.0 10.7 18.0RNase A: EC 28.3 20.0 43.9 44.0 18.6 19.0 9.2 16.0RNase A: ECG 33.2 22.5 42.7 44.0 11.3 16.0 6.9 17.0RNase A: EGC 21.4 14.5 51.4 52.0 16.9 20.0 9.5 13.0RNase A: EGCG 26.8 18.0 43.0 42.0 20.3 24.0 9.8 16.0
The numbers in bold are the values showing the most change in each column. Underlined values correspond to values lower that that of free RNase A.
Inhibition of RNase A by Green Tea Polyphenols
DOI 10.1002/prot PROTEINS 573
corresponding to the increase in the a-helix and random
structures at the expense of b-sheet region. It was found
that the polyphenols EC, ECG, and EGCG preferentially
stabilize the a-helix and EGC stabilizes b-sheet con-
formations of RNase A compensating from random
structure. The distinctive changes in secondary structural
elements are more prominent in the deconvoluted
spectra of RNase A–polyphenol complexes (Fig. 9). The
increase in a-helix content of RNase A has also been
observed when it interacts with 30-azido-30-deoxythymi-
dine (AZT).24
Quantitative analyses of the protein secondary struc-
ture for free RNase A and its polyphenol complexes in
D2O are given in Table I. For free RNase A, the maxi-
mum of the amide I band is at 1629 cm�1, which indi-
cates that the major contribution to the amide I band is
due to the b-sheet of RNase A, which is similar to a pre-
vious observation.38 The amide II band arises mainly
from an out-of-phase combination of N��H in plane
bending and C��N stretching vibrations of peptide link-
ages.39 Differences in the intensity of the amide II region
were observed at 1550 cm�1 for EC, 1546 cm�1 for ECG,
Figure 7Variation of Y222/Y208 with ligand concentration for (A) EC (B) ECG, (C) EGC and (D) EGCG. RNase A concentration: 11.2 lM for EC, ECG and EGC and 14.8 lM
for EGCG.
K.S. Ghosh et al.
574 PROTEINS DOI 10.1002/prot
1572 cm�1 for EGC, and 1544 cm�1 for EGCG at the
higher protein: ligand ratio. The increase in intensity
observed in the difference spectra between 1544 and
1550 cm�1 for all polyphenols can be attributed to the
interaction of the polyphenols with the backbone of the
protein. Interestingly the amide II regions are substan-
tially broadened for ECG and EGCG in comparison to
EC or EGC. The presence of the gallate moiety has previ-
ously been shown to have an inhibitory effect on several
proteins.40–42 A recent study reported that the ester-
bonded gallate catechins, EGCG and ECG, isolated from
green tea are potent inhibitors of dihydrofolate reductase
activity in vitro.43 We also note that the presence of the
gallate moiety of ECG and EGCG (ring D) plays a crucial
Figure 8FTIR difference spectra of RNase complexes with (A) EC, (B) ECG, (C) EGC and (D) EGCG at two different concentrations, RNase A : polyphenol 1:0.5 (���) and
1.1.5 (–*–) (left hand axis) with FTIR spectra of free RNase A (—) (right hand axis). RNase A concentration: 16 mg/mL.
Inhibition of RNase A by Green Tea Polyphenols
DOI 10.1002/prot PROTEINS 575
Figure 9Deconvoluted spectra of (A) RNase A; (B) RNase A-EC complex; (C) RNase A-ECG complex; (D) RNase A-EGC complex; (E) RNase A-EGCG complex. The percentage
of a-helix, b-sheet, and random coil are indicated in the legend for each panel.
K.S. Ghosh et al.
576 PROTEINS DOI 10.1002/prot
role in the interaction, which is reflected in the broaden-
ing of the amide II band in the difference spectra. This
implies that there is a substantial effect on the peptide
backbone N��H in plane bending and C��N stretching
vibrations on interaction with ECG and EGCG. This ob-
servation has also been correlated with docking studies
(discussed later). The overall observed spectral changes
are indicative of a nonspecific interaction signifying that
the binding involves different parts of the protein as the
concentration of the polyphenols is increased.
Attempts to crystallize the protein with the polyphe-
nols have been unsuccessful so far. Soaking experiments
of RNase A crystals with 100 mM solutions of the poly-
phenols as well as RNase A crystals grown in the presence
of either 5 or 10 mM of polyphenols did not show any
binding of the compounds to RNase A (Leonidas DD.
Personal communication.) Hence protein-ligand docking
studies were performed to obtain some insight into the
amino acid residues involved in the interactions of the
polyphenols. Model development for the rational design
of proteasome inhibitors based on docking studies of tea
polyphenols has been reported earlier in this journal.44
Our earlier studies with synthetic inhibitors of RNase A
revealed docking poses similar to those obtained from
structural studies (RMSD 0.47 A). Interestingly, these
inhibitors were competitive in nature and were found to
dock to the active site, which was further confirmed
from structural studies (PDB entry 2G8R).12,45 The
mode of binding observed in the docking experiments
for the polyphenols with RNase A is also correlated with
the experimental studies. The docking poses shown in
Figure 10 reveal that the polyphenols do not dock to the
ribonucleolytic site of RNase A (1FS3), which is in agree-
ment with our experimental observations. This could
also be the probable reason why crystallization of the
protein ligand complexes has not been possible. From
the docking results, we find that a nonregular region of
the protein comprising a turn and a loop encompassing
residues 34–39 is primarily involved in the interactions.
An inspection of the structure of the protein revealed
that Asn 34 and Asp 38 possess helical /,w angles that
can act as turn elements in the polypeptide chain.46 Asn
34 lies on the right hand side of the Ramachandran plot
and Asp 38 in the a-helical region. It may also be men-
tioned that the docking poses indicate interactions with
the backbone atoms of the nonregular region comprising
residues 34–39. This substantiates the observation made
in the difference spectra that the increase in intensity
between 1544 and 1550 cm�1 is due to the interaction of
the polyphenols with the backbone of the protein. Possi-
ble hydrogen bonding distances for the polyphenols with
the protein are given in Table II. For EC and EGC the
oxygen atoms of the A ring are involved in the interac-
tion whereas in ECG and EGCG the A ring is oriented
away from the protein and interactions are preferred
with the gallate moiety (ring D). In case of ECG and
EGCG docked to RNase A, the orientation of the D rings
are reversed. In ECG Arg 10 is close to the ester moiety
whereas the B ring oxygen atoms interact with Glu 2.
The D ring ��OH groups of ECG interact with Arg 39.
For EGCG, the D ring is close to Glu 2 and the B ring is
oriented such that it interacts with the side chains of Arg
10 and Arg 39. Lys 7 and Arg 10 are part of the cation
cluster of the binding subsite adjacent to the active site
and are known play an indirect role in C>p hydrolysis.11
The docking suggestions indicate interactions with Arg
10, which corroborates the noncompetitive nature of in-
hibition observed experimentally.
To further probe the nature of the ligand binding
pockets we calculated the change in ASA of the interact-
ing amino acid residues in the complexed and uncom-
plexed forms. The results of these calculations are given
in Table III. As expected there is no change in the ASA
of the catalytic residues, His 12 and His 119 that form
part of the P1 binding site. There is only a slight change
in ASA of Lys 41 for all the ligands. This indicates that
Figure 10Stereoview of the docked conformations of the polyphenols with Ribonuclease A
(1FS3). The protein conformations have been kept identical for comparison.
His12 and His 119 have been marked to indicate the location of the catalytic
site. Possible hydrogen bonds are shown as dashed lines. Panels correspond to A:
EC; B: ECG; C: EGC; and D: EGCG.
Inhibition of RNase A by Green Tea Polyphenols
DOI 10.1002/prot PROTEINS 577
the interactions observed with Arg 39 cause Lys 41 to be
less accessible to the solvent, which in turn may inhibit
the catalytic activity of RNase A indirectly. For EC, Asp
38 and Arg 39 lose maximum ASA on complex forma-
tion. The residues losing the most ASA on complex for-
mation with ECG are Asn 34 and Lys 37. For EGC, Arg
10, Asn 34, and Arg 39 seem to the most affected. For
EGCG which was found to inhibit the enzymatic activity
of RNase A with an inhibition constant of 81 lM, in
addition to changes observed at Lys 37 and Asp 38, addi-
tional changes in ASA are found at Lys 1. Lys 1 from the
B3 subsite of RNase A, which has purine specificity, (Fig.
2) plays a role in the hydrolysis of RNA.47,48 For RNA
the recognition preferentially occurs through B2R2 P1
(the main binding site). The phosphate of the second
nucleoside binds to P2 while the base and ribose interact
Table IIDistances (in A) Between Polar Neighbors of RNase A (PDB code 1FS3) and the Polyphenols
RNase A EC ECG EGC EGCG
Lys 1 Nf 2.96 [D(30-O)]Glu 2 Oe1 2.94 [B(30-O)] 2.79 [A(5-O)] 2.70 [D(30-O)]Glu 2 N 2.81 [D(40-O)]Lys 7 Nf 3.00 [A(7-O)]Arg 10 Nh1 2.70 [D(10-C¼O)] 2.59 [C(3-O)] 3.04 [B(50-O)]Arg 10 Nh2 2.65 [A(5-O)] 2.27 [D(10-C¼O)] 3.13 [C(1-O)]Gln 11 Oe1 2.40 [A(7-O)] 3.13 [B(40-O)]
3.25 [B(50-O)]Asn 34 Nd2 3.08 [A(7-O)]Asn 34 O 2.36[A(5-O)] 3.06 [D(10-C¼O)] 2.74 [C(1-O)]
3.39 [C(3-O)]Leu 35 O 3.06 [D(40-O)] 2.96 [B(40-O)] 3.05 [B(30-O)]Lys 37 N 2.73 [D(50-O)] 2.95 [B(50-O)]Asp 38 Od1 2.53 [B(30-O)]Asp 38 N 2.84 [D(50-O)] 2.48 [B(50-O)]Arg 39 Nh1 2.65 [C(1-O)] 2.88 [D(30-O)] 2.55 [B(30-O)] 2.72 [B(30-O)]
2.65 [D(40-O)] 2.90 [B(40-O)]Arg 39 Ne 3.16 [D(40-O)] 3.09 [B(40-O)]Arg 39 N 3.27 [D(50-O)] 2.87 [B(50-O)]Arg 39 O 2.89 [D(50-O)] 2.38 [B(40-O)]
2.82 [D(40-O)] 2.91 [B(50-O)]Lys 41 N 3.39 [B(40-O)]
Table IIIChange in Accessible Surface Area (DASA) in A2 of Interacting Residues of RNase A (Uncomplexed), and its Complexes With EC, ECG, EGC, and EGCG
Amino acid residues
DASA
Location of the residueEC ECG EGC EGCG
Lys 1 0.00 21.27 0.00 33.61 UnorderedGlu 2 0.00 17.85 22.97 16.99 UnorderedLys 7 16.59 21.33 12.24 20.54 H1Arg 10 7.89 25.63 37.17 16.71 H1His 12 0.00 0.00 0.00 0.00 H1Asn 34 9.69 35.93 32.35 21.56 T2Leu 35 12.53 12.53 12.53 12.53 Loop between T2 and S1Lys 37 17.10 39.98 13.27 34.19 Loop between T2 and S1Asp 38 36.03 19.43 8.72 32.35 Loop between T2 and S1Arg 39 39.49 24.08 25.43 24.66 Loop between T2 and S1Lys 41 7.50 3.74 3.63 3.90 UnorderedHis 119 0.00 0.00 0.00 0.00 S7SDASA 153.35 227.13 173.22 222.48SDASA/Mol.Wt 0.53 0.51 0.57 0.49Kd (lM) (PEARLS) 47.10 0.48 15.10 0.32Kd (klM) (CD) 141 � 9 80 � 3 112 � 12 40 � 1Kd (lM) (UV) 150 � 0.32 79 � 2.7 127 � 0.28 49 � 0.68
Total change in ASA (SDASA) of RNase A complexes and their calculated dissociation constants as obtained from PEARLS.34
Dissociation constants from experimental studies have also been included.
H: a-Helix; T: Turn; S: b-Sheet.
K.S. Ghosh et al.
578 PROTEINS DOI 10.1002/prot
with two additional binding sites, B3 and R3 respectively.
Interaction of the D ring 30 oxygen atom of EGCG with
Lys 1 may be a probable cause for the observed noncom-
petitive inhibition. The loss in ASA has been summed for
each polyphenol and normalized with respect to molecu-
lar weight. Calculations for the expected values of the
dissociation constants from docking studies have been
obtained using PEARLS.35 The order of Kd values calcu-
lated using PEARLS follows the same order as obtained
from our experimental studies. We find a direct correla-
tion between the experimental and theoretical data with
correlation constants ranging from 0.83 to 0.99.
The results presented in this paper have been com-
bined and summarized in Figure 11. The specific interac-
tions identified from docking studies that support our
experimental findings imply that the gallate moiety is
indeed a feature worth further investigation in terms of
design potential. The importance of the gallate moiety
has been further investigated by considering metal com-
plexes of ECG and EGCG with Cu(II) where we found
that the complexes also inhibit the ribonucleolytic activ-
ity of the protein.49 Our speculations about the interac-
tions and their effect on the activity of the protein may
be further substantiated by structural studies of the
RNase A–polyphenol complexes which is the subject of a
further study. With knowledge of the specific ligand
binding sites one may thus rationally design derivatives
of the compounds based on green tea that may act as
inhibitors of the proteins. Further studies exploiting the
multiple hydroxyl groups present in the gallate moiety
and their metal chelating properties is currently in pro-
gress. This study is expected to provide guidelines for the
design of more potent inhibitors of RNase A that could
in turn be extrapolated to members of the superfamily
with unusual biological properties.
ACKNOWLEDGMENTS
TKM and JD thank CSIR, New Delhi for fellowships.
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