Elucidating the interaction of γ-hydroxymethyl-γ-butyrolactone substituents with model membranes and protein kinase C-C1 domains

Article abstract of DOI:10.1039/c5mb00100e

The protein kinase C (PKC) family of proteins is an attractive drug target. Dysregulation of PKC-dependent signalling pathways is related to several human diseases like cancer, immunological and other diseases. We approached the problem of altering PKC activities by developing C1 domain-based PKC ligands. In this report γ-hydroxymethyl-γ-butyrolactone (HGL) substituents were investigated in an effort to develop small molecule-based PKC regulators with higher specificity for C1 domain than the endogenous diacylglycerols (DAGs). Extensive analysis of membrane-ligands interaction measurements revealed that the membrane-active compounds strongly interact with the lipid bilayers and the hydrophilic parts of compounds localize at the bilayer/water interface. The pharmacophores like hydroxymethyl, carbonyl groups and acyl-chain length of the compounds are crucial for their interaction with the C1 domain proteins. The potent compounds showed more than 17-fold stronger binding affinity for the C1 domains than DAG under similar experimental conditions. Nonradioactive kinase assay confirmed that these potent compounds have similar or better PKC dependent phosphorylation capabilities than DAG under similar experimental conditions. Hence, our findings reveal that these HGL analogues represent an attractive group of structurally simple C1 domain ligands that can be further structurally altered to improve their potencies.

Full text of DOI:10.1039/c5mb00100e

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Elucidating the interaction of c-hydroxymethyl-c-
butyrolactone substituents with model
membranes and protein kinase C–C1 domains†
Cite this: Mol. BioSyst., 2015,
11, 1389
Rituparna Borah,‡ Narsimha Mamidi,‡ Subhankar Panda, Sukhamoy Gorai,
Suraj Kumar Pathak and Debasis Manna*
The protein kinase C (PKC) family of proteins is an attractive drug target. Dysregulation of PKC-dependent
signalling pathways is related to several human diseases like cancer, immunological and other diseases.
We approached the problem of altering PKC activities by developing C1 domain-based PKC ligands. In this
report g-hydroxymethyl-g-butyrolactone (HGL) substituents were investigated in an effort to develop small
molecule-based PKC regulators with higher specificity for C1 domain than the endogenous diacylglycerols
(DAGs). Extensive analysis of membrane–ligands interaction measurements revealed that the membrane-
active compounds strongly interact with the lipid bilayers and the hydrophilic parts of compounds
localize at the bilayer/water interface. The pharmacophores like hydroxymethyl, carbonyl groups and acyl-
chain length of the compounds are crucial for their interaction with the C1 domain proteins. The
potent compounds showed more than 17-fold stronger binding affinity for the C1 domains than DAG
under similar experimental conditions. Nonradioactive kinase assay confirmed that these potent
compounds have similar or better PKC dependent phosphorylation capabilities than DAG under similar
experimental conditions. Hence, our findings reveal that these HGL analogues represent an attractive
group of structurally simple C1 domain ligands that can be further structurally altered to improve
their potencies.
Received 3rd February 2015,
Accepted 17th March 2015
DOI: 10.1039/c5mb00100e
www.rsc.org/molecularbiosystems
The reversible cellular translocation of classical PKC iso-
enzymes to the plasma membrane is initially arbitrated
Introduction
The protein kinase C (PKC) family of proteins are involved in signal by Ca2+ binding to the C2 domain, followed by DAG binding
transduction and various cellular events including, apoptosis, pro- to the C1 domain. On the contrary, DAG binding to the C1
liferation, metabolism and others.1,2 The PKC proteins contain a domain activates novel PKC isoenzymes. Membrane associa-
C-terminal catalytic domain and an N-terminal regulatory domain tion results in a conformational change and allows PKC iso-
with an auto-inhibitory sequence, and one or two membrane enzymes to penetrate further into the cellular membranes
targeting (C1 and C2) domains. Phosphorylation, increased concen- through DAG–C1 domain interactions. DAG binding results in
tration of diacylglycerol (DAG), and/or Ca2+ in the presence of folding-out of the N-terminal pseudo substrate sequence of the
anionic phospholipids activate these enzymes at the membrane PKC enzyme from its catalytic site, which allows access of
surface and transmit their signals by phosphorylating specific several substrates to the catalytic site of the PKC enzymes.5–9
proteins.3,4 DAG binds to the C1 domains of both the classical
PKC isoenzymes play a pivotal role in the pathology of several
(calcium-, DAG-, and phospholipid-dependent), and novel (calcium- diseases including cancer and Alzheimer’s diseases.10,11 Therefore,
independent, but DAG- and phospholipid-dependent) PKCs. PKC isoenzymes have been a subject of intensive research and drug
development. On the other hand, the catalytic domain of PKC
enzymes is highly homologous with several other protein kinases in
Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039,
human genome. Hence, regulation of PKC enzyme activity by
targeting its C1 domain provides a more conventional approach.
Several studies have already shown that the regulatory domain
of PKC isoenzymes might have independent biological func-
tions.2,9,12–16 The C1 domains are smaller in size (B50 residues),
retain conserved structure, and contain only one ligand binding
domain. In addition, the number of proteins containing this
India. E-mail: dmanna@iitg.ernet.in; Fax: +91 03 61258 2350;
Tel: +91 03 61258 2325
† Electronic supplementary information (ESI) available: Characterization data of
the synthesized compounds. Copies of 1H and 13C NMR spectra of the com-
pounds. Molecular docking results of the compounds. Monomeric binding assay
and FRET-based competitive binding assay of the compounds. See DOI: 10.1039/
c5mb00100e
‡ R.B. and N.M. contributed equally to this work.
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C1 domain is small. In consequence, a variety of PKC regulators Results and discussion
directed to the C1 domain have been developed as a new class
Design and synthesis
of antitumor agents.4,17,18 C1 domain ligands such as
Structure–activity relationship (SAR) studies showed that hydroxy-
methyl and carbonyl functional groups of DAGs, DAG-lactones
and phorbol esters play an important role for their interactions
with the backbone amide protons and carbonyl of PKC–C1
domains. However, all these ligands have differential C1 domain
binding potencies. Conformationally rigid phorbol esters have
more than 1000-fold stronger binding affinity for a C1 domain
than structurally simple and flexible DAGs. Several conformationally-
constrained DAG-lactones showed intermediate C1 domain binding
potency.25–27 Inspired by the structural rigidity and higher binding
affinity of the DAG-lactones, we selected (S)-g-hydroxymethyl-g-
butyrolactone (HGL) analogues for the devolvement of PKC–C1
domain modulators. The HGL moiety provides a rigid five-
membered ring as in DAG-lactones, but the HGL analogues are
structurally different due to the position of its ester linkage and
number of hydrophobic ‘tails’ (Fig. 1).
These compounds also retain necessary pharmacophores,
namely hydroxyl and carbonyl functionalities within the same
molecule, and hydrophobic tails important for membrane inter-
action (Fig. 1). Unlike most of the potent C1 domain ligands like
phorbol esters, bryostatins and others, HGLs are structurally
simpler and contain lesser rigidity. Structural modifications
and large sale productions of HGLs are comparatively easier
than those from potent natural products.25–27 The HGLs (1–3)
were synthesized in five to six steps from the L-glutamic acid
as a starting material. HGLs with long (palmitic acid) and
short chain (octanoic acid and propanoic acid) acids were
synthesized in order to study the impact of hydrophobicity on
the binding affinity. The synthesis was accomplished via a modified
reported reaction protocol.28 We initially prepared HGL analogous
1a and 1b in order to investigate the role of hydroxymethyl and
hydroxyl groups on the binding of C1b subdomains of PKC
isoforms. Compounds 1a and 1b were prepared in five steps from
L-glutamic acid. The (S)-g-hydroxymethyl-g-butyrolactone (5) was
synthesized from L-glutamic acid in two steps according previously
reported procedures and the hydroxyl group was protected with
bryostatin-1 are in clinical trials and ingenol-3-angelate is in
clinical use.19,20 Phorbol esters, which strongly bind with the
C1 domains, have provided pharmacological tools for studying
PKC function and activity. However, phorbol esters are known
to promote tumors.19–21 Compounds like prostratin, gnidima-
crin, and DPP (12-deoxyphorbol-13-phenylacetate) have also
shown PKC enzyme dependent antitumor activities in mice
and selected human cell lines.10,22
Phorbol esters, bryostatin and other reported high-affinity
C1 domain binding ligands are structurally complex, and they
bind competitively to the DAG-binding site of the C1 domain
with affinities several orders of magnitude higher than only
DAGs.23,24 The conformationally rigid scaffold of these C1
domain ligands made it difficult to alter their specificity and
large-scale production. However, conformationally restricted
DAG-lactones are reported to have intermediate potency
between DAG and phorbol esters.21–24 It is also deduced that
the structural rigidity of DAG-lactones reduce the number of
possible rotameric forms of DAGs, and one of the rigid rota-
mers mimics the actual conformation of physiologically active
DAGs. The stronger binding of a DAG-lactone to a PKC–C1
domain than only DAG apparently is governed by a smaller
decrease in entropy. Stronger binding is also governed by the
presence of required pharmacophores and proper orientation
of the ligands inside the binding pocket and their localization
at the membrane bilayer. Structural and functional studies of
the PKC–C1 domains have revealed that Thr-12, Leu-21, and
Gly-23 residues play a crucial role in ligand binding. The
hydrophobic residues present along the circumference of the
binding pocket interact with the hydrophobic moiety of the ligand
and facilitate insertion of the C1 domain into the membrane
after ligand binding, thereby stabilizing the formation of the
ternary (ligand–receptor–membrane) binding complex. In an
attempt to develop C1 domain ligands that are simple and
readily amenable to the introduction of a structural variation
template, we used (S)-g-hydroxymethyl-g-butyrolactone (HGL).
The other objective of designing these simple molecules is to
overcome the spread in potency between complex natural
product ligands and DAG.
The present study describes the design, synthesis, aggregation
behaviour in aqueous solution, interaction with liposomes, in vitro
binding properties of HGL substituents to the C1b subdomain
of PKCd and PKCy, and their PKC enzyme activity. The results
demonstrate that the C1 domain binding potencies of these
compounds correlates well with their effect on membrane
fluidity and hydration. The potent HGL analogues bind com-
petitively to the DAG-binding site of the C1 domains of PKC.
The g-hydroxymethyl group, carbonyl groups of lactone, and ester
of the active compounds play a crucial role in PKC–C1 domain
binding. At lower micromolar concentrations, these HGL analogs
also activate PKC enzyme. Overall, the results expand our under-
standing on the molecular parameters affecting PKC–C1 domain
binding to membranes by synthetic HGL analogues.
Fig. 1 Structures of DAG, DAG-lactones and g-hydroxymethyl-g-
butyrolactone (HGLs) indicating the crucial pharmacophores required for
PKC–C1 domain binding.
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Scheme 1 Synthesis of protected g-butyrolactone (6).
Scheme 2 Synthesis of a-hydroxylmethyl-g-lactone derivative (1).
and C5 protons in the COSY experiment. This reveals that these
two protons are on the same plane. The coupling constant of C3
proton and C7 proton, J = 7.7 Hz, indicates that these two protons
are trans to each other.28 The reaction was also performed with
two more aldehydes under this optimized reaction condition and
the results are summarized in Table 1. The synthesis of dialcohol
1a and 1b (in 80% yield) was accomplished via careful removal of
the trityl group under trifluoroacetic acid conditions as shown in
the Scheme 2. To verify the hydrophobicity effect of the ligands
on the biding affinity, we also prepared HGL ester derivatives
by introducing a long chain fatty acid (palmitic acid) and
short chain acids (octanoic acid, propanoic acid). The acyl
chains were then introduced into alcohol 10 using a standard
N,N0-dicyclohexylcarbodiimide (DCC)-mediated coupling reaction
with readily available carboxylic acids (palmitic acid/caprylic
acid/propanoic acid) to produce compounds 2 and 3 in good
yield as shown in Table 2. Finally, the trityl group was de-protected
under trifluoroacetic acid conditions to access HGL derivatives
in good yield.
Fig. 2 Stereochemical assignments of product 8b based on COSY and
NOE experiment.
tritylchloride in the presence of triethyl amine in good yield
(Scheme 1).28
After that, the model aldol-reaction was conducted between
the protected g-butyrolactone (6) and octanal (7b) in the
presence of various bases such as NaH, triethyl ammonia
(TEA), DIPEA, DBU and DABCO; but even after 24 h we did
not obtain the desired product. In order to screen the bases/
optimize the reaction conditions, we performed the same
reaction with LDA (1.5 equiv.) in the presence of CuCN/LiCl
(1.0 equiv.)/(2.0 equiv.) as an additive at ꢀ78 1C and obtained
the desired aldol adduct 8b in 70% isolated yield (Table S1,
ESI,† entry 6) whereas in case of n-BuLi (1.5 equiv.) the yields
were increased up to 80% (Table S1, ESI,† entry 7) in presence
of the same additive ratio. We used several other Lewis acids as
an additive to improve the yields of the products; unfortunately
no fruitful result was obtained. Therefore, n-BuLi (1.5 equiv.)
and CuCN/LiCl (1.0 equiv./2.0 equiv.) as an additive in dry THF
solvent (at ꢀ78 1C), was chosen as the best reaction condition
(with 80% yield within 20 min) for the aldol reaction. The
reaction generated only one diastereomer (8b) with (S, R, S)
configurations, which was confirmed by cosy and NOE experi-
ments (Fig. 2). There was a strong interaction between the C3
Aggregation studies of the compounds
To check the behaviour of the compounds in aqueous solution,
we first investigated their aggregation behaviours by measuring
the fluorescence properties of a polarity indicator, pyrene.
Concentration-dependent aggregation behaviours of the com-
pounds and simultaneous inclusion of pyrene molecules
into hydrophobic cores of the aggregates was reflected by an
increase in pyrene fluorescence intensity. The fluorescence
intensity ratio, I1/I3 of pyrene was considered as a measure of
the polarity of its microenvironment and was further used to
calculate critical aggregation concentration (CAC) values of the
compounds (Fig. S8, ESI†).29 The measured CAC values of the
compounds 1a, 2a, and 3a were 60, 22, and 43 mM, respectively
(Fig. 3). Above the CAC values, the higher and lower ratios of
I1/I3 indicate the polar (loose aggregates) and hydrophobic
(compact aggregates) environments. These results show that,
above 50–70 mM compound concentrations, the ratio of I1/I3
reached a plateau region. However, with further increase in
concentrations the I1/I3 ratio gradually reduced, signifying the
formation of loosely bound aggregates in aqueous solution with
an extensive concentration range. The results also indicate that
tested compounds aggregate at lower concentration ranges in
aqueous solution, possibly due to the absence of charge head
groups. Investigation of these CAC ranges of aggregate formation
is essential in understanding their interaction properties both
with the lipid bilayers and PKC–C1 domain under monomeric
form in aqueous solution.
Table 1 Aldol reactionsa
Aldehyde
Product
Yieldb (%)
CH3(CH2)14CHO (7a)
CH3(CH2)6CHO (7b)
CH3CH2CHO (7c)
8a
8b
8c
80
80
85
a
Performed with protected g-lactone (1.0 equiv.), n-buLi (1.5 equiv.),
CuCN/LiCl (1.0 equiv.)/(2.0 equiv.) and aldehyde (1.5 equiv.) in dry THF.
b
Isolated yields.
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Table 2 Synthesis of g-butyrolactone derivatives
Compound
R
R0
Compound
R
R0
10ac
10ab
10bc
10ba
CH3(CH2)14 (8a)
CH3(CH2)14 (8a)
CH3(CH2)6 (8b)
CH3(CH2)6 (8b)
CH3CH2 (9c)
CH3(CH2)6 (9b)
CH3CH2 (9c)
2a
3a
2b
3b
CH3(CH2)14
CH3(CH2)14
CH3(CH2)6
CH3(CH2)6
CH3CH2
CH3(CH2)6
CH3CH2
CH3(CH2)14 (9a)
CH3(CH2)14
subsequent LE–liquid condensed (LC) region and a more con-
densed phase during the compression process. The LE phase of
these pure compounds, and compounds in the presence of
DPPC lipid, begin at a much higher molecular area of 250 Å2
indicating a very loose configuration of the molecules within
the fluid monolayer. The presence of LE–LC is indicated by the
plateau region in the isotherm. The additional ordering in p–A
isotherms and significant change in the LE–LC region of these
compounds in the presence of DPPC lipid may be due to their
interaction properties between their head groups and hydrophobic
‘tails’ (Fig. 4). It is also important to note that the presence of acyl
groups demonstrate significant effects on the monolayer properties
of the compounds; however, alternative hydrophobic groups show
almost no effect on the monolayer properties of the compounds.
Nevertheless, monolayer properties of the compounds in the
presence of DPPC lipid clearly depict their strength of interaction.
Therefore, the hydroxyl-lactone moiety of the compounds strongly
affects the assembly properties and its interactions with the DPPC
molecules, and also lipid interactions play a significant role in the
monolayer organization of the molecules.
Fig. 3 Measurement of critical aggregation concentration of compounds
1a, 2a, and 3a in aqueous solution. Plot of pyrene fluorescence intensity
ratio I1/I3 against increasing concentration of compounds. [Pyrene] = 2 mM,
lex = 380 nm.
Membrane–ligand interaction measurements: orientation of
ligands at the interface
Surface pressure (p)–area (A) isotherms provide an insight into
how chemical structure and physical behaviour, including intra-
molecular and intermolecular interactions, modify the assembly
of the amphiphilic molecules at the air/water interface.30,31 The
p–A isotherms of the pure compounds displayed a slightly
different assembly pattern from compounds in the presence of
DPPC lipid (Fig. 4). However, the assembly properties of the pure
compounds 2a and -3a are significantly different from pure DPPC
lipids under similar experimental conditions. The isotherms also
indicate that these compounds become partly soluble with an
increase in lateral pressure. The p–A isotherms of these pure
compounds show a profound liquid-expanded (LE) phase and a
Measurement of change in fluidity and hydration of the
lipid bilayer
To recognize the consequences of membrane active compounds
upon the fluidity of the lipid bilayer and dynamics of lipid mole-
cules, we measured the fluorescence anisotropy of the membrane
active 1,6-phenyl-1,3,5-hexatriene (DPH) molecules under liposomal
environment. The hydrophobic DPH molecules are known to embed
within the hydrophobic core of the lipid bilayers, allowing estimat-
ing the modulation of lipid-bilayer fluidity induced by membrane-
active compounds.29 Fig. 5A represents the anisotropy values of DPH
molecules in the presence/absence of the compounds under lipo-
somal environment. The anisotropy value of DPH in the presence of
compound 2a is slightly smaller than with the other compounds,
indicating increase in bilayer fluidity. Changes in membrane surface
were monitored by a dansyl-PE probe. The fluorescence signal of
dansyl is environmentally sensitive and efficiently gets quenched
by water.32,33 The increase in % mole of membrane active HGL
compounds induces an increase in dansyl fluorescence quenching
(Fig. 5B). The increase in quenching can be correlated with the
increase in dansyl-PE head group hydrations at the membrane
surface, which correlates well with the increase in membrane fluidity
in the presence of these compounds.
Fig. 4 Structure of the membrane active compounds (A). Surface pressure
(p)–molecular area (A) isotherm of the saturated hybrid lipids (B).
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¨
Forster resonance energy transfer (FRET)-based competitive
binding assay.
Interaction with soluble ligands
Trp-fluorescence properties of the proteins are widely used to
examine the ligand-induced changes in protein conformation
and/or microenvironment. The presence of a single Trp-residue
(Trp-252 in delta, Trp-253 in theta) close to the ligand binding
site makes these C1b subdomains a model system to monitor
ligand binding properties. The ligand-induced Trp-fluorescence
quenching data in the native state (insignificant or no shifting in
emission peak position observed) was used to determine the
binding affinity of the C1b subdomains for the ligands in mono-
meric form. The compound concentrations used during the
spectroscopic measurements were well below their CAC values,
indicating their monomeric form under the experimental condi-
tions. The ligand binding can cause an environmental change for
Trp-residue, exposing it to a hydrophilic environment. This could
lead to a decrease in the Trp-emission signal with increasing
concentrations of the compounds (Fig. 7 and Fig. S9, ESI†).
The calculated binding parameters revealed that compounds 2
and 3, with different chain lengths, strongly interact with the
C1b subdomains. In particular, monomeric ligands 2b and 3b
show more than 9- and 15-fold stronger binding affinity than
DAG8 for PKCd–C1b subdomain (Table 3). To understand the
importance of hydrophobicity of the compounds in protein
binding, a similar analysis was performed with the long-chain
HGL derivatives. Although there is a distinct difference in
hydrophobicity between compounds like 3a and 3b, they show
only a subtle difference in their protein binding affinities. This
could be due to their binding orientations with the C1b sub-
domains. The binding of these compounds with the C1b
subdomains is mostly governed by hydrogen bonding with
the amino acid backbone through their g-hydroxymethyl and
carbonyl functionalities. In addition, hydrophobic interaction
among the compounds and amino acid residues at the circum-
ference of the binding pocket may play an important role in
Fig. 5 Membrane fluidity and hydration change measurements. Fluorescence
anisotropy of DPH embedded in PC/cholesterol/ligand (60/20/20) liposomes.
Control: no ligand was added to the liposomes (A). Effect of compounds
on the fluoresce intensity of dansyl-PE embedded in PC/cholesterol/
dansyl-PE (79/20/1) liposomes (B). Values represent the mean ꢁ SD from
triplicate measurements.
Extent of membrane localization
The structural analysis reveals that the head groups of DAG
and HGLs are anticipated to be placed near the bilayer/water
interfacial region. However, the extent of localization of these
pharmacophores containing head groups at the bilayer/water
interface is crucial for their capability to interact with the C1
domains. For this reason we determined sodium dithionite-induced
fluorescence quenching rates of the NBD moiety using DPPC/ligand/
NBD-PE liposomes. The NBD moiety of NBD-PE lipid is reported
to be embedded close to the bilayer interface, providing a useful
marker for surface interactions of membrane-active C1 domain
ligands.34–36 The measured NBD fluorescence quenching rates
propose that the NBD moiety became more ‘‘shielded’’ from the
soluble dithionite quencher because of the presence of ligands
within the liposomes (Fig. 6). Therefore, membrane environment
modification by these compounds may enhance the ability of a C1
domain to insert into the membrane. The results also suggest that
these compounds are more localized at the bilayer/water interface
and more accessible for protein binding than DAG16.
Protein–ligand interaction measurements
In the present study, the C1b subdomains of PKCd, PKCy iso-
enzymes were used to measure the binding parameters of HGLs.
These C1b subdomains have sufficiently strong DAG binding
affinities and are easy to purify from bacterial cells. The binding-
potencies of the compounds were measured by a Trp-fluorescence
quenching method, steady-state fluorescence anisotropy, and
Fig. 7 Binding of compounds with the PKCd–C1b subdomain. Represen-
tative plot of Trp-fluorescence intensity of PKCd–C1b (1 mM) in buffer
(20 mM Tris, 160 mM NaCl, 50 mM ZnSO4, pH 7.4) in the presence of
varying concentration of 1–3, where F and F0 are fluorescence intensity in
the presence and absence of the ligands, respectively. The solid lines are
nonlinear least-squares best-fit curves. Values represent the mean ꢁ SD
from triplicate measurements.
Fig. 6 Fluorescence quenching of NBD-PE embedded in PC/ligand16/NBD-PE
(89 : 10 : 1) liposomes. Sodium dithionite = 0.6 mM. Control: no ligand.
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Table 3 Ligand binding parameters of PKCd C1b and PKCy C1b proteinsa at room temperature
KD(ML)/(mM)
KD(ML)/(mM)
PKCd C1b
Compound
PKCd C1b
PKCy C1b
Compound
PKCy C1b
DAG16
1a
2a
6.57 ꢁ 0.23
0.89 ꢁ 0.11
0.40 ꢁ 0.14
0.38 ꢁ 0.09
7.14 ꢁ 0.39
0.81 ꢁ 0.12
0.33 ꢁ 0.08
0.98 ꢁ 0.09
DAG8
1b
2b
11.37 ꢁ 0.69
1.33 ꢁ 0.12
1.02 ꢁ 0.15
0.73 ꢁ 0.19
6.92 ꢁ 0.39
0.68 ꢁ 0.11
0.30 ꢁ 0.09
1.19 ꢁ 0.19
3a
3b
a
Protein, 0.25 mM in buffer (20 mM Tris, 160 mM NaCl, 50 mM ZnSO4, pH 7.4). Values represent the mean ꢁ SD from triplicate measurements.
ligand binding affinities of the protein. The long alkyl chains
facilitate the compounds to interact with the membrane bilayer,
where PKC proteins interact with the membrane through the C1
domain for activation and regulation of various cellular pathways.
A similar trend in binding affinities was also observed with the
reported C1 domain ligands. However, these binding para-
meters of the compounds in monomeric form do not show a
clear specificity for either PKCy or PKCd–C1 domains.
Molecular docking analysis
Fig. 8 Structures of ligand-bound PKCd–C1b subdomains. Modelled struc-
ture of 1b (A), 2b (B) and 3b (C) docked into PKCd–C1b (1PTR) subdomain.
In order to better understand the possible binding mode of the
compounds and the interacting residues of the proteins, we
performed molecular docking analysis using the crystal coordi-
nates of the PKCd–C1b in complex with phorbol-13-O-acetate
(1PTR).37 The molecular models revealed that the HGLs were
anchored to the binding site in a similar fashion as phorbol
esters and DAG-lactones.26 The most effective compound, 2b,
showed three possible hydrogen bonds with the amino acid
backbone. The hydroxymethyl group was hydrogen bonded to
the carbonyls of L251. The carbonyl group of the lactone moiety
formed a hydrogen bond with the backbone amide proton of
G253. An additional hydrogen bonding was also observed between
side chain amine of Q257 with the carbonyl group of ester moiety.
A similar hydrogen bonding network was also observed between
compound 3b and the PKCd–C1b protein. Whereas, the hydroxyl
group of compound 1b showed five hydrogen bondings with the
backbone amide proton and carbonyls of T242 and L251 and the
side chain amide proton of Q257 (Fig. 8). However, the binding
affinity measurements and molecular docking analysis do not
corroborate. This could be due to the strength of interaction and
the conformational adjustments of the protein and compounds
under experimental conditions and also the contribution of
bridging water molecules between HGLs and C1 domains could
generate stronger interactions.
The modelled structures were generated using the Molegro Virtual Docker,
version 4.3.0. The oxygen atoms and nitrogen atoms are shown in red and
blue, respectively. The dotted line (black) indicates possible hydrogen bonds.
of compounds increase the rigidity of the surrounding environ-
ment of the protein.
Interaction with ligand associated liposomes
The PKC–C1 domains contain a membrane binding groove
along with its DAG-binding site. Under physiological condi-
tions DAG-dependent membrane binding and/or penetration of
the C1 domain in the presence of anionic phospholipids
activates PKC isoenzymes and regulates its cellular activities.
Consequently, C1 domain binding properties of the com-
pounds were measured under membrane environment using
protein to membrane FRET-based competitive binding assay.
Trp-residue of the C1b subdomains acts as the FRET donor and
a low density of membrane-inserted dansyl-PE (dPE) lipid
provides the acceptor. DAG8 was used as a competitive inhibitor
for this assay. Anionic phospholipids, in particular phosphati-
dylserine, is known to induce the DAG-dependent membrane
binding of the C1 domain through its interaction with the
cationic groove.12,26,34,38 Hence, PS was incorporated into the
Steady-state anisotropy measurements
We also performed steady-state fluorescence anisotropy mea- liposomes. Additional zwitterionic lipid PE was used to form
surements of the proteins in absence or presence of the ligands stable liposomes. The reduction in the protein-to-membrane
to gain more information of the ligand–protein interaction. FRET signal (Fig. S10, ESI†) was inspected to determine
The degree of anisotropy of pure PKCd C1b protein increases the competitive displacement of C1b subdomains from the
from 0.0439 in buffer to 0.0874 and 0.0850 on interactions with liposomal surface to the bulk solution and apparent inhibitory
10-fold excess of ligands 2b and 3b, respectively (Table S2, constant [KI(DAG8)app] calculation (Fig. 9 and Fig. S11, ESI†).
ESI†). Similar increases in anisotropy values were also observed The measurements were performed for membrane active com-
for proteins in presence of DAGs and other compounds. pounds 1a, 2a and 3a. The binding parameters demonstrated
Although the increases in anisotropy values were different for that compound 2a has stronger binding affinity for the PKCy–
the compounds, this experiment still suggests that the presence C1b subdomain than the other compounds under the liposomal
1394 | Mol. BioSyst., 2015, 11, 1389--1399
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Fig. 10 The PKC activity in the absence and presence of DAG16 and potent
compounds (8 mM). Representative UV-illuminated agarose gel image of the
product of reactions run with PKC full-length enzyme. Control with no PS
containing PKC activator solution and activator (lane a). Activity assay in the
presence of DAG16 (lane b) and potent compounds 1a (lane c), 2a (lane d) and
3a (lane e) (A). Relative amounts of phosphorylated PepTag C1 peptide were
calculated from fluorescence spectral analysis (B).
Fig. 9 Competitive displacement assay for the PKCd–C1b subdomains (1 mM)
bound to liposome containing ligands 1a, 2a and 3a. The bound complex was
titrated with the DAG8.
Table 4 Equilibrium binding parameters for PKCd C1b and PKCy C1b
proteina with the ligand-associated liposomesb at room temperature
PKC activity assay did not show any significant differences
in their phosphorylation capabilities among the potent com-
pounds. Therefore, further studies are required to resolve their
potency differences. However, the activity assay confirms that
KI(DAG8)app/(mM)
Compound PKCd C1b PKCy C1b
KD(L16)/(nM)
PKCd C1b
PKCy C1b
1a
2a
3a
33.39 ꢁ 1.95 34.12 ꢁ 1.19 72.02 ꢁ 5.91 63.95 ꢁ 5.12
36.95 ꢁ 2.01 51.75 ꢁ 3.74 28.78 ꢁ 7.89 16.88 ꢁ 1.95
22.19 ꢁ 1.68 25.90 ꢁ 2.57 45.82 ꢁ 7.01 103.43 ꢁ 6.98 the synthesized potent HGL compounds activate PKC enzyme
under the experimental conditions.
a
Protein, 1 mM in buffer (20 mM Tris, 150 mM NaCl, 50 mM ZnSO4, pH 7.4).
b Active liposome composition, PC/PE/PS/dPE/ligand (55/15/20/5/5). Values
represent the mean ꢁ SD from triplicate measurements.
These studies show that the membrane active HGL deriva-
tives interact with phospholipids and influence their mono-
layer/bilayer properties. The pharmacophores-containing moiety,
g-hydroxymethyl-g-butyrolactone of the compounds is preferably
localized at the water/bilayer interface and accessible for PKC–C1
domain binding. The binding parameters and molecular docking
analysis of the compounds highlight the importance of pharmaco-
phores, hydrophobicity, and binding orientations of the ligands.
The binding properties also suggest that membrane active
compounds can differentially influence the in vitro membrane
interaction properties of the C1 domains of PKCd and PKCy
enzymes. The compound 2a has a little stronger binding
affinity for both the C1 domains than other compounds. We
hypothesize that this modest stronger binding affinities of
compound 2a could be because of the presence of a propio-
nate group along with a membrane-active hydrophobic ‘tail’,
which allow the compound to interact strongly with hydro-
phobic residues surrounding the ligand binding site of the C1
environment. This competitive displacement assay also verifies
that the potent compounds preferably interact with the C1b
subdomains through its DAG/phorbol ester binding site. We have
also calculated the equilibrium dissociation constant (KD(L)) for
the C1b subdomains binding to the liposome-associated targeted
ligand using eqn (4). Comparison of the equilibrium dissociation
constant also revealed that C1b subdomains have higher binding
affinity for the compound 2a associated liposomes (Table 4).
Therefore, the in vitro C1 domain binding measurements pointed
out that higher concentration of DAG8 was required for the displace-
ment of C1b subdomains from the compound 2a-associated
liposomes. Direct protein binding to the compound containing
liposomes also showed strong binding affinities (Table S3, ESI†).
PKC activity assay
To verify the feasibility of the synthesized HGL compounds in domains or the effect of compound 2a on the lipid bilayer
activating PKC enzymes, PepTag-nonradioactive kinase activity organization. We also presume that the presence of these
assay was performed in the presence of PS.39,40 In this assay compounds makes the bilayer structure more loosely packed,
PKC enzyme-specific florescent peptide was used as substrate. allowing the C1 domains to bind with the HGLs and penetrate
PKC-dependent phosphorylation of the fluorescent peptide changes into the lipid bilayer, which is critical for the PKC enzyme
the net charge from +1 to ꢀ1, allowing the phosphorylated activation at the inner-plasma membrane surface. However,
and non-phosphorylated florescent peptide to be separated by the negligible binding potency difference between PKCd- and
an agarose gel electrophoresis method. The phosphorylated PKCy–C1b domains could be because of the dissimilarities in
fluorescent peptide migrated toward the positive electrode and surface areas and the residues present within the binding site.
a relative amount of phosphorylated fluorescent peptide was Our nonradioactive kinase activity assay clearly showed that
further quantified by fluorescence spectral analysis according HGLs activate the PKC enzymes in a similar manner as that of
to the manufacturer’s protocols. The results showed that under DAG. PKC activating capabilities of these compounds could be
the similar experimental condition the potent compounds have lower than a phorbol ester or other structurally complex
similar or better PKC activating capabilities than DAG (Fig. 10 molecules under similar experimental conditions; however,
and Fig. S12, ESI†). In contrast to our C1 domain binding affinities we successfully developed structurally restricted g-butyrolactone-
for both monomeric form and under liposomal environment, based simple C1 domain ligands.
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Conclusion
residue. This crude product was directly used for the next step
without further purifications.
In this study we demonstrated that g-hydroxymethyl-g-butyrol-
actone substituents strongly interact with the model membrane
and C1 domain of novel PKC isoenzymes. Strong interaction
between compounds and the model membrane alter its monolayer/
bilayer properties including fluidity and hydration. The protein
binding properties suggest that hydroxymethyl and acyl-groups of
the compounds are important for C1 domain binding through its
DAG/phorbol ester binding site. The enhanced fluidity of the lipid
bilayer structures could allow the PKC–C1 domains to interact
strongly with the HGL derivatives in the presence of anionic
phospholipids. Protein kinase activity assay also confirmed that
these potent compounds can activate PKC enzymes. Therefore,
our findings suggest that these hydroxymethyl g-butyrolactone
substituents are potential regulators of PKC isoforms and can be
used in PKC-based drug development processes.
General procedure for the reduction of c-butyrolactone
carboxylic acid
To a stirring solution of g-butyrolactone carboxylic acid (1.0 equiv.)
in THF (5.0 mL) was added BH3ꢂSMe2 (1.2 equiv.) drop-wise at room
temperature and stirring was continued for another 6 h.28 After the
completion of the reaction the solvent was removed under reduced
pressure to yield a residue which was purified by silica gel column
chromatography using a gradient solvent system of 3–6% methanol
in dichloromethane to yield g-hydroxymethyl-g-butyrolactone.
General procedure for the trityl protection of c-hydroxymethyl-
c-butyrolactone
To a stirring solution of TrCl (1.1 equiv.) and triethylamine
(1.2 equiv.) in dry dichloromethane, g-hydroxymethyl-g-butyrol-
actone (1.0 equiv.) in dry dichloromethane was added. The
resulting mixture was stirred at room temperature for 12 h.28
After completion of the reaction, the solvent was removed under
reduced pressure to yield a residue. The residue was dissolved in
dichloromethane (20 mL) and washed with a saturated solution of
NaHCO3. The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography and a gradient solvent system
of 20–30% ethyl acetate to hexane to give target compound.
Experimental section
General information
All chemicals were purchased from Sigma (St. Louis MO), SRL
(Mumbai, India) and used for synthesis without further purification.
Dry solvents were obtained as per reported procedures. Reactions
were performed under nitrogen atmosphere and monitored using
thin layer chromatographic (TLC) plates prepared from silica
gel 60 F254 (0.25 mm). Compounds were purified through
column chromatography using 60–120 mesh silica gel. NMR
spectra were recorded using CDCl3 (d = 7.24 for 1H and d = 77.23
for 13C NMR) with Varian 400 MHz and Bruker 600 MHz General procedure for the aldol reaction
spectrometers. Coupling constants ( J values) are reported in
To a stirring solution of CuCN (1.0 equiv.) and LiCl (2.0 equiv.) in
hertz, and chemical shifts are reported in parts per million
(ppm) downfield from tetramethylsilane using residual chloro-
form (d = 7.24 for 1H NMR, d = 77.23 for 13C NMR) as an internal
standard. Multiplicities are reported as follows: s (singlet), d
(doublet), t (triplet), m (multiplet), and br (broadened). Mass
spectra were recorded using a Waters Q-TOF Premier mass
spectrometer system, and data were analysed using the built-in
software. 1,2-Dipalmitoyl-sn-glycerol (DAG16), 1,2-dioctanoyl-sn-
glycerol (DAG8), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dipalmitoyl-
sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl)
(NBD-PE) and N-[5-(dimethylamino)naphthalene-1-sulfonyl]-1,2-
dihexadecanoyl-sn-glycero-phosphoethanol -amine (Dansyl-PE) were
purchased from Avanti Polar Lipids (Alabaster, AL). The PepTag-
nonradioactive kinase activity assay kit was obtained from Promega
(Madison, WI). Ultrapure water (Milli-Q system, Millipore, Billerica,
MA) was used for the preparation of buffers.
dry THF was added protected g-hydroxymethyl-g-butyrolactone
(1.0 equiv.) under a N2 atmosphere and the reaction mixture was
stirred for 5 min at ꢀ78 1C, then n-BuLi (1.5 equiv., 1.5 M solution
in THF) was added. Stirring was continued for 10 min at ꢀ78 1C
and then aldehyde (1.5 equiv.) in dry THF was added drop-wise.41
After completion of the reaction, the reaction was quenched with
methanol/water and the solvent was removed under reduced
pressure. The residue was dissolved in ethyl acetate and washed
with saturated NaHCO3 followed by brine solution. The organic
layer was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Column chromatography with silica gel and a
gradient solvent system of ethyl acetate to hexane yielded the
target protected alcohol.
General procedure for the preparation of esters
Carboxylic acid (1.1 equiv.), dicyclohexylcarbodiimide (1.1 equiv.)
and N,N-dimethylaminopyridine (0.1 equiv.) were added to a
stirring solution of the protected alcohol (1.0 equiv.) in anhydrous
dichloromethane (5 mL) under a N2 atmosphere.34 Stirring was
continued for 12 h at room temperature. After completion of the
General procedure for the synthesis of c-butyrolactone
carboxylic acid
To a stirring solution of L-glutamic acid (1.0 equiv.) in water was reaction, the reaction mixture was filtered and washed with
added HNO2 (1.2 equiv.) followed by HCl (1.2 equiv.) at 0 1C and dichloromethane. The filtrate was concentrated under reduced
stirring was continued for 8 h. After completion of the reaction pressure and column chromatography was performed with silica
the solvent was removed under reduced pressure to yield a gel and a gradient solvent system of 2–5% ethyl acetate to hexane.
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General procedure for the removal of trityl-group
concentration of 1.25 mM. After 30 min of incubation at room
temperature DPH fluorescence anisotropy was measured at
430 nm (excitation 355 nm). The concentration of compounds was
2.9 mM. To measure the change of membrane hydration, dansyl-PE
doped liposomes (PC/cholesterol/dansyl-PE (79/20/1)) were pre-
pared separately with and without doping of membrane active
HGL compounds at different concentrations (0–80 mol%) and
the change in dansyl fluorescence signal was recorded.32,33
To a stirring solution of trityl-protected compound (1.0 equiv.)
in dichloromethane (5.0 mL) was added TFA (2.0 equiv.) at
room temperature and stirring was continued for 1 h.28 After
completion of the reaction, the solvent was removed under
reduced pressure to yield a residue. The residue was dissolved
in EtOAc (20 mL) and washed with a saturated solution of sodium
bicarbonate. The organic layer was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography using 7–10%
ethyl acetate to hexane to yield the target compound.
Extent of membrane localization
The extent of localization of the compounds at the liposome–water
interface was compared by the NBD fluorescence quenching
method using PC/ligand16/NBD-PE liposomes (89/10/1) in 50 mM
Tris buffer, pH 8.2, containing 150 mM NaCl, according to the
reported procedure.43 The NBD fluorescence quenching reaction
was initiated by adding sodium dithionite solution (a stock solution
of 0.6 M sodium dithionite in 50 mM Tris buffer, pH 11, and
containing 150 mM NaCl, was used to obtain a final concentration
of 1 mM). The change in NBD fluorescence emission intensity at
530 nm (lex = 469 nm) was recorded for 3 min at room temperature.
Aggregation studies
The aggregation behaviours of the compounds in aqueous solution
were examined by fluorescence measurements at room temperature,
using a Fluoromax-4 spectrofluorometer. The fluorescence proper-
ties of pyrene were monitored to measure the critical aggregation
concentrations (CAC) of the compounds.35,42 The stock solutions of
compounds were freshly prepared by first dissolving them in
spectroscopic-grade dimethylsulfoxide (DMSO) and then diluted
with water. The amount of DMSO was kept less than 1% (by volume)
for each set of experiments and had no effect on any experimental
results. For fluorescence measurements, a saturated ethanolic
solution of pyrene (2 mM) and varying concentrations of ligands
were incubated in water at room temperature. Pyrene was excited
at 335 nm, and emission spectra were recorded from 345 to
550 nm. Pyrene produces five intense fluorescence peaks, but
only I1 (373 nm) and I3 (383 nm) were considered for measure-
ments of CAC values.
Protein purification
The C1b subdomain of PKCd and PKCy isoforms were
expressed in E. coli cells and purified by using methods similar
to those reported earlier.35,43
Fluorescence-based protein binding analysis
To compute the binding parameters under a membrane-free system,
ligand-induced Trp-fluorescence quenching measurements were
performed on a Fluoromax-4 spectrofluorometer at room tempera-
ture.34,35 The stock solutions of compounds were freshly prepared by
first dissolving complexes in spectroscopic-grade DMSO and then
diluted with buffer. The amount of DMSO was kept less than 3%
(by volume) for each set of experiments and had no effect on any
experimental results. For fluorometric titration, protein (1 mM) and
varying concentrations of compounds were incubated in a buffer
solution (20 mM Tris, 150 mM NaCl, 50 mM ZnSO4, pH 7.4) at room
temperature. Protein was excited at 284 nm, and emission spectra
were recorded from 300 to 550 nm. Proper background corrections
were made to avoid the contribution of buffer and dilution effects.
The resulting plot of Trp-fluorescence as a function of ligand
concentration was subject to nonlinear least-squares best-fit
analysis to calculate apparent dissociation constants for the
monomeric ligands (KD(ML)), using eqn (1), which describes
binding to a single independent site.
Interfacial behaviour measurement by Langmuir trough
techniques
The air–water interfacial behaviours of the amphiphilic compounds
were investigated using the Langmuir trough technique (NIMA
technology Ltd) according to the Wilhelmy plate method.30,36 Stock
solutions of the compounds were prepared in a chloroform/
methanol mixture [4 : 1 (v/v)]. The sub-phase consisted of Milli-Q
water (Millipore, Bedford, MA) and had a resistance of 18.4 MO
cm. Before each experiment the cleanliness of the sub-phase was
verified by repeated compression without lipids. If the surface
pressure changed by less than 0.2 mN mꢀ1 then the surface was
considered to be clean. Lipid solutions (5 mL of 0.5 mg mLꢀ1
stock solution) were deposited onto the surface of the sub-phase.
After the solvent had been allowed to evaporate for 10 min, the
monolayer was compressed at a constant rate of 10 mm minꢀ1
and the surface pressure (p)–area (A) isotherm was continuously
recorded. All experiments were conducted at room temperature
(25 1C). The p–A isotherm was analysed by inbuilt software.
,
ꢀ
ꢁ
½xꢃ
ðF0 ꢀ FÞ ¼ DFmax
þ C
(1)
½xꢃ þ KDðMLÞ
where F and F0 represented the fluorescence intensity at 339 nm
in the presence and the absence of ligand respectively. The DFmax
represents the calculated maximal fluorescence change; [x]
Measurement of the change in bilayer fluidity and hydration
To measure the change in membrane fluidity, anisotropy of DPH represents the total monomeric ligand concentration.
under liposomal environment was measured according to the
Fluorescence anisotropy measurements were also performed on
reported procedure.43 The fluorescence probe DPH was incorporated the same fluorimeter using reported method.34,35 All anisotropy
into the liposomes by adding the dye dissolved in THF (1 mM) values of the proteins in the absence or presence of compounds are
to liposomes (PC/cholesterol/ligand (60/20/20)) up to a final the mean values of three individual determinations. The degree (r)
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of anisotropy in the tryptophan fluorescence of the proteins was intensity values were plotted against the liposomes containing
calculated using eqn (2), at the peak of the protein fluorescence ligand concentrations to generate the binding isotherms
spectrum, where IVV and IVH are the fluorescence intensities of using eqn (1).
the emitted light polarized parallel and perpendicular to the
excited light, respectively, and G = IVH/IHH is the instrumental
Molecular modelling
grating factor.
Molecular docking analysis was performed using the crystal struc-
ture of PKCd–C1b (Protein Data Bank code: 1PTR) subdomain.37
The generation of energy minimized three-dimensional struc-
tures of ligands and ligand–protein docking was performed
using methods similar to those described earlier. Briefly, the
energy-minimized three-dimensional structure of ligands was
prepared by using the GlycoBioChem PRODRG2 Server.44 Ligand–
protein docking was performed using the Molegro Virtual Docker
software, Version 4.3.0 (Molegro ApS, Aarhus, Denmark).45 The
binding site was automatically detected by the docking software
and restricted within spheres with a radius of 15 Å. During
docking analysis the following parameters were kept unchanged:
number of runs 10, population size 50, crossover rate 0.9, scaling
factor 0.5, maximum iteration 5000 and grid resolution 0.30. The
docking results were examined on the basis of moledock and
re-rank scores. The docking poses were exported and re-examined
with PyMOL visualization software.
ðIVV ꢀ GIVH
Þ
r ¼
(2)
ðIVV þ 2GIVH
Þ
¨
Protein-to-membrane Forster resonance energy transfer
(FRET) based binding assay was used to measure the binding
affinity and specificity of the selected compounds under the
liposomal environment.34,35 In this assay, membrane-bound
C1 domain was displaced from liposomes by the addition
of the DAG8. The liposomes composed of PC/PE/PS/dPE
(60/15/20/5) and PC/PE/PS/dPE/ligand (55/15/20/5/5) were
used as control and for ligands, respectively. The stock
solution of DAG8 was titrated into the sample containing C1
domain (1 mM) and excess liposome (100 mM total lipid) in a
buffer solution (20 mM Tris, 150 mM NaCl, 50 mM ZnSO4,
pH 7.4) at room temperature. The competitive displacement
of protein from the membrane was quantitated using a
protein-to-membrane FRET signal (lex = 280 nm and lem
=
PKC activity analysis
505 nm). Control experiments were performed to measure the
dilution effect under similar experimental conditions and
the increasing background emission arising from direct dPE
excitation. Protein-to-membrane FRET signal values as a
function of DAG8 concentration were subjected to nonlinear
least-squares-fit analysis using eqn (3) to calculate apparent
equilibrium inhibition constants (KI(DAG8)app) for DAG8.
Where, [x] represents the total DAG8 concentration and DFmax
represents the calculated maximal fluorescence change.
The PKC enzyme activity was measured using a PepTag
non-radioactive protein kinase assay kit in the presence of
DAG16 and potent compounds with a modified assay protocol.
The assay was stopped after 30 min by incubating the sample
vials at 95 1C for 10 min and then 2 mL of 80% glycerol
was added to the samples. Agarose gel (0.8%) electrophoresis
was run at 110 V for 20 min and the gel image was captured
under UV light. The phosphorylated fluorescent peptide
bands were excised from agarose gel, and PKC activity was
quantitatively estimated by fluorescence spectral analysis
(lex = 540 nm).
!
½xꢃ
F ¼ DFmax 1 ꢀ
þ C
(3)
½xꢃ þ KIðDAG8Þapp
The equilibrium dissociation constant (KD(L)) for the binding
of the C1 domains to the ligand-associated liposomes was
calculated from eqn (4), using KD(ML) and KI(DAG8)app
values,34,35 where [L]free is the free ligand concentration
(2.63 ꢁ 0.04 mM). During calculations the ligand concentra-
tions in the liposome interior were ignored because of their
inaccessibility for the protein. Thus, the protein accesses
about half of lipids in the liposomes. The ligand concen-
tration was used in excess relative to the protein. The free
ligand concentration was calculated by assuming that most of
the protein would bind to the liposome and an equimolar
amount of ligand can be subtracted from the accessible
ligand.
Control Reactions
Reagents for the assay
(mL)
(mL)
1
2
3
4
5
6
PepTag PKC reaction 5ꢄ buffer
PepTag C1 peptide (0.4 mg mLꢀ1
PKC activator 5ꢄ solution
5
5
0
0
1
4
5
5
5
2.5
1
)
Compounds (DAG/1a/2a/3a; 80 mM)
Peptide protection solution
Protein kinase C (2.5 mg mLꢀ1 in
PKC dilution buffer)
4
7
Deionized water for final reaction
volume of 25 ml
5
2.5
ꢀ
ꢁ
½Lꢃfree
KIðDAG8Þapp¼ KDðMLÞ 1 þ
(4)
KDðLÞ
Acknowledgements
We also measured direct liposome binding of PKC C1b sub-
domains using a Trp-fluorescence quenching method. The The authors acknowledge their sincere gratitude to the Depart-
protein (0.25 mM) was added to PC/PE/PS/ligand16 (55/20/20/5) ment of Chemistry, Centre for the Environment, Central Instru-
liposomes prepared by an extrusion method. Fluorescence mentation Facility, Indian Institute of Technology Guwahati,
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