Synthesis and single enzyme activity of a clicked lipase-BSA hetero-dimer

Article abstract of DOI:10.1039/b516551b

Click chemistry is used to construct a novel lipase-BSA hetero-dimer, in which the latter protein acts as a foot enabling the anchoring of the enzyme onto the surface for single enzyme studies. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b516551b

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Synthesis and single enzyme activity of a clicked lipase–BSA
hetero-dimer{
a
Nikos S. Hatzakis, Hans Engelkamp, Kelly Velonia, Johan Hofkens, Peter C. M. Christianen,
a
b
c
a
d
Allan Svendsen, Shamkantr A. Patkar, Jesper Vind, Jan C. Maan, Alan E. Rowan* and
d
a
a
a
a
Roeland J. M. Nolte*
Received (in Cambridge, UK) 22nd November 2005, Accepted 19th January 2006
First published as an Advance Article on the web 9th February 2006
DOI: 10.1039/b516551b
Click chemistry is used to construct a novel lipase–BSA hetero-
dimer, in which the latter protein acts as a foot enabling the
anchoring of the enzyme onto the surface for single enzyme
studies.
In order to further extend the scope of our studies and to
overcome difficulties arising from non-specific adsorption of
the enzyme molecules onto the glass surface, we have developed
a novel general protocol for studying single enzymes using a
so-called protein foot. The latter is based on bovine serum albumin
(BSA), which is a 67 kDa carrier protein often used for
Single enzyme experiments provide new means to obtain
information regarding the properties and behaviour of enzymes.
Although the first investigations in this direction were performed
6
stabilization of enzymes, proteins and antibodies. Due to its
strong adsorption to both hydrophilic and hydrophobic
surfaces, BSA is also used as a biocompatible surface coating
1
in the early sixties, it is only with the advances in fluorescent
7
agent. For our purpose the BSA protein was coupled to
microscopy and scanning probe technologies that major progress
2
has been made. Recent studies have shown that it is possible to
8
Thermomyces Lanuginosa Lipase (TLL) by the Cu(I) catalyzed
9
probe the conformational motions in enzymes and to obtain
unique aspects of the catalytic activity of single enzyme molecules,
[3 + 2] Huisgen cycloaddition reaction, (commonly referred
to as ‘‘click chemistry’’) to give a unique protein hetero-dimer.
TLL is a well characterized 30 kDa fungal lipase which is widely
used as a workhorse enzyme due to its stability at high
temperatures.
3
which are otherwise masked in ensemble-averaged measurements.
4
Recently we reported on the direct and real time observation of
a single-enzyme-catalyzed reaction employing the technique of
confocal fluorescent microscopy (CFM). Using the lipase B from
Candida Antarctica, immobilized on a hydrophobic glass surface,
we were able to monitor single enzyme turnovers of the hydrolysis
of a pro-fluorescent substrate for long periods of time (up to
10,11
A TLL mutant
with only one free solvent accessible lysine
(Lys 46) was thought to be the ideal candidate for our experiments.
We utilized this single reactive lysine group for the specific
1
2
coupling of TLL to BSA. The lower affinity of TLL for
hydrophobic surfaces, as compared to that of CALB, used in our
previous experiments, should enable BSA to be preferentially
adsorbed on the hydrophobic surface, acting as a protein foot.
This would in turn allow the lipase to remain in solution and retain
its native conformation, thereby helping overcome problems that
arise from non-specific absorption such as denaturation and low
reproducibility.
3
hours). This study allowed us to unravel hidden characteristics in
the catalytic behaviour of this lipase, which were explained using a
model in which the single enzyme molecule exhibits distinctive
catalytic activities arising from a number of slowly interconverting
4
conformations.
The main limitation of the above mentioned studies is the
random orientation of the enzyme molecules physisorbed on the
surface. As a result of this, enzymes may be adsorbed with active
sites facing the surface making them inaccessible for substrate
molecules or even denaturation of the enzyme molecules may
occur. In order to overcome these limitations various techniques
The acetylene functionalized TLL (1) (Scheme 1) was obtained
by coupling 4-pentynoic acid to the free lysine function of the
13
enzyme using standard peptide coupling conditions.
Confirmation that the acetylene function was attached to TLL
was obtained by a reaction of the enzyme with an azide substituted
coumarin dye (2) (see Supporting Information). Reaction of the
5
have been developed such as immobilization of the enzyme in a
5
c
2b
BSA layer, the use of an agarose gel and furthermore specific
5b
14
attachment of enzymes on a surface.
free, solvent accessible thiol of BSA with 3-azidopropyl-1-
maleimide (3) afforded the complementary azide functionalized
BSA (4). Coupling of the acetylene functionalized TLL and the
azide modified BSA was performed in 20 mM phosphate buffer
(pH 7.2) by mixing equimolar quantities of the BSA and TLL
a
Institute for Molecules and Materials, Radboud University Nijmegen,
Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands.
E-mail: A.Rowan@science.ru.nl; R.Nolte@science.ru.nl;
Fax: (+31) 24 365 2929; Tel: +31 (0)24 36 52323
components. The reaction, with CuSO /ascorbic acid was allowed
4
b
to proceed for three days at room temperature and was followed
by separation of the hetero-dimer (5) from unreacted BSA and
TLL using gel filtration chromatography (yield of product > 80%).
The formation and purity of the hetero-dimer were ascertained
by FPLC analysis and further confirmed by SDS PAGE
(see Supporting Information).
Department of Organic Chemistry, University of Geneva, 30 quai
Ernest Ansermet, Geneva 4, CH-1211, Switzerland
Department of Chemistry, Katholieke Universiteit Leuven,
c
Celestijnenlaan 200 F, 3001 Heverlee, Belgium
NoVo-Nordisk a/s, NoVo Alle, Bagsvaerd, DK-2880, Denmark
d
{ Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b516551b
2
012 | Chem. Commun., 2006, 2012–2014
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488
Fig. 1 Fluorescence emission experiments on individual Alexa -labeled
TLL–BSA hetero-dimers. A) Confocal Fluorescence Microscopy micro-
4
88
Scheme 1 Synthesis and characterization of the TLL–BSA hetero-dimer.
graph of Alexa -labeled TLL–BSA molecules. B) Bleaching of the
488
Alexa dye molecules attached to the BSA of the TLL–BSA dimer. The
bleaching took place in the presence of substrate molecules. C) Time trace
measured at the location marked in white in Fig. 1A, showing the local
formation of fluorescent molecules. D) Time trace measured at the
location marked in red in Fig. 1A, showing the background noise level.
Catalytic activity studies on the TLL–BSA hetero-dimer and the
mutant TLL were performed in solution using the pro-fluorescent
substrate 5-(and-6)-carboxyfluorescein diacetate (5(6)-CFDA)-
mixed isomers. The unfunctionalized TLL displayed an activity
of 8 nmol/min in the hydrolysis of the CFDA ester (see Supporting
Information). For the TLL covalently connected to BSA the
relative activity, under identical conditions, was found to be twice
as fast as the activity of the unmodified enzyme (y 16 nmol/min).
This increase in activity is tentatively attributed to the opening of
the lid of this lipase by the conjugated hydrophobic BSA, a
Positioning the laser spot at a location where no 5 is present, i.e.
the spot marked with a red circle in Fig. 1A, allowed for
1
7
independent measurement of the background fluorescence signal
Fig. 1D). This was found to be similar to the background signal
measured in between the peaks in Fig. 1C.
(
2
1
15
From the time traces an average turnover number of 17 s per
single hetero-dimer on the surface was determined. Furthermore,
all visible enzyme molecules were now on average equally active,
highlighting the favourable effect of anchoring the lipase onto the
glass surface with the help of the BSA foot avoiding non-specific
absorption. Further analysis of the waiting times of the individual
turnovers showed that consecutive turnovers were not completely
random. Fig. 2 shows the two-dimensional histograms of the time
durations of the turnover cycles n events apart (n = 1, n = 15 and
n = 100). For n = 1, that is events directly following each other, a
mechanism analogous to that of the normal enzyme.
The enzymatic activity of individual hetero-dimer molecules was
examined by single molecule experiments (Fig. 1). To this end the
BSA molecules in the hetero-dimers 5 were labelled with Alexa-
4
88
16
Fluor dye molecules allowing the direct visualization of the
29
hetero-dimers by CFM. A solution (10 M) of thus modified
4
88
Alexa –5 in water was deposited on a hydrophobic cover slip.
After allowing the hetero-dimer to adsorb, the solution was
removed and the location of the labelled single hetero-dimer
molecules was determined by CFM (Fig. 1A). Rinsing the cover
slip repeatedly with water did not alter the CFM image, which
indicates that adsorption of 5 to the surface was indeed strong. A
2
6
solution of the pro-fluorescent substrate CFDA (10 M) was
subsequently added, and a single molecule of 5 was positioned in
488
the focus of the CFM. Fig. 1B shows that the Alexa labels
attached to 5 became bleached within 50 seconds. At the same
time, peaks of fluorescence emission started to appear{ (Fig. 1C).
These peaks are the result of the enzymatic formation of the
fluorescent product within the active site of the enzyme, followed
by rapid diffusion into the bulk solution. Each peak is the result of
Fig. 2 Two-dimensional histograms of the event durations separated by
i
n events, p(t , ti+n). For n = 1, a clear diagonal feature is present, showing
4
a single enzyme turnover cycle. The time between each fluorescent
the correlation between consecutive enzymatic events. This diagonal is still
visible for n = 15. Events separated by 100 events do not show any
correlation. Scales range from 2 to 300 ms (logarithmic); the intensity
range spans 0–350 occurrences (linear).
peak corresponds to a single turnover event. The analysis of these
peaks allows the direct measurement of the exact kinetics of a
single TLL enzyme.
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R. J. M. N.), and the Royal Netherlands Academy of Arts and
Sciences (grant to R. J. M. N.). Dr Joseph Sly is acknowledged for
the kind supply of the fluorescent coumarine.
Notes and references
{ Individual TLL enzymes, without the BSA foot, when deposited on the
same hydrophobic surface exhibited no catalytic activity.
1
2
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Fig. 3 The fluctuating enzyme model, in which the enzyme adopts a
variety of interconverting conformations, each one exhibiting different
4
catalytic activity.
clear diagonal feature is present, which can be explained as a
2
b
memory effect. Long turnovers are more often followed by long
turnovers and short turnovers are more often followed by short
turnovers. Interestingly, even after 15 events, this memory effect is
still present as there is still some correlation visible for n = 15. For
n = 100, a correlation is no longer observed. The distribution of the
turnover times was found to be in agreement with our recently
proposed fluctuating enzyme model for the single enzyme
5
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4
behaviour of CALB. In this model the enzyme moves slowly
8
D. M. Lawson, A. M. Brzozowski, S. Pety, C. Verma and G. G. Dodson,
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through several conformations, each exhibiting its own activity
4
and corresponding turnover times. The memory is lost once the
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enzyme changes conformation. It is likely that this model of
3
,4
enzyme fluctuations also applies to other enzymes and may be of
more general nature (Fig. 3).
In conclusion we have shown that the copper catalyzed Huisgen
10 A. Svendsen, I. G. Clausen, S. A. Patkar, K. Borch and M. Thellersen,
[2 + 3] cycloaddition reaction can be efficiently used to prepare
Methods Enzymol., 1997, 284, 317.
1 Mutant TLL (TLL-1823) possessing only one free solvent accessible
protein dimers. The resulting TLL–BSA hetero-dimer was shown
to possess a higher activity than TLL, which is tentatively
attributed to the ‘‘interfacial activation’’ of the lipase caused by the
presence of the hydrophobic BSA. Furthermore, all studied dimers
1
lysine was provided by Novo enzymes.
12 K. Wannerberger and T. Arnebrant, Langmuir, 1997, 13, 3488.
1
3 K. K. W. Wong, H. Colfen, N. T. Whilton, T. Douglas and S. Mann,
J. Inorg. Biochem., 1999, 76, 187.
4 L. Tao and A. M. English, Biochemistry, 2004, 43, 4028.
1
8
were active and had similar long term activity. This observation
suggests that the above described anchoring approach is an ideal
method for constructing active enzyme surfaces.
1
1
5 (a) U. Derewenda, A. M. Brzozowski, D. M. Lawson and
Z. S. Derewenda, Biochemistry, 1992, 31, 1532; (b) A. M. Brzozowski,
H. Savage, C. S. Verma, J. P. Turkenburg, D. M. Lawson, A. Svendsen
The initial single enzyme studies described strongly support the
3,4
and S. Patkar, Biochemistry, 2000, 39, 15071.
recently proposed fluctuating enzyme model. The anchored TLL
enzyme remains active for more than 40,000 turnovers. It slowly
fluctuates between different conformations, each with its own
catalytic activity. Our approach allows the study of interesting
characteristic features of the catalytic behaviour of enzyme
molecules at the single molecule level, such as denaturation and
the effect of temperature. These studies are currently underway in
our laboratory.
488
1
6 As we reported earlier (ref. 4a), labelling enzymes with Alexa fluor
renders focusing of the laser beam easier. In this case the labelling occurs
selectively on BSA due to the absence of lysines on TLL. In this way the
native conformation and catalytic activity of TLL remain intact.
17 The non-specific hydrolysis of CFDA ester in the presence of BSA was
488
studied using CFM. BSA was labelled with Alexa fluor and was
immobilized on the hydrophobic glass under otherwise identical
conditions. The observed hydrolysis was negligible (see supporting
information).
1
8 The initial activity of the dimer on the surface was found to be 80 times
higher than in solution. However the substrate concentration in the
single enzyme studies was above saturation level, which could not be
accomplished in solution.
These investigations were supported by the Research School
NRSC-Catalysis (grant to R. J. M. N.), the European Union
Bioscope program and Bionics program (grants to A. E. R. and
2
014 | Chem. Commun., 2006, 2012–2014
This journal is ß The Royal Society of Chemistry 2006