Suite of activity-based probes for cellulose-degrading enzymes

Article abstract of DOI:10.1021/ja309790w

Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, h

Full text of DOI:10.1021/ja309790w

Article
pubs.acs.org/JACS
Suite of Activity-Based Probes for Cellulose-Degrading Enzymes
Lacie M. Chauvigne-Hines,‡ Lindsey N. Anderson,‡ Holly M. Weaver, Joseph N. Brown, Phillip K. Koech,
́
Carrie D. Nicora, Beth A. Hofstad, Richard D. Smith, Michael J. Wilkins, Stephen J. Callister,
and Aaron T. Wright*
Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Blvd, Richland, Washington 99352, United States
S
* Supporting Information
ABSTRACT: Microbial glycoside hydrolases play a dominant
role in the biochemical conversion of cellulosic biomass to
high-value biofuels. Anaerobic cellulolytic bacteria are capable
of producing multicomplex catalytic subunits containing cell-
adherent cellulases, hemicellulases, xylanases, and other
glycoside hydrolases to facilitate the degradation of highly
recalcitrant cellulose and other related plant cell wall
polysaccharides. Clostridium thermocellum is a cellulosome-
producing bacterium that couples rapid reproduction rates to
highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl
aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the
direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes
to harness the utility and multimodal possibilities of click chemistry and to increase enzyme active site inclusion for liquid
chromatography−mass spectrometry (LC−MS) analysis. We directly analyzed ABP-labeled and unlabeled global MS data,
revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-
containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the
functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of
the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling.
Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater
understanding of the organismal role associated with biofuel development.
enzymes synergistically degrades the recalcitrant polysaccharides
of cellulosic materials. The cellulosome assembly is mediated by
a noncatalytic scaffoldin (CipA) protein, which through high-
affinity interactions can bear up to nine catalytic enzyme units,
and contains a family 3 cellulose-binding module (CBM3) for
attachment to cellulosic materials and a type II dockerin module
for attachment of the cellulosome units to the cell surface.6 The
cellulosomal enzyme units possess a type I dockerin module that
binds strongly to cohesin modules on scaffoldin.7
The C. thermocellum genome encodes 72 proteins with type I
dockerin modules.5 These include cellulases, β-glucanases,
xylanases, pectinases, mannanases, xyloglucanases, and protei-
nases, matching the complex diversity of the cellulosic material
the bacterium acts upon.5 These catalytic activities can be
generally categorized as: (i) endoglucanases, which hydrolyze
internal sites on the cellulose chain, (ii) exoglucanases, which
hydrolyze the reducing or nonreducing free chain ends of
cellulose and can act in a continuous, processive manner, and (iii)
β-glucosidases, which hydrolyze soluble cellodextrins and
cellobiose to glucose.2 These enzymes hydrolyze glycosidic
bonds resulting in an inversion or retention of the stereochemical
INTRODUCTION
■
The desire to identify biofuel alternatives to liquid fossil fuels has
kindled worldwide interest in converting cellulosic biomass to
high-value fuels and other small carbon compounds.1 Cellulosic
biomass is the most abundant renewable source of carbon and
energy on the planet, with estimates of 180 million tons of
feedstocks from agriculture and forest litter available per year.2
Harnessing the catalytic prowess of glycoside hydrolases (GHs)
within microbial cellulose-degrading machinery for biofuel
development is of high value and high interest.1 Cellulolytic
bacteria and fungi secrete complex suites of cellulases, hemi-
cellulases, and accessory enzymes for hydrolysis of highly
recalcitrant cellulose and other related plant cell wall
polysaccharides.
The rate-limiting step in converting cellulosic material to
biofuels is the hydrolysis of polysaccharides by glycoside
hydrolases. A gram-positive, thermophilic, anaerobic bacterium,
Clostridium thermocellum, has the highest rate of cellulose
utilization of any bacterium, making it of particular interest for
biofuel production.3 Among living organisms, C. thermocellum
belongs to a unique group of bacterium that can utilize cellulosic
materials to generate ethanol directly.4 C. thermocellum secretes a
cell surface-bound macromolecular multienzyme complex,
termed the cellulosome.5 This extensive consortia of extracellular
Received: October 5, 2012
© XXXX American Chemical Society
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configuration of the anomeric center. Numerous glycoside
hydrolase families within the catalytic machinery of cellulosomes
fall within these categories.8,9
Activity-based protein profiling (ABPP) has been successfully
applied to the identification of enzymatic activities within
complex proteomes.10 Glycoside hydrolases play a crucial role in
biological functions from human health to biofuel generation,
thus leading to the development of activity-based probes (ABPs)
to profile these valuable enzymes.11−22 Herein, ABPs have been
developed as affinity-based or mechanism-based irreversible
inhibitors. Our affinity-based ABPs contain a region to impart
specificity for glycoside hydrolases alongside a reactive group
that forms a covalent bond to catalytic or noncatalytic regions of
the enzyme. Mechanism-based ABPs form a covalent enzyme−
ABP bond upon catalytic reaction.23 Although multiple ABPs
have been developed for GHs, no large-scale comparative
analysis has been performed. Additionally, the array of diversity
and variation of catalytic mechanisms found within GH families
limit the profiling scope of a single ABP in a complex biological
system.
To address the reactivity and selectivity of multiple GH-ABPs,
we describe herein the synthesis and functional characterization
of a suite of ABPs based on known scaffolds for affinity and
mechanism-based inhibition of glycoside hydrolases (Figure 1A).
All probes are click-chemistry compatible to reduce probe size
and allow simultaneous fluorescent and liquid chromatography−
mass spectrometry (LC−MS) characterization of ABP−protein
labeling events.24 The ABP suite is applied directly to the
complex cellulose-degrading proteome of C. thermocellum. Not
only do we show how these probes have complementary and
distinct enzyme labeling profiles, but also we demonstrate the
utility of these probes for characterizing the cellulose-degrading
machinery of biofuel-relevant organisms.
EXPERIMENTAL SECTION
■
General Procedures and Materials. NMR spectra were recorded
at 25 °C on a Varian Oxford 500 MHz spectrometer at the following
frequencies: 499.8 MHz (1H) and 125.7 MHz (13C); spectra were
calibrated to the chemical shift of tetramethylsilane (δ = 0 ppm). Spectra
were assigned with appropriate 1H and 13C NMR experiments.
Chemical shifts are reported in parts per million, coupling constants
in Hertz (Hz), and multiplicities indicated with: singlet (s), doublet (d),
triplet (t), doublet of doublets (dd), doublet of triplets (dt), doublet of
doublet of doublets (ddd), and multiplet (m). ESI mass spectra were
obtained with an LCQ Deca spectrometer. High-resolution mass spectra
were obtained with an Exactive Orbitrap mass spectrometer (Thermo
Scientific). Protein concentration measurements were made using a
SpectraMax 96-well UV/vis microplate reader. Fluorescent ABP-labeled
proteins were separated by SDS-PAGE on Invitrogen 10% Tris-Glycine
precast gels and imaged with a Protein Simple FluorchemQ: 534 nm
excitation, 606 nm emission filter. Unless otherwise noted, a Grace
Davison Discovery Sciences Reveleris medium pressure liquid
chromatographer fitted with commercial silica cartridges was used for
purification of ABPs and intermediates. Starting mono- and disaccharide
material for the synthesis of all GH-ABPs was purchased from
Carbosynth Ltd. Other reagents were purchased from Sigma-Aldrich,
Acros, or Alfa Aesar and used as received unless stated otherwise. Dry
solvents, if not purchased, were obtained via an LC Technology
Solutions, Inc., SP-1 solvent drying system; reactions were carried out in
an inert nitrogen or argon environment.
Figure 1. (A) Structures of glycoside hydrolase-directed activity-based
probes (GH-ABPs). (B) C. thermocellum secretome samples, containing
cellulosome proteins, were incubated at 1 mg/mL with individual GH-
ABPs (75 μM). Click chemistry was used to append the fluorophores
rhodamine-azide and -alkyne, and proteins were separated by SDS-
PAGE and imaged to reveal the fluorescent ABP-labeled proteins.
Control gels and protein abundance stains are shown in the Supporting
Information.
frozen upon pelleting with liquid nitrogen. Spent growth media,
containing secreted and cellulosome proteins (“cellulosomal secre-
tome”), was concentrated using a Millipore Amicon centrifugal device.
The buffer was exchanged to NaOAc (50 mM, pH 6) and the
cellulosomal secretome collected. After initial protein content was
determined by BCA, protein concentrations were normalized and stored
at −80 °C until further analysis.
Enzyme Assays. Cellulase (endo-1,4-β-glucanase) and xylanase
(endo-1,4-β-xylanase) activity were determined according to manu-
facturer (Megazyme Intl. Ireland) specifications. Both assays release
Synthesis of GH-ABPs. See Supporting Information.
Bacterial Culture. Clostridium thermocellum (ATCC 27405) was
grown in a modified Dehority medium,25 supplemented with 4 g L−1
microcrystalline cellulose (without the addition of volatile fatty acids),
and incubated at 60 °C under strict anaerobic conditions. Biomass was
harvested between mid- and late-log phases by centrifugation and flash
B
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remazolbrilliant blue R coupled substrate, which is measured by
absorbance at 590 nm; cellulase activity: 140 milliU/mL; xylanase
activity: 60 milliU/mL.
Proteome Labeling and Fluorescent Gel Analysis. Proteome
samples (35 μL) were adjusted to a protein concentration of 1 mg/mL
in NaOAc (50 mM, pH 6 containing SDS (0.42 μM)) buffer, labeled
with an individual GH-ABP (GH1 (300 μM), GH2a-d (75 μM), GH3a-
b (75 μM), and GH4a-b (75 μM)), and incubated at 60 °C for 3 h with
mild agitation.
Following ABP incubation, proteome samples treated with an azide-
containing probe were treated with a rhodamine-alkyne fluorescent
reporter group (30 μM, prepared in DMSO), dithiothreitol (1.4 mM,
prepared fresh in water), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]
amine (TBTA) (171 μM, prepared in DMF), and CuSO4 (2.86 mM,
prepared in water). Proteome samples treated with an alkyne-containing
probe were treated with a rhodamine-azide fluorescent reporter group
(30 μM), tris(2-carboxyethyl)phosphine (TCEP) (429 μM, prepared
fresh in water), TBTA (87 μM, prepared in 4:1 tert-butanol:DMSO),
and CuSO4 (857 μM, prepared in water). Samples were vortexed and
incubated in the dark at rt for 1 h, at which time SDS-PAGE loading
buffer (reducing) was added and samples were heated at 85 °C for 2 min
prior to being loaded into precast 10% Tris-Glycine gels.
CuSO4 (0.5 mM, prepared in water). Samples were vortexed and
incubated at rt for 1.5 h.
Following click chemistry, samples were concentrated using Amicon
Ultra-0.5 centrifugal filter devices at 14 000g (RCF × g) for 20 min at 4
°C and washed with PBS (10 mM, pH 7.4, 2 × 300 μL) before
reconcentrating to 20 μL. PBS (0.5 mL, containing 1.2% SDS) was
added to the filter device and then inverted into a clean recovery tube
and collected at 1000g for 4 min at 4 °C. Samples were heated at 95 °C
for 2 min and centrifuged at rt for 4 min at 6000g, removing particulates.
Protein concentrations were determined for each sample using a BCA
assay; all samples were normalized to 300 μg prior to enrichment. For
the analysis to result in the strict evaluation of protein function on a per
unit mass basis, protein content was normalized prior to loading
proteome samples onto streptavidin resin.
For enrichment of ABP-labeled proteins, a 100 μL aliquot of
streptavidin agarose resin (Thermo Fisher), per prepared sample, was
placed in a BioRad Bio-Spin Chromatography Column using a vacuum
manifold. The resin was washed with PBS (3 mL) and transferred to a 15
mL tube using two 0.5 mL aliquots of PBS. An additional 1.5 mL of PBS
was added to each tube followed by the normalized proteome sample.
Each falcon tube contained a final SDS concentration of ∼0.2%. Tubes
were then rotated for 4 h at rt.
Global Sample Prep and LC−MS Analysis. Protein concen-
trations were determined by Coomassie assay (Thermo Scientific), then
denatured and reduced by adding a final concentration of 8 M urea and
fresh dithiothreitol (DTT) to a final concentration of 10 mM. Samples
were incubated at 60 °C for 30 min, then diluted 8-fold with NH4HCO3
(100 mM, pH 8.4), to reduce salt concentration. CaCl2 was added to the
diluted samples to a final concentration of 1 mM and digested for 3 h at
37 °C using sequencing grade trypsin (Promega) at a ratio of 1 unit of
trypsin per 50 units of protein. Following incubation, digested samples
were desalted using an appropriately sized C-18 SPE column (Supelco).
Final peptide concentrations were determined by BCA assay.
Digested proteins from spent growth media and cell pellet fractions
were analyzed using a custom-built 2D HPLC system coupled to an
LTQ Orbitrap Velos mass spectrometer (Thermo Fisher). The first
dimension of separation occurred by way of a strong cation exchange
(SCX) column (15 cm × 360 μm outer diameter (o.d.) × 150 μm
internal diameter (i.d.)) containing 5 μm of Polysulfethyl A (PolyLC
Inc.) coupled to a trapping column (4 cm × 360 μm o.d. × 150 μm i.d.)
containing 5 μm of Jupiter C18 (Phenomenex). Fifteen fractions were
trapped and individually separated by a reverse-phase column (35 cm ×
360 μm o.d. × 75 μm i.d.) containing 3 μm of Jupiter C18. Mobile
phases consisted of 0.1 mM NaH2PO4 and 0.3 M NaH2PO4 for the first
dimension and 0.1% formic acid in water and 0.1% formic acid in
acetonitrile for the second dimension. Columns were manufactured in-
house by slurry packing media into fused silica (Polymicro Technologies
Inc.) using a 1 cm sol−gel frit for media retention.26
Instrument operating conditions included a heated capillary temper-
ature of 200 °C and a spray voltage of 2.2 kV, respectively. Data were
acquired for approximately 100 min per fraction, beginning 10 min into
the reverse-phase gradient. Orbitrap spectra (automatic gain control
(AGC) 1 × 106) were collected from 400 to 2000 m/z at a resolution of
100k followed by data-dependent ion trap MS/MS spectra (AGC 3 ×
104) of the six most abundant ions using a collision energy of 35%.27 A
dynamic exclusion time of 60 s was implemented to discriminate against
previously analyzed ions.
Proteome Labeling and LC−MS Analysis. Proteome samples
were adjusted to a protein concentration of 0.5 mg/mL in NaOAc buffer
(50 mM, pH 6, containing SDS (0.42 μM)) and labeled using individual
GH-ABPs: GH1 (300 μM), GH2a-d (75 μM), GH3a-b (75 μM), and
GH4a-b (75 μM). “No ABP” control samples were prepared by addition
of DMSO (2 μL) rather than probe. All samples were incubated at 60 °C
for 3 h with mild agitation. Following probe incubation, azide-containing
GH-ABP labeled proteome samples were treated with biotin-alkyne (30
μM), dithiothreitol (DTT; 1.67 mM, prepared fresh in water), TBTA
(100 μM, prepared in DMF), and CuSO4 (1.65 mM final, prepared in
water). Proteome samples treated with alkyne-containing GH-ABPs
were treated with biotin-azide (30 μM), TCEP (250 μM, prepared fresh
in water), TBTA (51 μM, prepared in 4:1 tert-butanol:DMSO), and
Following streptavidin capture of probe-labeled proteins, each sample
containing resin solution was transferred into a Bio-Spin column on the
vacuum manifold. The resin was washed with 0.5% SDS in PBS (1 mL,
repeat 3×), 6 M urea in PBS (1 mL, repeat 3×), Milli-Q water (1 mL,
repeat 3×), and PBS (1 mL, repeat 5×). The resin was then transferred
to sterile 1.5 mL tubes using two 0.5 mL aliquots of ammonium
bicarbonate (25 mM, pH 8) and the supernatant discarded.
To obtain peptides for MS analysis, 200 μL of NH4HCO3 (25 mM,
pH 8) was added to the resin for each sample and treated with a trypsin
solution (2 μL, trypsin was reconstituted in 40 μL of NH4HCO3). Resin
solutions were heated at 37 °C for 15 h with agitation. Following trypsin
digestion, supernatant was collected by centrifugation at 6000g and
placed in a sterile tube. An additional amount of NH4HCO3 (150 μL)
was added to sample resin and agitated on a thermal mixer at 37 °C for
10 min. Supernatants were again collected at 6000g and added to
corresponding tubes from prior collection. Volatiles were removed from
tryptic peptide solutions by a speedvac concentrator, and peptides were
reconstituted in 40 μL of NH4HCO3 and heated for 10 min at 37 °C
with mild agitation. Samples were centrifuged at 122000g in a Beckman
TLA 120.1 rotor for 20 min at 4 °C then placed in vials for subsequent
MS analysis using 25 μL aliquots.
LC−MS Proteomic Analysis of ABP-Labeled Proteins. Peptides
were separated by high-resolution, reversed-phase, constant-pressure
capillary liquid chromatography as previously described.28,29 Briefly, MS
analysis was performed using a Thermo Electron ion trap LTQ MS
outfitted with a custom ion funnel and electrospray ionization (ESI)
interface. Data were acquired for 100 min, beginning 65 min after
sample injection (15 min into gradient). LTQ spectra were collected
from 400 to 2000 m/z at a resolution of 100k, followed by data-
dependent ion trap MS/MS spectra of the six most abundant ions using
a 35% collision energy.29 A dynamic exclusion time of 30 s was used to
discriminate against previously analyzed ions.
Data Analysis. LTQ-MS raw data were extracted using
Decon_MSn30 and analyzed with the SEQUEST algorithm (V27,
revision 12)31 by aligning experimental MS spectra with theoretical
spectra generated from the C. thermocellum annotated genome (C.
thermocellum ATCC 27405 Genbank Accession CP000568; down-
loaded_2008−04−07).
Data filtering criteria based on the MS-GF score and precursor ion
mass accuracy were used to limit false positive identifications to <1% at
the peptide level, estimated employing a reverse database search.32
Using peptides that match the given criteria, we then stipulated that to
classify a protein as specifically labeled by a GH-ABP we required the
following criteria: (i) ≥2 unique peptides per protein; (ii) ≥2 peptides
measured per protein in at least two replicates; (iii) the protein exhibits
≥3-fold more abundance in the GH-ABP-labeled samples relative to the
“No probe” control. All filter passing peptide identifications were tallied
to provide quantitative spectral count data for each protein. Relative
C
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Scheme 1. GH-ABP Syntheses
protein abundance was estimated by averaging peptide spectral counts
for a protein across the three GH-ABP-labeling replicates, with standard
deviation under 10%.30,31,33
At the onset of our studies, we evaluated a number of known
classes of affinity and mechanism-based inhibitors for compar-
ison, paying particular attention to the likelihood of the probe
class to capture elements of the cellulosome. 2-Deoxy-2-
fluoroglycosides were first reported as glycosidase inhibitors in
1987 by Withers and co-workers34 and first as ABPs by Vocadlo
and Bertozzi.15 These inhibit retaining GHs with discriminating
specificity for the active site. The 2-deoxy-2-fluoro substitution
slows the formation and hydrolysis of the covalent glycosyl-
enzyme adduct by stabilizing the oxocarbenium like transition
state.23,35 However, the glycosyl-enzyme adduct can slowly
hydrolyze with a lifetime ranging from seconds to months; thus it
may be described as a very slow enzymatic substrate rather than a
mechanism-based inhibitor.23 Despite contrasting interpreta-
tions, the long life of the adduct was deemed suitable for our
proteomic experiments; in turn, we synthesized the glucose-
based GH1-ABP (Scheme 1A) according to literature
procedures15,18,19 and utilized selective protection and reactivity
manipulation of the carbohydrate functional groups.
RESULTS AND DISCUSSION
■
Probe Design and Synthesis. Activity-based probes GH1−
GH4b (Figure 1A) are designed to facilitate two-step bio-
orthogonal chemistry, whereby probes first label enzymes,
followed by Cu(I)-catalyzed cycloaddition (click chemistry
(CC)) to a reporter group. Our CC-enabled GH-ABPs are
composed of three general elements to facilitate capture and
identification of cellulosomal GHs: (1) a reactive group as either
an affinity-based or mechanism-based inhibitor for targeting
active enzymes, (2) a mono- or disaccharide as the binding group
that facilitates probe-enzyme adduction, and (3) a latent alkyne
or azide handle for CC attachment of a reporter group to
visualize binding events by fluorescence or identify ABP targets
by biotin enrichment and MS measurement.24 Incorporation of a
CC-compatible alkyne or azide to the GH-ABP replaces bulky
biotin or fluorophores that would not normally be part of the
natural substrate for GH enzymes.
Probes GH2a−d-ABPs are affinity-based inhibitors, in which
the glycosyl-binding group imparts selectivity toward enzymes
acting on sugar moieties; however, these probes can label the
D
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acid/base catalytic residues of retaining β-glycosidases, as well as
amino acid residues within the active site of inverting and
retaining enzymes through nucleophilic displacement of the
acetylhalide.23,36 N-Bromo- and N-iodoacetylglycosylamines
have been used to label and inactivate several glycosidases and
cellulases and show sufficient stability toward decomposi-
tion.36,37 For broad characterization of the components of the
cellulosome, it can be said that these probes offer an advantage by
covering inverting and retaining enzymes and without specific
activity toward a single GH family. In the design of probes
GH2b- and GH2d-ABP the acetyl group was retained, as the
lipophilic property of the acetylated sugar probes may be
efficacious for binding to certain GH families.37 In a single-step
synthesis of probes GH2a-ABP and GH2c-ABP, 6-azido-1,6-
dideoxy-β-D-glucopyranosylamine was reacted directly with
either bromo- or iodoacetic anhydride, respectively (Scheme
1B).38 To obtain the acetylated GH2b-ABP and GH2d-ABP
probes, 2,3,4-tri-O-acetyl-1-amino-6-azido-1,6-dideoxy-β-D-glu-
cose was reacted directly with bromo- or iodoacetic anhydride,
respectively (Scheme 1B).
Probes of structure GH3-ABP and GH4-ABP are activated
difluoromethylphenyl aglycones, which upon glycoside hydro-
lase cleavage of the glycosidic bond release an activated aglycone
that subsequently irreversibly reacts with the GH. Unlike the
GH1- and GH2-ABPs, these probes are solely activated after GH
enzymatic cleavage.12,13 Hydrolysis of the glycosidic bond by a
GH liberates a difluoromethyl phenolate, followed by rapid
elimination of a fluorine atom to form a reactive quinone
methide. Any nucleophile within the active site, including
catalytic residues, can perform a Michael reaction with the
quinone methide, yielding a covalent adduct. A drawback from
prior studies, but an advantage to broadly identifying cellulosome
activity, is that the intermediate quinone methide can transiently
diffuse from the GH active site and label a reactive GH outside
the active site or label neighboring cellulosome components.
However, biotin and fluorophore containing ABPs have been
successfully applied to the labeling of GHs and in biologically
relevant systems.12,13,16,39 We designed our CC enabled phenyl-
dimethyl aglycone probes as mono- and disaccharides, with and
without acetylation. Beginning with 1-bromo-1-deoxy-2,3,4,6-
tetra-O-acetyl-α-D-galactopyranose (GH3-ABPs) or 1-bromo-1-
deoxy-2,3,4,2′,3′,4′,6′-hepta-O-acetyl-α-D-lactopyranose (GH4-
ABPs), the compounds were reacted with nitrophenol, 10,
followed by fluorination of the aldehyde, 11/15,39a and reduction
of the nitro moiety to an amine, 12/16.39a An alkyne for click
chemistry was appended to form GH3b-ABP and GH4b-ABP.
Removal of the acetyl groups provided GH3a-ABP and GH4a-
ABP. Prior studies have demonstrated that background labeling
of proteins during click chemistry is heightened when the probe
contains the azide and the reporter group the alkyne.21,24 Due to
the ease of installation, we appended the alkyne groups.
Figure 2. Organization of the Clostridium thermocellum cellulosome
structure. The scaffoldin protein (CipA) contains nine type I cohesins to
which nine enzymes bearing type I dockerin groups can bind and a
carbohydrate binding module for direct attachment of the entire
cellulosome complex to the cellulosic substrate. The scaffoldin also
contains a type II cohesin for direct attachment to cell surface anchoring
proteins (SdbA, OlpB, and Orf2). OlpB and Orf2 contain multiple type
II cohesin domains, permitting the assembly of polycellulosome
components with as many as 63 enzymatic subunits. Direct adherence
of enzymes to the cell surface is mediated by anchoring proteins OlpA
and OlpC, which contain a single type I cohesin.
nases, and other minor enzyme activities exhibited. To determine
probe reactivity and selectivity toward GHs, including those
associated with the cellulosome, we screened our suite of GH-
ABPs against the C. thermocellum spent growth culture medium,
which contains secreted proteins and cellulosome proteins
(a.k.a., “cellulosomal secretome”). Probes were incubated with
the cellulosomal secretome sample for 3 h at 60 °C at a final
concentration of 75 μM with the exception of GH1-ABP. Due to
the lower reactivity of GH1-ABP and its potential for hydrolysis,
the postenzyme reaction was used at final concentration of 300
μM instead.15 Following GH-ABP incubation, proteome
samples were treated with either rhodamine-azide or rhod-
amine-alkyne under click chemistry conditions, separated by
SDS-PAGE, and visualized by fluorescence (Figure 1B).
The probe labeling profiles observed by fluorescent gel
analysis (Figure 1B) show diverse protein reactivity mediated by
the reactive group selection, acetylation status of the hydroxyl
groups on the sugar binding groups, and GH-ABP size.
Unfortunately, GH1-ABP displayed no observable binding by
fluorescent gel. Increases to the concentration and alterations to
Probe Reactivity and Gel Analysis in C. thermocellum.
Effective degradation of lignocellulosic materials for production
of ethanol and biofuel precursors is a hallmark of the anaerobic
bacterium C. thermocellum. This organism produces an extensive
consortium of proteins and enzymes that work synergistically to
degrade cellulosic matter. Some proteins are secreted directly to
the medium, while others contain a dockerin module, which
strongly binds cohesin modules on scaffoldin (CipA), forming a
macromolecular “cellulosome” structure adherent to the cell
surface (Figure 2). C. thermocellum secretes a large number of
GHs from multiple inverting and retaining families, including:
exoglucanases, endoglucanases, hemicellulases, xylanases, pecti-
E
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Table 1. Spectral Count (SC) and Protein Count Data for Labeling by Each Individual GH-ABP and for the Global Analysis of the
C. thermocellum Cellulosomal Secretome
activity-based probe
data category
total protein count
GH2a
GH2b
GH2c
GH2d
GH3a
GH3b
GH4a
GH4b
Global
74
783
36
91
1044
41
38
338
24
84
1061
39
219
4035
60
177
2872
52
41
345
17
23
200
12
142
9
499
42254
84
total peptide SC
carbohydrate active protein count
a
carbohydrate active SC
471
27
599
27
284
20
630
25
1344
33
1101
30
211
9
6997
41
dockerin-containing protein count
b
dockerin-containing SC
303
25
358
27
188
21
374
27
667
33
534
31
100
9
71
9
1705
52
GH family protein count
c
GH family SC
301
373
191
410
757
630
100
71
2233
a
Includes all enzyme families that act upon glycoside substrates, including glycoside hydrolases, glycosyl transferases, and carbohydrate kinases.
Includes all enzymes that contain a type I dockerin module for binding within the cellulosome macromolecular structure. Includes all glycoside
b
c
hydrolase enzymes, including those that are secreted but do not contain a type I dockerin module.
labeling conditions including time and temperature consistently
failed to produce any labeling with GH1-ABP. Within GH2-
ABPs, reactivity was increased by acetylation (GH2b- and
GH2d-ABPs), but the effect of a Br or I in the reactive group had
minimal impact. Within GH3- and GH4-ABPs, acetylation
decreased reactivity, and the larger disaccharide GH4-ABPs were
considerably less reactive. It is interesting that the effect of
acetylation upon protein labeling is reversed between GH2-
ABPs and GH3- and GH4-ABPs. GH3- and GH4-ABPs require
enzymatic cleavage of the glycosidic bond to release the reactive
quinone methide for enzyme labeling. Acetylation likely disrupts
specific binding events required for glycosidic cleavage by the
enzyme’s catalytic amino acid residues. However, the GH2-ABPs
only require the presence of a nucleophilic amino acid residue to
displace the halogen from the reactive group for covalent protein
binding. Alternatively, the difference may be due to the bulky
reactive group of GH3- and GH4-ABPs; combination with the
added steric bulk from the acetyl moieties may preclude active-
site inclusion; whereas the smaller reactive groups of GH2-ABPs
may mitigate this effect. Intriguingly, the labeling profiles for the
most reactive probes, GH2b/d-ABPs and GH4a-ABP, are nearly
identical. This suggests that the protein targets are identical and
that acetylation facilitates GH2-ABP labeling but contributes to
an overall steric bulk detrimental to GH3- and GH4-ABP
enzyme labeling.
Probe Reactivity and LC-MS/MS Analysis in C.
thermocellum. To quantitatively assess the differences in GH-
ABP labeling profiles and selectivity with higher sensitivity and
resolution: (i) the cellulosomal secretomes were treated with
biotin-azide or biotin-alkyne under click chemistry conditions
and (ii) ABP-labeled proteins were enriched on streptavidin
agarose resin, (iii) digested with trypsin, and (iv) analyzed by
LC−MS/MS. Measured spectral counts were averaged across
replicates for all peptides unique to a single protein to determine
protein abundance. At least two unique peptides were measured
per protein, and the average of spectral counts for that protein
was required to be ≥2-fold the average of the spectral counts for
that same protein in the no probe control samples. Data passing
these filter criteria were used to compare probe reactivity and
selectivity (Table 1; see also Supporting Information Figure S2).
Probe reactivity was varied considerably, particularly between
N-bromo- or -iodoacetylglycosylamines (GH2-ABPs) and the
activated difluoromethylphenyl aglycone (GH3- and GH4-
ABPs). As with the gel results, no statistically significant probe
labeling was identified with GH1-ABP, and therefore no data are
shown in Table 1. Correlating with gel-based fluorescent
analyses, GH2-ABPs have increased reactivity when acetylated
(2b, 2d). This difference is most pronounced between GH2c-
and GH2d-ABPs, with nearly 70% reduction in peptide spectral
counts from the acetylglycoside to the free glycoside ABPs. The
reduction between GH2a- and GH2b-ABPs is subtler, only
showing a 25% difference in measured peptides and 20%
difference in proteins (Table 1). Differences in the size of the
halogen displaced by the enzymatic reaction resulted in an ∼50%
difference in reactivity between brominated glycoside GH2a-
ABP and iodinated glycoside GH2c-ABP. Acetylated glycoside
ABPs GH2b and GH2d do not follow this trend; rather, they are
similarly reactive. The halogen’s strong impact on free glycoside
probe labeling, versus no impact on acetylglycoside probe
labeling, may be attributed to two potential factors: (i)
acetylation drives active-site alignment of the probe, regardless
of the halogen, and/or (ii) the higher reactivity of iodinated GH-
ABPs, particularly toward hydrolysis, is limited by the presence
of acetyl groups that confer greater hydrophobicity to the
probe.40
The activated difluoromethylphenyl aglycones (GH3- and
GH4-ABPs) require mechanism-based glycosidic cleavage to
release a reactive quinone methide, subsequently forming a
covalent bond to a reactive amino acid residue within the active
site. Alternatively, the reactive quinone methide may transiently
diffuse and label a neighboring protein. The diffusion of the
quinone methide is a concern, as non-ABP targets are
subsequently labeled. The distribution of total spectral counts
(Table 1) reveals that GH3a- and GH3b-ABPs are most reactive.
High correlation was found between protein observations and
the fluorescent gel data in Figure 1B. As observed, acetylation
(GH3b and GH4b) decreases probe reactivity, opposite to the
halogenated acetylglycosylamine probes. However, the size of
the probe severely impacts reactivity, such that the disaccharide
GH4-ABPs are >10-fold less reactive than the monosaccharide
GH3-ABPs. The larger size restricts access to the active site and
limits access to only the enzymes with activity toward cellobiose
or cellulose (polysaccharides), reducing the concentration of
quinone methide generated.
To validate that ABP labeling is selective, rather than driven
solely by labeling of highly abundant proteins, ABP labeling was
compared directly to the global MS measurement of the C.
thermocellum cellulosomal secretome (Table 1). The global MS
resulted in the identification of 499 proteins (42 254 spectral
counts), notably greater than labeling by any GH-ABP.
Probe Selectivity for Cellulosome and Secreted
Carbohydrate Active Enzymes. To determine selectivity of
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Figure 3. (A) Percentage of total spectral counts for GH-ABP-labeled and global samples attributed to: (i) Dockerin Containing Enzymes − any
enzyme containing a type I dockerin module involved in cellulosome interaction, (ii) GH Enzymes − glycoside hydrolase enzymes with and without a
type I dockerin module, (iii) Carbohydrate-active Enzymes − all enzymes with and without type I dockerin modules that are active toward glycosides,
including GHs, glycosyl transferases, and carbohydrate kinases. The final group, (iv) Dockerin-GH vs GH − represents the distribution of spectral
counts attributed to type I dockerin-containing GH enzymes as a percentage of all measured GH enzyme spectral counts. (B) Percent distribution of
total spectral counts for GH-ABP labeled and global samples attributed to glycoside hydrolase (GH) enzymes. Peptide spectral counts were averaged for
each GH family; GH families were summed and distributed according to each GH-ABP. Percent distribution was calculated by dividing the average
peptide spectral counts for a GH family by the total spectral counts measured for all GH families, for a particular GH-ABP. See Supporting Information
for raw data. All measurements represent three sample replicates per GH-ABP.
GH-ABPs for carbohydrate active enzymes, we evaluated the
spectral count distribution of total spectral counts from global
proteomics measurements and ABP-labeled spectral counts
(Figure 3A). Selectivity for carbohydrate active enzymes,
including GHs, glycosyl transferases, and carbohydrate kinases
(see Supporting Information for a complete list of proteins
identified in GH-ABP and global measurements), was
determined by calculating the percentage of carbohydrate active
peptide spectral counts versus the total measured peptide
spectral counts. The probes with the most selectivity for
carbohydrate-active enzymes were predominantly found in the
GH2c-ABP (84%) and GH4b-ABP (71%). The less reactive
GH-ABPs were far more selective than the highly reactive GH3-
ABPs (3a, 33%; 3b, 38%). All other GH-ABPs are ∼60%
G
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selective for carbohydrate-active enzymes; even the least
selective probes are >2-fold more selective than the distribution
revealed by the global MS (only 17% carbohydrate active)
measurements.
We also evaluated the selectivity of GH-ABPs for GH
enzymes, including: cellulases, xylanases, and hemicellulases. The
GH enzymes represent our primary interest for our probe suite,
and given the reactive nature of these probes we anticipated
cross-reactivity, primarily with cellulosome components. Cover-
age of GH enzymes as a percentage of total peptides reveals the
same trends as carbohydrate-active enzymes, with less reactive
probes more selective for GH enzymes (e.g., GH2c-ABP −
56%). GH3-ABPs were least selective, ∼20%. However, only 5%
of the total peptides measured in the global analyses are for GHs,
revealing a 4- to 10-fold increase in selectivity for GHs using
ABPs. Next we evaluated the percent peptide spectral counts for
proteins containing a type I dockerin (Figure 3A), which is
indicative of a role in the cellulosome. The percentiles mirror the
GH distribution; however the more reactive probes (GH2d-, 3a-,
3b-ABPs) also labeled type I dockerin-containing carbohydrate
esterases and a serpin protease. When compared to the global
data, GH-ABPs are 4- to 14-fold more selective for type I
dockerin-containing proteins.
Lastly, we determined the distribution of type I dockerin-
containing GHs as a percentage of all measured GHs. The less
reactive probes (2a-, 2b-, 2c-, 4a-, and 4b-ABPs) labeled almost
exclusively (∼100%) cellulosome type I dockerin-containing
GHs. The more reactive probes ranged from ∼85 to 90%; the
remaining 10−15% of labeling is of secreted GHs, known to
closely interact with cellulosome components. Approximately
three-quarters of all spectral counts for GHs in the global data
contain the type I dockerin module. All-in-all, the data in Figure
3B reveal selectivity for cellulosome components, including GH
enzymes, with a significant distribution of peptide spectral counts
assigned to these categories when compared to the global
distribution.
Characterization of Glycoside Hydrolase Enzymes
Identified in GH-ABP and Global MS Measurements.
Activated difluoromethylphenyl aglycones and N-bromo- and N-
iodoacetylglycosylamines are both able to react with glycoside
hydrolases, showing inverting- or retaining-type mecha-
nisms.13,23b This dual specificity is an advantage for high-
throughput characterization of the GH complement of a
proteome. Within the C. thermocellum cellulosomal secretome,
GH enzymes are prevalent in both the cellulosome complexes
and as freely secreted enzymes. All GH-ABPs showed an
approximate 60:40 inverting:retaining labeling distribution
(Figure 4). This is the result of a large array of GH9 (inverting)
cellulase enzymes, a number of GH5 (retaining) endoglucanases,
and GH10 and GH11 (retaining) xylanases (Figure 3B and
Supporting Information). The global MS measurement revealed
a near 50:50 inverting:retaining distribution, due to the
identification of a large number of secreted retaining GHs. The
lack of labeling of these secreted and retaining GHs by any GH-
ABP likely indicates they were in an inhibited or zymogen
(proenzyme) nonfunctional state.
Figure 4. Percent distribution of catalytic reactivity of GH enzymes
resulting in an overall inversion or retention of the glycoside anomeric
configuration according to each GH-ABP. Three replicates per probe.
glucosidic linkages in cellulose, is the dominant GH family
represented. All 12 GH9 enzymes expressed in C. thermocellum
were labeled by GH-ABPs (e.g., GH3a-ABP; see Supporting
Information for all GHs labeled by individual probes). GH9
enzymes are unique in that they display a processive mechanism,
in which they are able to cleave internal regions of cellulose, and
continue to degrade processively down the cleaved chain. GH
family 5, which catalyzes the endohydrolysis of (1→4)-β-D-
glucosidic linkages in cellulose (EC 3.2.1.4) and hydrolysis of
(1→4)-β-D-glucosidic linkages in cellulose and cellotetraose to
release cellobiose from the nonreducing ends of the polymeric
chain (EC 3.2.1.91), was the second most prevalent GH family
labeled for all ABPs, with labeling of 5 of the 9 proteins encoded
in the genome. It has been demonstrated previously that GH5
and GH9 enzymes make up half the catalytic components of the
cellulosome, and the abundance of GH5 enzymes increases when
the carbon source for growth is crystalline cellulose.41
The order of labeling followed with GH94, cellobiose
phosphorylases (EC 2.4.1.20), and GH families 10 and 11,
xylanases that catalyze the endohydrolysis of (1→4)-β-D-
xylosidic linkages in xylans (EC 3.2.1.8). It is compelling to
note that C. thermocellum cannot grow on xylan as a carbon
source but contains a suite of xylanases (hemicellulases). Other
families labeled were GH48 processive cellulases (EC 3.2.1.4),
GH74 xyloglucanases (EC 3.2.1.151), and GH26 mannanases.
An additional six GH families were identified, but with less than
5% peptide spectral count distribution when compared to the
total GH peptide spectra measured (Figure 3B).
The distribution of GH family labeling was near identical
across the suite of GH-ABPs. This distribution was different
from the global data, which had less measurement of the primary
lignocellulose degrading enzyme families (GH5, 9, and 10).
Differences in probe reactivity are particularly evident within the
disaccharide GH4-ABPs; labeling of GH families 9 and 10 are
dominant with these probes. Acetylation (GH4b-ABP) severely
limited the labeling of cellobiase GH family 5 when compared to
the free glycoside (GH4a-ABP), but this trend is only within
GH4-ABPs. Labeling of GH94 cellobiose phosphorylases is
clearly reactivity driven, with GH2d-, GH3a-, and GH3b-ABPs
having the largest peptide distribution within this family.
Profiling the C. thermocellum Cellulosome. C. thermo-
cellum is a remarkable anaerobic cellulosome producing
organism, with a specific activity against crystalline cellulose
that is 50-fold higher than that of Trichoderma reesei, a
commercially employed fungal strain for biofuel production.2
A closer inspection of the GH enzymes labeled reveals broad
coverage of cellulose-degrading GH families (Figure 3B). We
determined the total average peptide spectral counts for only GH
family enzymes in the GH-ABP labeled and global MS data. We
then normalized the contribution of each individual GH family to
the total (Figure 3B). GH9, endoglucanases (enzyme classi-
fication (EC) 3.2.1.4) that catalyze the hydrolysis of (1→4)-β-D-
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Figure 5. Activity-based probe and global profiling of the C. thermocellum cellulosome. Components include scaffoldin (CipA), cell wall anchoring
proteins (OlpA, OlpB, SdbA, Orf2p), and type I dockerin-containing enzymes. Heatmap data are normalized within each column (i.e., each GH-ABP or
global data) and represent the average spectral counts for each protein normalized on a 0−100% scale. The associated table shows protein IDs and
names (if previously assigned), functions within the cellulosome, GH families, inverting or retaining catalytic mechanisms, carbohydrate binding
modules (CBM), and EC number. See Supporting Information for associated raw data.
a
Table 2. Spectral Counts for Groups of Cellulosome Functions for Each GH-ABP and Global Data
activity-based probe
cellulosome function
GH2a
GH2b
GH2c
GH2d
GH3a
GH3b
GH4a
GH4b
global
carbohydrate esterase
endoglucanase
exoglucanase
3
108
105
5
122
135
2
141
140
7
274
211
2
6
188
187
2
11
557
524
5
62
80
37
50
7
44
hemicellulase
mannanase
5
3
5
3
2
6
4
10
4
6
21
pectinase
2
2
serpin
6
8
xylanase/xyloglucanase
scaffoldin - cell anchoring
undefined
62
79
18
66
101
24
38
61
7
63
106
17
86
221
66
94
174
50
12
57
2
20
44
452
543
125
a
Spectral counts represent the sum of the average peptide SCs for all proteins within a category for a given ABP or the global data. See the
Supporting Information for raw data for individual proteins.
The cellulosome macromolecular structure is organized by the
association of cell wall proteins that bind extracellular
components, including GH enzymes, carbohydrate esterases,
and carbohydrate binding modules (CBMs) (Figure 2). Binding
between cell wall proteins and extracellular components is
mediated by type I and II dockerin and cohesin modules, with
high affinity protein−protein interactions (>109 M−1).42 An
extracellular scaffoldin (CipA) protein contains nine type I
cohesins to which nine enzymes bearing type I dockerin groups
can bind and a carbohydrate binding module for direct
attachment of the entire cellulosome complex to the cellulosic
substrate. Scaffoldin also contains a type II cohesin for direct
attachment to cell surface anchoring proteins (SdbA, OlpB, and
Orf2). OlpB and Orf2 contain multiple type II cohesin domains,
permitting the assembly of polycellulosome components with as
many as 63 enzymatic subunits. Direct adherence of enzymes to
the cell surface is mediated by anchoring proteins OlpA and
OlpC, which contain a single type I cohesin (Figure 2).
C. thermocellum produces 72 cellulosome enzymes (poly-
peptides with type I dockerins), although only approximately half
have been identified when the organism is cultured on
microcrystalline cellulose (Avicel).43 Prior global MS-based
I
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proteomic analysis identified 35 cellulosome type I dockerin-
containing proteins when grown on Avicel;43 in a series of seven
growth conditions 59 type I dockerin-containing proteins were
found.41 Both prior experiments employed targeted MS analyses
in which they only measured an enriched cellulosome. Our ABPP
approach focused on characterizing the cellulosomal secretome
and other secreted noncellulosomal GH enzymes. We identified
35 cellulosome type I dockerin-containing enzymes, scaffoldin
(CipA), and four cell anchoring proteins with our GH-ABP suite
(Figure 5).
The type I dockerin-containing enzymes measured by GH-
ABPs are primarily endoglucanases, exoglucanases, and
xylanases. Minor selectivity was shown for accessory enzymes
such as hemicellulases, mannanases, and carbohydrate esterases,
though these are minor components as seen in the global data.
Given this array of cellulosome functions, differences in coverage
are seen between ABPs (Table 2). Several GH-ABPs had a
peptide spectral count ratio of ∼2:2:1 endoglucanase:exogluca-
nase:xylanase labeling, which is significantly different than the
∼1:1:1 global MS distribution (Table 2). GH4a-ABP, a
disaccharide, showed the highest reactivity toward exoglucanases
but little xylanase coverage. The acetylated GH4b-ABP showed
high selectivity for exoglucanases, with nearly no labeling of
endoglucanases, and marginal coverage of xylanases.
Spectral counts measured for unique peptides assigned to
CipA (scaffoldin) are concomitant with coverage for either endo-
or exoglucanases for both global and GH-ABP data (Table 2).
Scaffoldin does not have specific GH activity but rather contains
nine type I cohesin domains to bind nine type I dockerin-
containing enzymes and a specific CBM for cellulose. However,
CipA has been shown to enhance the specific activity of
cellulosome enzymes upon binding, which includes CelS, a major
cellulosome exoglucanase (GH48).44 Our probes labeled CipA
at a higher level than the 9:1 ratio of enzymes:CipA. The strong
enzyme−CipA complex likely mediated this. In the case of GH2-
ABPs the tight enzyme−CipA interactions likely facilitated a
close proximity between the probes and nucleophilic amino acid
residues on CipA. Alternatively, for GH3- and GH4-ABPs, a
reactive quinone methide transiently diffused from the enzyme to
label CipA. This is made evident as the less reactive GH4-ABPs
labeled CipA far less than GH3-ABPs, but at a level concomitant
with endo- and exoglucanase activities.
Significant differences were seen between global and GH-
ABP-labeled cellulosome components, providing evidence of
selectivity. Evaluation of a particularly interesting set of
cellulosome proteins, the cellobiohydrolases (CelK, CbhA,
CelO, and CelS), reveals global versus ABP commonalities and
significant differences. CelK and CbhA are highly homologous
cellobiohydrolases, encoding CBM4 domains, an Ig-like domain,
a GH9, and a type I dockerin. A 50:50 abundance identified in the
global MS data mirrors the activity-dependent ABP data.
However, CelO, encoding a CBM3, a GH5, and a type I
dockerin, was not in the global data and only identified by GH2b-
and GH3a-ABPs. CelS, with a CBM3, a GH48, and a type I
dockerin, was the most abundant cellobiohydrolase in the global
data but was only half as reactive as CelK and CbhA when ABP-
labeled (Figure 5 and Supporting Information). Two other
highly abundant proteins in the global data were Cthe_0821,
encoding a CBM32, GH35, and type I dockerin, and XynC,
encoding a CBM22, GH10, and type I dockerin. All probes label
both of these proteins less extensively. Additionally, three
proteins were found only in the ABPP data: Cthe_0279, CelW,
and CelO. Our data show that selectivity is found for GH-ABPs
when compared against the global analyses.
Tuning the GH-ABPs with different reactive groups,
acetylation of the glycosides, and mono- versus disaccharides,
resulted in disparities in reactivity toward cellulosomal
components. The reactivity differences are complementary to
those discussed earlier, with the difluoromethylphenyl aglycones
being highly reactive. However, GH-ABP derivations do result in
selectivity (Figure 5). Compared to monosaccharide GH3-
ABPs, disaccharide GH4-ABPs are particularly selective toward
exoglucanase cellobiohydrolases (CelK, CelS, and CbhA) and
xylanases. Comparing the deacetylated GH4a-ABP to acetylated
GH4b-ABP shows GH4a as selective for two endoglucanases
(CelW, Cthe_0821) and GH4b as selective for CelS, which acts
in a processive fashion starting from the reducing end of the
cellulose chain.
A final examination of the cellulosome data reveals labeling of a
number of proteins for which specific function is hypothetical or
has not been identified (“undefined” in Figure 5) and enzymes
that do not contain type I dockerin units (see Supporting
Information for the specific targets of each GH-ABP). However,
employing STRING (Search Tool for the Retrieval of Interacting
Genes) to identify protein−protein interactions revealed that
enzymes without type I dockerins are closely integrated with
cellulosome components (see Supporting Information for
STRING interactions).45 Four glycoside hydrolases, BglA,
LicA, Cthe_1787, and Cthe_2119, have high scores for
interactions with cellulosomal cellulases and xylanases. There-
fore, the ABPP approach applied to cellulose-degrading systems
can identify key proteins with undefined functions and
cellulosome and cellulosome-associated enzymes.
Together, our research reveals the dynamic assembly and
reactivity of the C. thermocellum cellulosome. As we have
identified by GH-ABP labeling, a diverse array of type I dockerin-
containing GH enzyme families and additional accessory
proteins function in concert. The high rate of cellulose
degradation by this anaerobic bacterium is clearly a function of
the sum of its parts.
CONCLUSIONS
■
Here, we have developed and applied a suite of difluoromethyl-
phenyl aglycone, 2-deoxy-2-fluoroglycoside, and N-halogenated
glycosylamine GH-ABPs to the direct labeling of the C.
thermocellum cellulosomal secretome. These GH-ABPs were
synthesized with alkynes to harness the utility and multimodal
possibilities of click chemistry and to increase enzyme active site
inclusion. Comparing GH-ABP-labeled to global MS data reveals
probe selectivity for GH enzymes, including a large array
included in the cellulosome. Derivatization of the GH-ABPs,
including reactive groups, acetylation of the glycoside binding
groups, and mono- and disaccharide binding groups, resulted in
considerable variability in protein labeling. Our probe suite is
applicable to aerobic and anaerobic cellulose-degrading systems
and facilitates a greater understanding of roles for organisms in
biofuel development. Future work will include the optimization
of the GH-ABPs to increase selectivity and application of the
probes to aerobic bacteria and fungal cellulose-degrading
organisms. Clearly, the combination of GH-ABPs and MS-
based proteomics provides a high-throughput avenue to
characterizing GH enzymes in complex lignocellulose degrading
systems.
J
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(10) For concise reviews: (a) Li, N.; Overkleeft, H. S.; Florea, B. L.
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J. M. F. G.; Overkleeft, H. S. Org. Biomol. Chem. 2011, 9, 5908−5926.
(b) Stubbs, K. A.; Vocadlo, D. J. Aust. J. Chem. 2009, 62, 521−527.
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(13) Kurogochi, M.; Nishimura, S.; Lee, Y. C. J. Biol. Chem. 2004, 43,
5338−5342.
ASSOCIATED CONTENT
* Supporting Information
Probe syntheses and characterization. Gel with controls. NMR
spectra for probes. Heatmap showing all carbohydrate active
proteins labeled by GH-ABPs. Data for all ABP and global
measurements. STRING interactions. Unique peptides identi-
fied for proteins. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
(14) Romaniouk, A. V.; Silva, A.; Feng, J.; Vijay, I. K. Glycobiology 2004,
14, 301−310.
AUTHOR INFORMATION
Corresponding Author
aaron.wright@pnnl.gov
■
(15) Vocadlo, D. J.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2004, 43,
5338−5342.
(16) Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura, S.
Biochemistry 2005, 44, 11669−11675.
Present Address
(17) Williams, S. J.; Hekmat, O.; Withers, S. G. ChemBioChem 2006, 7,
116−124.
†Battelle Charlottesville, 1001 Research Park Blvd, Town Center
Two, Suite 400, Charlottesville, Virginia 22911.
(18) Hekmat, O.; Florizone, C.; Kim, Y. −W.; Eltis, L. D.; Warren, R. A.
J.; Withers, S. G. ChemBioChem 2007, 8, 2125−2132.
(19) Stubbs, K. A.; Scaffidi, A.; Debowski, A. W.; Mark, B. L.; Stick, R.
V.; Vocadlo, D. J. J. Am. Chem. Soc. 2008, 130, 327−335.
(20) Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K. Y.; Strijland, A.;
Donker-Koopman, W. E.; van den Nieuwendijk, A. M.; Bleijlevens, B.;
Kramer, G.; Florea, B. I.; Hooibrink, B.; Hollak, C. E.; Ottenhoff, R.;
Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. Nat.
Chem. Biol. 2010, 6, 907−913.
Author Contributions
‡These authors contributed equally. The manuscript was written
through contributions of all authors, and all authors have given
approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(21) Witte, M. D.; Walvoort, M. T. C.; Li, K. −Y.; Kallemeijn, W. W.;
Donker-Koopman, W. E.; Boot, R. G.; Aerts, J. M. F. G.; Codee, J. D. C.;
van der Marel, G. A.; Overkleeft, H. S. ChemBioChem 2011, 12, 1263−
1269.
(22) Gandy, M. N.; Debowski, A. W.; Stubbs, K. A. Chem. Commun.
2011, 47, 5037−5039.
■
We thank the Biological Separations & Mass Spectrometry group
for helpful discussions and critical reading of the document. This
work was supported by the Laboratory Directed Research and
Development Program at Pacific Northwest National Laboratory
(PNNL), a multiprogram national laboratory operated by
Battelle for the U.S. DOE under contract DE-AC05-
76RL01830. This work used instrumentation and capabilities
developed under support from the National Institutes of Health
(NIH) National Center for Research Resources
(5P41RR018522) and the National Institute of General Medical
Sciences (8P41GM103493-10), and the U.S. DOE Office of
Biological and Environmental Research (DOE-BER). Mass
spectrometry-based proteomic measurements were performed
in the Environmental Molecular Sciences Laboratory, a DOE-
BER national scientific user facility at PNNL.
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