New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities

Article abstract of DOI:10.1021/jacs.5b07812

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single high

Full text of DOI:10.1021/jacs.5b07812

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Article
New Tricks for Old Proteins: Single Mutations in a Non-
enzymatic Protein Give Rise to Various Enzymatic Activities
Yurii S. Moroz, Tiffany T. Dunston, Olga V. Makhlynets, Olesia V. Moroz,
Yibing Wu, Jennifer H. Yoon, Alissa B. Olsen, Jaclyn M. McLaughlin, Korrie
L. Mack, Pallavi M. Gosavi, Nico A.J. Van Nuland, and Ivan V Korendovych
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b07812 • Publication Date (Web): 10 Nov 2015
Downloaded from http://pubs.acs.org on November 12, 2015
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Journal of the American Chemical Society
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New Tricks for Old Proteins: Single Mutations in a Non-
enzymatic Protein Give Rise to Various Enzymatic Activities
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Yurii S. Moroz,1,‡,† Tiffany T. Dunston,1,‡ Olga V. Makhlynets,1,‡ Olesia V. Moroz,1,‡,† Yibing Wu,2
Jennifer H. Yoon,1 Alissa B. Olsen,1 Jaclyn M. McLaughlin,1 Korrie L. Mack,1 Pallavi M. Gosavi,1 Nico
A. J. van Nuland3 and Ivan V. Korendovych1,*
1 Department of Chemistry, Syracuse University, 111 College Place, Syracuse NY, 13244
2 Department of Pharmaceutical Chemistry University of California – San Francisco 555 Mission Bay Blvd. South, San
Francisco, CA 94158, USA
3 Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
ABSTRACT: Design of a new catalytic function in proteins, apart from its inherent practical value, is important
for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic apꢀ
proach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted
a 74ꢀresidueꢀlong Cꢀterminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the reꢀ
sulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previousꢀ
ly reported de novo designed esterases. The simplicity of our design protocol should complement and expand the
capabilities of current stateꢀofꢀart approaches to protein design. These results show that even a small nonꢀ
enzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolyꢀ
sis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away
from becoming entry points for subsequent evolution.
Introduction. Nature has developed the means to facilitate an
enormous variety of chemical transformations with exceptionꢀ
al regioꢀ and stereoꢀselectivity starting with simple reactants in
water. Moreover, the repertoire of enzymes for new reactions
continues to grow. Just during past decades new proteins
emerged to degrade synthetic chemicals introduced into the
environment.1ꢀ6 Much effort has been dedicated to understand
how proteins evolve to adapt new functions and the consensus
emerged that new function originates from latent, promiscuous
enzymes or broad specificity enzymes that serve as starting
points for further evolution.2,7ꢀ9 Indeed, redesigning existing
proteins to perform new reactions starting with chemically
similar substrates has been very fruitful.10ꢀ13 Over the past
years principles and methods of directed evolution have been
perfected to allow for substantial improvement of very modest
initial activity by screening relatively small focused combinaꢀ
torial libraries.14,15 While optimization of existing enzymes has
been successful, it does require measurable initial activity and
is often limited by substrate scope, enzyme size, preparation
issues, etc. Achieving reasonable initial activity is especially
difficult for reactions with no obvious natural analogs. Comꢀ
putational design lifts this fundamental limitation by providing
such starting points.16,17 Combination of sophisticated compuꢀ
tational algorithms with directed evolution has been shown to
successfully create catalysts for a wide range of chemical
transformations.18ꢀ22 Importantly, initial configuration of the
computationally introduced active site can be far from optiꢀ
mal; Hilvert et al. showed that directed evolution may even
completely reshape the catalytic site during the optimization of
a computationally designed retroaldolase.23
Current methods for computational enzyme design rely on
precise positioning of multiple functional groups deemed to be
necessary for catalysis into the rigid protein scaffold to miniꢀ
mize a scoring function that predicts thermodynamic stability
of a protein. Despite successes of this approach to create good
starting points for subsequent directed evolution, computaꢀ
tionally designed enzymes still have fairly modest activity.24,25
Moreover, simultaneous introduction of multiple (often more
than 10) mutations is often so detrimental to the protein stabilꢀ
ity that additional rounds of mutagenesis may be required to
ensure that the designed catalysts are properly folded.26
While we are still far away from being able to create efficient
catalysts de novo, lessons from successes and failures of curꢀ
rent state of the art methods of enzyme design27 present us
with an opportunity. Indeed, if directed evolution is so powerꢀ
ful, can we focus on introducing a single highly reactive funcꢀ
tional group into a protein without extensive engineering of
the protein scaffold to achieve function? Such a minimalist
strategy offers highꢀthroughput and low computational cost.
Here we took on a task of de novo designing a catalyst for
ester hydrolysis, a reaction that finds many practical applicaꢀ
tions ranging from fine chemical synthesis to biofuel producꢀ
tion. We chose pꢀnitrophenyl esters as substrates due to the
abundance of data for catalysts developed by various apꢀ
proaches including computational design,28,29 combinatorial
screening30ꢀ35 and catalytic antibody selection.36ꢀ40 While low
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rates of pꢀnitrophenyl ester hydrolysis are relatively easy to
to establish the feasibility of transition state geometry as deꢀ
achieve,41 it is profoundly humbling that the best artificial
esterase that doesn’t show significant product inhibition is
only modestly active with a kcat value of just 3.3 minꢀ1 (Table
S1, Supporting Information).36
scribed in detail in the Experimental Section. Only a single
mutant (cꢀCaM M144H) satisfied all parameters described
above, importantly, the R enantiomer of pNPP provided a
better fit in the hydrophobic cavity of cꢀCaM (Fig. 1, Step 4).
By analogy with our previous work44,45 we named cꢀCaM
M144H AlleyCatE, where E stands for “esterase”.
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Scheme 1. Overall reaction (top) and the mechanism of
histidine catalyzed ester hydrolysis.
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Step 2
Step 1
Ensure that the substrate
may bind to the protein
Identify potential sites (orange)
for the active residue
40
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20
10
0
It is well known that common folds are quite frequent in proꢀ
teins catalyzing a variety of different reactions (e.g. TIM barꢀ
rel) and successful computationally designed catalysts take
advantage of existing enzymatic folds to achieve new funcꢀ
tion. But does that mean that nonꢀenzymatic proteins lack such
ability? Calmodulin (CaM), an allosterically regulated protein,
uses metal binding to achieve high fold stability. Stability has
been long postulated to play a critical role for designability,42
thus we set out to explore whether calmodulin could assume
esterase activity. We chose chromogenic pꢀnitrophenylꢀ(2ꢀ
phenyl)ꢀpropanoate (pNPP, Scheme 1) as a representative
ester substrate to compare our studies with previous design
work.29
-10
-20
Residue mutated to His
Step 3
Step 4
Check whether the mutation can
be accommodated by the protein
Test whether transition state
geometry is possible
Figure 1. Overview of the design methodology.
Naturally occurring esterases often rely on multiple amino
acid residues (arranged in dyads and triads) aided by transition
state stabilization provided by oxyanion holes to achieve effiꢀ
cient catalysis. However, it has been demonstrated that a histiꢀ
dine residue in a protein scaffold may catalyze ester hydrolyꢀ
sis, albeit with modest activity.28 Therefore we decided to enꢀ
gineer a single histidine residue into the calmodulin scaffold to
hydrolyze pNPP.
AlleyCatE was then recombinantly expressed in Escherichia
coli (E. coli) and purified by affinity chromatography. Alleyꢀ
CatE shows the classic “burst phase” dependence of product
formation on time (Fig. 2), consistent with the acyl intermediꢀ
ate formation described in Scheme 1. The acyl intermediate
was directly observed by MALDIꢀTOF mass spectrometry
(Figs. S1ꢀS4, Supporting Information). The MALDI spectrum
for unmutated cꢀCaM shows no acylation in the presence of
pNPP (Fig. S5, Supporting Information) confirming that Alꢀ
leyCatE is acylated at the newly introduced H144. However, it
should be noted that after several turnovers MALDIꢀTOF
spectra of AlleyCatE in the presence of substrate show multiꢀ
ple acylation events (up to 4 total) suggesting that the initially
formed acylꢀH144 intermediate can acylate AlleyCatE (Fig.
S6, Supporting Information). AlleyCatE showed significant
esterase activity with a k2/Ks of 70,000 ± 17,000 Mꢀ1minꢀ1 and
an apparent kcat/KM of 4,800 ± 300 Mꢀ1minꢀ1 for hydrolysis of
RꢀpNPP (Fig. 2, Table 1). AlleyCatE undergoes at least 12
turnovers and shows no signs of inhibition by pꢀnitrophenol or
2ꢀphenylpropionic acid (Figs. S7, S8, Supporting Inforꢀ
mation). In agreement with the design, AlleyCatE is enantioseꢀ
lective: RꢀpNPP is hydrolyzed ~3ꢀfold faster than its enantioꢀ
mer (Table 1). This level of activity, achieved with a single
mutation in a nonꢀenzymatic scaffold, is to our knowledge
higher than that of any previously reported de novo designed
Results and Discussion. The design process is schematically
outlined in Fig. 1. First, we ensured that the hydrophobic caviꢀ
ty of the Cꢀterminal domain of CaM (cꢀCaM) could accomꢀ
modate the substrate. Docking both enantiomers of pNPP into
cꢀCaM using AutoDock Vina43 shows that the substrate fits
well in the hydrophobic cavity of the protein (Fig. 1, Step 1).
Next, we identified all the residues facing the substrate (shown
in orange in Fig. 1, Step 2) and computationally evaluated the
effect of mutating these residues to histidines using Rosetta.
The protein backbone was fixed and the obtained models were
sorted by score (Fig. 1, Step 3). Most of the mutants were able
to accommodate histidine’s side chain without clashes; one of
the mutants (A128H) that showed relatively high Rosetta score
was removed from further consideration. The second step of
optimization was to determine the possibility of Michaelis
complex formation. Both enantiomers of pNPP were docked
into the computational models obtained in the previous step.
The poses found from the docking studies were then evaluated
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esterases (Table S1, Supporting Information). The observed
preserved in this mutant (Fig. 2a). AlleyCatE’s activity drops
kcat values (up to 0.24 minꢀ1) for AlleyCatE are comparable to
the values obtained for the less active catalytic antibodies
(0.33ꢀ0.4 minꢀ1)38,39 for this reaction and are two orders of
magnitude lower than the kcat values shown by best catalytic
antibodies for pꢀnitrophenyl ester hydrolysis under the same
conditions.36,37,46,47 While the observed degree of enantioselecꢀ
tivity is low when compared to promiscuous esterase activity
found in natural enzymes,48 it does represent a starting point
for subsequent improvement by directed evolution.
by at least 100ꢀfold in the absence of Ca2+.
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Introducing a histidine residue into the hydrophobic region of
CaM resulted in a small loss of the free energy of folding, well
tolerated by the highly stable calmodulin fold as shown by
chemical denaturation studies (Fig. S9, Table S2, Supporting
Information). The NMR structure of AlleyCatE in the calciꢀ
umꢀbound state is very similar (backbone rmsd 0.78 Å) to that
of designed model; importantly, the observed rotameric state
of the active histidine residue is the same as predicted (Fig.
2b).
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The activity of AlleyCatE to hydrolyze pNPP is well above the
background of natural esterases in E. coli crude lysate (Fig.
S10, Supporting Information) making directed evolution to
further improve catalytic efficiency simple and practical.
Reetz et al. showed that saturation mutagenesis of residues
around the active site offers the highest probability of sucꢀ
cess,49 therefore we employed this approach to show the feasiꢀ
bility of AlleyCatE’s further improvement. Saturation mutaꢀ
genesis performed on nine residues around the active residue
produced several proteins with higher activity. The best variꢀ
ant (cꢀCaM M144H A128T, named AlleyCatE2) was characꢀ
terized in detail. The k2/Ks value of AlleyCatE2 is 2ꢀfold highꢀ
er and the overall apparent kcat/KM is nearly 40% higher than
those of AlleyCatE (Table 1). The allosteric regulation of the
activity is fully preserved in AllyCatE2 (Fig. S11, Supporting
Information). These results obtained from a very limited liꢀ
brary of mutants show that AlleyCatE is evolvable and addiꢀ
tional rounds of directed evolution may improve the reactivity
even further. Previously we have shown that catalytic efficienꢀ
cy of AlleyCat, a calmodulinꢀbased Kemp eliminase, can be
easily improved by 200ꢀfold.50
b
a
Figure 2. a) Hydrolysis of RꢀpNPP (50 µM) catalyzed by Alleyꢀ
CatE (2 µM) in the presence (blue) and in the absence (red) of 10
mM CaCl2. b) NMR structure of the AlleyCatE. The substrate was
docked into the structure using AutoDock Vina. The side chain
for the active residue (M144H) is shown in color. The side chains
forming the hydrophobic cavity are shown in grey, the rest of the
side chains are omitted for clarity. PDB code 4bya.
Proper positioning of the histidine residue in the cavity is critiꢀ
cal, introducing histidine in any of the other eight positions
facing the hydrophobic pocket resulted in no significant esterꢀ
ase activity.
One of the key features of calmodulin is its allosteric regulaꢀ
tion. Binding of calcium ions to the apo cꢀCaM results in a
drastic conformational change that exposes protein’s hydroꢀ
phobic region to solvent. AlleyCatE is active only in the presꢀ
ence of Ca2+ confirming that the allosteric regulation is still
Calmodulin provides a relatively featureless hydrophobic caviꢀ
ty for catalyst design, thus we have probed the degree to which
such protein fold can provide substrate selectivity. We emꢀ
ployed pꢀnitrophenyl acetate (pNPA), a common substrate for
measuring esterase activity of various enzymes, as our referꢀ
ence point.
Table 1. Kinetic parameters for pNPP AlleyCatE and AlleyCatE2.
Protein
Substrate
k2/KS,
k2 x 10-2,
min-1
KS,
kcat/KM,
M-1 min-1
kcat x 10-2,
min-1
KM (app) ,
M-1 min-1
ꢀM
ꢀM
AlleyCatE
R-pNPP
S-pNPP
pNPA
70,000±17,000
15,000±200
1,950±170
140±45
140±40
75±13
160±30
220±30
90±9
22±6
86±26
400±70
13±3
4,800 ± 300
1,700 ± 200
330 ± 45
8.8±0.7
3.7±0.4
24±10
18±1
21±3
700 ±300
42 ±4
AlleyCatE2
R-pNPP
S-pNPP
pNPA
120,000±27,000
26,400±1,000
4,600±500
6,600 ± 600
2,400 ± 100
360 ± 30
28 ± 1
6.5±0.3
7.0±0.5
83 ± 11
200±24
27 ± 2
200 ±15
The enzymatic efficiencies of pNPA hydrolysis catalyzed by
cꢀCaM mutants are substantially lower (more than 35ꢀfold)
that those for pNPP despite similar kuncat values for the two
substrates (Table 1). This change in the kcat/KM value origiꢀ
nates mostly from the increase in KM, suggesting that even the
simplest of design principles can provide a certain degree of
substrate recognition.
Analysis of the properties of artificial esterases designed by
various approaches (Table S1, Supporting Information) is
quite instructive. It is clear that by properly arranging a histiꢀ
dine nucleophile in a protein or a peptide assembly using any
of the described approaches it is possible to achieve rapid forꢀ
mation of the acyl intermediate. In the case of peptide assemꢀ
blies the subsequent hydrolysis of the acyl intermediate is fast
thus k2 is the apparent kcat. The major advantage of computaꢀ
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tional designs is their ability to utilize existing protein scafꢀ
using Rosetta. The Rosetta score for the mutants were comꢀ
folds for substrate recognition and/or allosteric regulation.51,52
At the same time the increased hydrophobicity of the substrate
binding pocket slows down the subsequent hydrolysis and
affects the overall turnover.
pared to that of the original structure and reported in Fig. 1.
The models of R and S isomers of 4ꢀnitrophenyl 2ꢀ
phenylpropanoate were created with MarvinSketch (ChemAxꢀ
on) and optimized with UCSF Chimera59 and ADT 1.5.6.60
These models were then nonꢀcovalently docked into the comꢀ
putationally derived models of CaM mutants using Autodock
Vina software.61 At least 20 docking for each mutant were
generated. Then the distances between the δ and ε atoms (both
nitrogen and carbon atoms were considered to account for a
possibility of a 180 degree rotation around the CβꢀCγ bond) in
the histidine ring and the carbonyl carbon were computed. The
poses for the substrates were considered capable of supporting
catalysis only if a) the distance between carbonyl carbon of the
ester and the nitrogen atom of imidazole ring (either Nε or Nδ)
was less than 3.5 Å AND b) the O=CcarbonylꢀNimidazole angle was
between 100 and 140 degrees. The sequences for all proteins
discussed in this paper are given in Table S3 (Supporting Inꢀ
formation).
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Conclusion. In this paper we demonstrate how easily a miniꢀ
malistic approach to protein design can be applied to different
types of chemical reactions: introducing a single histidine resꢀ
idue into calmodulin confers esterase activity onto this nonꢀ
enzymatic protein. The obtained kinetically enantioselective,
allosterically controlled esterase is active enough to compete
against the background of natural enzymes in the E. coli crude
lysate, which allowed for further activity improvement using
directed evolution. We show that enzyme promiscuity, considꢀ
ered to be a major driving force in evolution of new functions,
has profoundly wider meaning. Even a small 74ꢀresidue nonꢀ
enzymatic protein can efficiently attain catalytic activities in
various reactions (Kemp elimination,44 ester hydrolysis, retroꢀ
aldol reaction53) as a result of just a single mutation. In other
words, proteins can be just one mutation away from becoming
entry points for subsequent evolution. It remains to be estabꢀ
lished whether other proteins can be as easily modified to cataꢀ
lyze novel chemical transformations, but there are no reasons
to a priori assume that this latent enzymatic promiscuity is
limited to calmodulin.
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Cloning and mutagenesis. All genes were cloned into
pMCSG49 expression vector using ligase independent cloning
(LIC).62 This vector contains an Nꢀterminal His6ꢀtag and a
tobacco etch virus (TEV) protease recognition site
(ENLYFQ/S). Site directed mutagenesis was performed using
Pfu Turbo polymerase (Promega) using manufacturer’s standꢀ
ard protocols. The plasmids encoding AlleyCatE and Alleyꢀ
CatE2 have been deposited in AddGene.
In addition to providing insight into understanding how proꢀ
teins evolve to adopt new functionality this work has practical
implications for protein design. It is reassuring that the miniꢀ
malist approach can accurately predict productive mutations.
The simplicity of the docking procedure allows for very quick
screening of potential catalyst candidates and to identify opꢀ
portunities that may be discarded by more stringent requireꢀ
ments for the transition state geometry. The advent of tools to
shape proteins for binding new substrates54 will enable this
approach to be applied to more complex reactions and protein
scaffolds. As various iterative methodologies of design and
directed evolution have been highly successful,55,56 subsequent
stepwise improvement of the catalyst candidates identified by
minimalist approach is likely to produce efficient catalysts
using simple tools at low computational cost following a path
that is similar to the one observed in nature.
Protein expression and purification. The vectors containing
the genes of interest were transformed into E. coli BL21(DE3)
pLysS cells and expressed overnight at 37 °C using ZYMꢀ
5052 autoꢀinduction medium supplemented with ampicillin
(100 ug/mL) and chloramphenicol (34 mg/L).63 The cells were
lysed in a buffer containing 25 mM Tris (pH 8.0), 20 mM imꢀ
idazole, 10 mM CaCl2, and 300 mM NaCl on ice with protease
inhibitor (phenylmethylsulfonyl fluoride) added. The lysate
was then centrifuged at 20,000 g for 1 hour at 4 °C to remove
the cell debris and the supernatant was applied onto a NiꢀNTA
column (Clontech). The column was washed multiple times
with the lysis buffer and the protein was eluted with buffer
containing 25 mM TRIS, 300 mM imidazole, 10 mM CaCl2,
and 300 mM NaCl (pH 8.0). To remove a polyhistidine tag,
the proteins were subjected to subsequent TEV digestion. The
protein buffer was changed to TEV digestion buffer (50 mM
TRIS pH 8.0, 75 mM NaCl) using a BioRad 10 DG desalting
column and then dithiothreitol (DTT) and ethylenediaminetetꢀ
raacetic acid (EDTA) were added to a final concentration 1
mM and 0.5 mM respectively. TEV protease was added to the
protein, using 1:10 ratio of OD280(TEV) to OD280(protein). The
solution was mixed, sterilized using a 0.22 ꢁm filter, and incuꢀ
bated overnight at 34°C. Following the digest, DTT and
EDTA were removed with a desalting column. The obtained
protein solution in 20 mM HEPES, 10 mM CaCl2, 100 mM
NaCl (pH 7.5) was passed through a NiꢀNTA column to reꢀ
move the undigested protein and the cleaved Hisꢀtag. The
SDSꢀPAGE gel of the pure proteins is presented in Fig. S12
(Supporting Information).
Experimental section.
General. Reagents and buffers were purchased from Biꢀ
oBasic, Inc. and Santa Cruz Biotechnology, Inc. DNA oligoꢀ
nucleotides were purchased from Integrated DNA Technoloꢀ
gies. Enzymes for cloning were purchased from Promega and
New England Biolabs. Racemic 4ꢀnitrophenyl 2ꢀ
phenylpropanoate was prepared from 2ꢀphenylꢀpropionic acid
and 4ꢀnitrophenol using previously reported procedure.29 The
enantiomerically pure (R)ꢀ and (S)ꢀ4ꢀnitrophenylꢀ(2ꢀphenyl)ꢀ
propanoates were prepared from the corresponding commerꢀ
cially available enantiomers of 2ꢀphenylꢀpropionic acid using
the same protocol.
Computational studies. A high resolution crystal structure of
calmodulin (PDB entry 1CLL)57 served as a starting point of
the design. The side chain placement was computationally
optimized with fixed backbone (fixbb protocol) using Rosetꢀ
ta.58 Mutations in the resulting model were computationally
introduced by changing the amino acid identity in positions
A88, F92, L105, V108, M109, M124, A128, F141, M144,
M145 to a histidine and side chain placement was again comꢀ
putationally optimized with fixed backbone (fixbb protocol)
Expression and purification of isotopically labeled pro-
teins. Uꢀ15N,13Cꢀlabeled AlleyCatE was expressed with double
antibiotic resistance following the previously described proꢀ
cedure.64 The plasmid encoding the protein sequence with a Nꢀ
terminal His6ꢀtag followed by the TEV protease recognition
site (ENLYFQ/S) was transformed into BL21(DE3) pLysS
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cells. The next day, a single colony was inoculated into a
ꢀ
ꢊ
ꢍ
ꢎꢏꢐꢑ
ꢁ − ꢂꢃꢄꢅꢆꢁℎꢇꢂꢆꢈꢉꢄꢇ = ꢋꢄ + ꢌ 1 − ꢇ
(1)
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50 mL Luria Bertani (LB) culture and was grown overnight at
30 °C. The next morning the overnight culture was added to
1 L of 2x LB and incubated at 37 °C and 230 rpm until OD600
reached 0.6 – 0.7. The cell were harvested by centrifugation
for 8 minutes at 1,000 g and 30 °C, washed first with unlaꢀ
beled M9 minimal medium then with 25 mL of M9 minimal
medium that contains 15NH4Cl and uniformly 13Cꢀlabeled gluꢀ
cose. The cell pellets were resuspended in 300 mL of M9 minꢀ
imal medium solution containing 15NH4Cl as a nitrogen
source, 13Cꢀlabeled glucose as a carbon source and incubated
at 37 °C for 2.0 hours to allow for the recovery of growth.
Protein expression was induced by addition of isopropyl βꢀDꢀ
1ꢀthiogalactopyranoside (IPTG) to a final concentration of
1 mM. After induction for 2 hours the cells were harvested by
centrifugation and proteins were purified using NiꢀNTA affiniꢀ
ty chromatography as described above.
where t is time. Plotting reciprocal values of A and b vs. 1/[S]0
at various initial substrate concentrations (Figs. S13ꢀS18, Supꢀ
porting Information) allows for straightforward determination
of kcat, KM(app), k2 and Ks.66
CD spectroscopy. Circular dichroism experiments were perꢀ
formed on a JASCO Jꢀ715 CD spectrometer in a step scan
mode (4 s averaging time) averaging over three runs using a
quartz cuvette with a 1 mm path length. Samples were preꢀ
pared in a buffer containing 4 mM HEPES (pH 7.5), 2 mM
CaCl2 and 30 mM NaCl with final protein concentration mainꢀ
tained at 25.0 ꢁM. The sample absorbance never exceeded 1.5
at all wavelengths in order to obtain reliable MRE values. The
ellipticity of proteins was monitored at 222 nm for chemical
denaturation experiments in the presence of varying concenꢀ
trations of guanidinium hydrochloride (0 – 6 M). The data
collected for cꢀCaM and AlleyCatE were fitted to the equation
below.
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Crude cell lysate screening and directed evolution. To test
the activity of various proteins to catalyze ester hydrolysis in
crude cell lysates, the genes for cꢀCaM, AlleyCatE, CaM and
CaM M144H (full length protein) mutant were transformed
into E. coli BL21 (DE3) pLysS cells. Individual colonies were
cultured into 200 ꢁL of LB containing 100 ꢁg/mL of ampicilꢀ
lin in 96ꢀwell plates and grown for 6ꢀ7 hours at 37 °C. 10 ꢁL
of the resulting culture was used to inoculate ZYMꢀ5052 autoꢀ
induction media containing 100 ꢁg/mL of ampicillin and
grown overnight at 37 °C. The following day, cells were harꢀ
vested and lysed with cell lysis buffer containing 20 mM
HEPES, 10 mM CaCl2, 100 mM NaCl, 0.2% Triton Xꢀ100, pH
8.0. Hydrolysis of 25 ꢁM 4ꢀnitrophenyl 2ꢀphenylpropanoate
(pNPP) by the crude cell lysates was monitored spectrophotoꢀ
metrically at 405 nm. For directed evolution studies, the
screening was done with the full length CaM to improve exꢀ
pression levels. The presence of the Nꢀterminal domain does
not affect catalytic activity. Saturation mutagenesis to generate
all possible single mutants in positions D80, L105, H107,
V108, L112, A128, A88, F89, and V91 was done by following
a standard mutagenesis protocol with Pfu Turbo DNA polyꢀ
merase (Stratagene) using an NNK codon and AlleyCatE as a
template. The resulting library of about 900 mutants was
screened for hydrolytic activity and the mutant that showed
the best steadyꢀstate activity in the crude lysate (AlleyCatE2)
was chosen for further characterization.
MREf +(MREu + yu[D])e−ꢁG −m [D]
RT
MRE =
1+e−ꢁG −m[D]
RT
(2)
where MRE is the observed mean residue ellipticity; MREf,
MREu are mean residue ellipticities representing the folded
and unfolded states for the Cꢀterminal domain, respectively;
ꢂG is the free energy of unfolding; [D] is the concentration of
the denaturant and yu is the slope for the unfolded state. CD
spectrum of AlleyCatE is shown in Fig. S19 (Supporting Inꢀ
formation). The thermodynamic parameters yielded by the fit
of chemical denaturation curve are summarized in Table S2
(Supporting Information).
NMR Spectroscopy and Structure Calculations. The NMR
sample of Uꢀ15N,13C ꢀlabeled protein were prepared at a conꢀ
centration 1.1 mM in a buffer containing 5 % D2O, 0.05 %
DSS, 0.01 % NaN3. All experiments were conducted at 30°C
on a Varian 600 MHz spectrometer equipped with cryogenic
probe. Chemical shift assignments were made using 15Nꢀ and
13CꢀHSQC,
HNCO,
HNCACB,
CBCA(CO)HN,
HBHA(CO)HN, 3D (H)CCHꢀTOCSY. The assignments were
further confirmed by 3D 15Nꢀ and 13Cꢀedited NOESY spectra
with the mixing time of 100 ms. NMR data were processed,
analyzed and visualized using NMRPipe67 and CARA. H
1
UV-vis spectroscopy kinetic assays. Kinetic assays were
performed in a 1 cm path length quartz cuvette on the Agilent
Cary 60 UVꢀVis spectrophotometer monitoring absorbance of
the product at 405 nm. The extinction coefficient for pꢀ
nitrophenol (12,700 Mꢀ1cmꢀ1) under these conditions was taken
from the literature data.65 Proteins were diluted to a final conꢀ
centration of 2.0 ꢁM or 6.0 ꢁM for hydrolysis of pNPP (R and
S isomers) and pNPA respectively. Substrate stock solutions
of pNPP (1 mM) and pNPA (100 mM) were prepared in aceꢀ
tonitrile. The reaction was monitored in triplicate for 900 seꢀ
conds (0.100 s averaging time) in 200 ꢁL of buffer containing
20 mM HEPES (pH 7.5), 10 mM CaCl2, 100 mM NaCl. 150
ꢁL of substrate solution in buffer was added first to the cuvette
and subsequently 50 ꢁL of the protein solution was added. The
final content of acetonitrile was 1.5% in reaction mixtures
containing pNPA and 6% in the reaction mixtures containing
pNPP. The obtained kinetic traces were analyzed using the
method of Kezdy and Bender.66 Briefly, the kinetic traces were
fit to
chemical shifts were referenced to internal DSS and 13C and
15N chemical shifts were referenced indirectly via gyromagnetꢀ
ic ratios. Backbone dihedral angles constraints were derived
from chemical shifts using TALOS+.68 1Hꢀ1H upper distance
constraints for structure calculations were obtained from the
analysis of the 3D 13Cꢀ and 15Nꢀedited NOESY spectra. Autoꢀ
mated NOE assignment and structure calculation was done
with CYANA 3.069 together with dihedral angle constraints
and metal ions constraints, which were taken from the crystal
structure of CaM (PDB code 1CLL).57 The resulting assignꢀ
ments were verified and corrected by interactive spectral analꢀ
ysis. Subsequently the structure calculation was performed
iteratively with CYANA, with iterations used to verify and
complete resonance assignments, refine NOESY peak lists and
optimize the distance calibration constants. The first set of
refined structures was obtained using XPLORꢀNIH.70 The
final set of structures was further refined by restrained moꢀ
lecular dynamics in explicit water71 using the program CNS
1.272 with the OPLSX force field. Structural statistics and
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Journal of the American Chemical Society
Page 6 of 7
global quality factors were computed with PSVS 1.5.73ꢀ77
Chemical shifts, NOESY peak lists, and raw timeꢀdomain data
for AlleyCatE were deposited in the BioMagResBank (BMRB
ID 19376); the coordinates were deposited in the Protein Data
Bank (PDB code 4BYA). The structure statistics are summaꢀ
rized in Table S4 (Supporting Information).
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Mass Spectrometry. MALDIꢀTOF spectrometry was done on
a Bruker Autoflex III instrument. Prior to each experiment the
instrument was calibrated with standard samples provided by
the manufacturer. Dihydroxybenzoic acid (DHB) or sinapinic
acid was used as a matrix. For acylated samples the substrate
was added to the proteins and the samples were immediately
dried and analyzed.
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ASSOCIATED CONTENT
Supporting Information. Details of NMR structural characteriꢀ
zation. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* ikorendo@syr.edu
Present Addresses
†Department of Chemistry, Taras Shevchenko National Universiꢀ
ty of Kiev, 64 Volodymyrska St., Kiev, 01601, Ukraine.
Author Contributions
The manuscript was written through contributions of all authors.
‡These authors contributed equally.
Funding Sources
No competing financial interests have been declared. This work
was supported in part by an NSF IGERT fellowship to T.T.D.,
grant number 1332349 from NSFꢀEFRI, ORAU Ralph E. Powe
Junior Faculty Enhancement award and a Humboldt Fellowship to
I.V.K.
ACKNOWLEDGMENT
This paper is dedicated to the memory of Prof. Anatoly I. Brusiloꢀ
vets. We thank Dr. Alexander N. Volkov for insightful discusꢀ
sions and Prof. Robert P. Doyle for providing access to CD inꢀ
strument.
ABBREVIATIONS
CaM, Calmodulin; pNPA, pꢀnitrophenyl acetate; pNPP, 4ꢀ
nitrophenylꢀ(2ꢀphenyl)ꢀpropanoate.
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TOC graphic
Wild type - No catalysis
Glu92 - Kemp Elimination
His144 - Ester hydrolysis
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