Inscribing the perimeter of the PagP hydrocarbon ruler by site-specific chemical alkylation

Article abstract of DOI:10.1021/bi1011496

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP selects palmitate chains using its β-barrel-interior hydrocarbon ruler and interrogates phospholipid donors by gating them laterally through an aperture known as the crenel. Lipid A palmitoylation provides antimicrobial peptide resistance and modulates inflammation signaled through the host TLR4/MD2 pathway. Gly88 substitutions can raise the PagP hydrocarbon ruler floor to correspondingly shorten the selected acyl chain. To explore the limits of hydrocarbon ruler acyl chain selectivity, we have modified the single Gly88Cys sulfhydryl group with linear alkyl units and identified C10 as the shortest acyl chain to be efficiently utilized. Gly88Cys-S-ethyl, S-n-propyl, and S-n-butyl PagP were all highly specific for C12, C11, and C10 acyl chains, respectively, and longer aliphatic or aminoalkyl substitutions could not extend acyl chain selectivity any further. The donor chain length limit of C10 coincides with the phosphatidylcholine transition from displaying bilayer to micellar properties in water, but the detergent inhibitor lauryldimethylamine N-oxide also gradually became ineffective in a micellar assay as the selected acyl chains were shortened to C10. The Gly88Cys-S-ethyl and norleucine substitutions exhibited superior C12 acyl chain specificity compared to that of Gly88Met PagP, thus revealing detection by the hydrocarbon ruler of the Met side chain tolerance for terminal methyl group gauche conformers. Although norleucine substitution was benign, selenomethionine substitution at Met72 was highly destabilizing to PagP. Within the hydrophobic and van der Waals-contacted environment of the PagP hydrocarbon ruler, side chain flexibility, combined with localized thioether-aromatic dispersion attraction, likely influences the specificity of acyl chain selection.

Full text of DOI:10.1021/bi1011496

9046 Biochemistry 2010, 49, 9046–9057
DOI: 10.1021/bi1011496
Inscribing the Perimeter of the PagP Hydrocarbon Ruler by Site-Specific
Chemical Alkylation†
M. Adil Khan,‡ Joel Moktar,‡ Patrick J. Mott,‡ Mary Vu,‡ Aaron H. McKie,§ Thomas Pinter,§ Fraser Hof,§ and
Russell E. Bishop*,‡
‡Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster
University, Hamilton, ON, Canada L8N 3Z5, and Department of Chemistry, University of Victoria, Victoria, BC, Canada V8W 3 V6
§
Received July 19, 2010; Revised Manuscript Received September 16, 2010
ABSTRACT: The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP selects
palmitate chains using its β-barrel-interior hydrocarbon ruler and interrogates phospholipid donors by gating
them laterally through an aperture known as the crenel. Lipid A palmitoylation provides antimicrobial
peptide resistance and modulates inflammation signaled through the host TLR4/MD2 pathway. Gly88
substitutions can raise the PagP hydrocarbon ruler floor to correspondingly shorten the selected acyl chain.
To explore the limits of hydrocarbon ruler acyl chain selectivity, we have modified the single Gly88Cys
sulfhydryl group with linear alkyl units and identified C10 as the shortest acyl chain to be efficiently utilized.
Gly88Cys-S-ethyl, S-n-propyl, and S-n-butyl PagP were all highly specific for C12, C11, and C10 acyl chains,
respectively, and longer aliphatic or aminoalkyl substitutions could not extend acyl chain selectivity any
further. The donor chain length limit of C10 coincides with the phosphatidylcholine transition from
displaying bilayer to micellar properties in water, but the detergent inhibitor lauryldimethylamine N-oxide
also gradually became ineffective in a micellar assay as the selected acyl chains were shortened to C10. The
Gly88Cys-S-ethyl and norleucine substitutions exhibited superior C12 acyl chain specificity compared to that
of Gly88Met PagP, thus revealing detection by the hydrocarbon ruler of the Met side chain tolerance for
terminal methyl group gauche conformers. Although norleucine substitution was benign, selenomethionine
substitution at Met72 was highly destabilizing to PagP. Within the hydrophobic and van der Waals-contacted
environment of the PagP hydrocarbon ruler, side chain flexibility, combined with localized thioether-
aromatic dispersion attraction, likely influences the specificity of acyl chain selection.
PagP is an outer membrane palmitoyltransferase that catalyzes
the transfer of a palmitate chain (16:0) from the sn-1 position of a
glycerophospholipid to the free hydroxyl group of the (R)-3-
hydroxymyristate chain at position 2 of lipid A (endotoxin)
(Figure 1A) (1-3). In Escherichia coli and related pathogenic
Gram-negative bacteria, PagP evades host immune defenses by
resisting antimicrobial peptides (4-7) and attenuating the in-
flammatory response to infection triggered by lipopolysaccharide
through the TLR4/MD2 pathway (8-10). Transcription of the
pagP gene is governed by the PhoP/PhoQ regulon, which con-
trols expression of bacterial lipid A modification enzymes
induced during infection by host antimicrobial peptides (11, 12).
PagP is thus a target for the development of anti-infective agents
and a tool for the synthesis of novel vaccine adjuvants and
endotoxin antagonists (2, 3).
preceded by an N-terminal amphipathic R-helix (13-15). Like
other β-barrel membrane proteins, the center of the PagP β-barrel
is relatively rigid with more flexible external loops where the
active site residues are localized. Although the catalytic mechan-
ism remains to be elucidated, PagP alternates between two
dynamically distinct states likely representing latent and active
conformations in the outer membrane environment (16). PagP
activity is triggered by outer membrane lipid asymmetry pertur-
bations that enable the phospholipid and lipid A substrates
to each have access to the active site from the external leaflet
(17-19). Phospholipid and lipid A access occurs by lateral
diffusion through two gateways in the β-barrel wall known as
˚
the crenel and embrasure, respectively (20) (Figure 1A). A 1.9 A
resolution X-ray study of PagP inhibited by the detergent
lauryldimethylamine N-oxide (LDAO)1 revealed a buried LDAO
molecule within the rigid β-barrel core, a site corresponding with
the palmitoyl group binding pocket known as the hydrocarbon
The structure of E. coli PagP has been determined both by
solution nuclear magnetic resonance spectroscopy and by X-ray
crystallography, revealing an eight-strand antiparallel β-barrel
1Abbreviations: βME, β-mercaptoethanol; CD, circular dichroism;
†This work was supported by CIHR Operating Grant MOP-84329
awarded to R.E.B. and an NSERC Discovery Grant awarded to F.H. A.
H.M. is a Pacific Century Scholar, and F.H. is a Career Scholar of the
Michael Smith Foundation for Health Research and a CIHR New
Investigator.
*To whom correspondence should be addressed: Department of
Biochemistry and Biomedical Sciences, McMaster University, Health
Sciences Centre 4H19, 1200 Main St. W., Hamilton, ON, Canada L8N
3Z5. Telephone: (905) 525-9140, ext. 28810. Fax: (905) 522-9033.
E-mail: bishopr@mcmaster.ca.
Kdo, 3-deoxy-
D
-manno-oct-2-ulosonic acid; DDM, n-dodecyl β-D-mal-
toside; ESI-MS, electrospray ionization mass spectrometry; EDTA,
ethylenediaminetetraacetic acid; Gdn-HCl, guanidine hydrochloride;
IPTG, isopropyl β-D-thiogalactopyranoside; LDAO, lauryldimethyla-
mine N-oxide; MNBS, methyl p-nitrobenzenesulfonate; MPD, 2-meth-
yl-2,4-pentanediol; Nle, norleucine; PDB, Protein Data Bank; PtdCho,
phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PBS, phos-
phate-buffered saline; SeM, selenomethionine; SDS, sodium dodecyl
sulfate; Tu, thermal unfolding temperature; TLC, thin layer chroma-
tography; UV, ultraviolet.
r
pubs.acs.org/Biochemistry
Published on Web 09/20/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 42, 2010 9047
F
IGURE 1: Structural relationships among PagP, the hydrocarbon ruler, and E. coli outer membrane lipids. (A) E. coli PagP transfers a palmitoyl
group (red) from the sn-1 position of a phospholipid, such as phosphatidylethanolamine (PtdEtn), to the free hydroxyl group of the N-linked
(R)-3-hydroxymyristate chain atposition2 onthe proximalglucosamine unitoflipid A. One ofthe simplestlipopolysaccharideacceptors forPagP
in the outer membrane is known as Kdo2-lipid A or Re endotoxin. PagP is an eight-strand antiparallel β-barrel preceded by an N-terminal
amphipathic R-helix (colored green). The amino (N) and carboxyl (C) termini are labeled with black letters, and the β-strands are labeled with
white letters (A-H). Aromatic belt residues (shown in wireframe format) demarcate the membrane interfaces between the extracellular and
periplasmic spaces. Abound molecule ofthe detergent(colored magenta) delineatestheinteriorhydrocarbon ruler. Pointsfor lateral phospholipid
access via the crenel (flanked by Pro127 and Pro144) and for lateral lipid A access via the embrasure (flanked by Pro28 and Pro50) are indicated.
Although the catalytic mechanism is unknown, several key residues (shown in stick format) have been mapped to the extracellular surface loops
(L1-L4). Loop L4 is disordered in the crystal structure (Protein Data Bank entry 1THQ) but has been introduced and energy minimized in the
model shown. (B) A sagittal section reveals the bound LDAO molecule and residues flanking the walls of the hydrocarbon ruler. A nondegenerate
exciton interaction between Tyr26 and Trp66 manifests a couplet during far-UV CD spectroscopy. PagP far-UV CD exhibits positive exciton
ellipticity at 232 nm, and the negative ellipticity at 218 nm that arises more strongly from the β-barrel is also enhanced by the exciton. The adjacent
Gly88 can be substituted to modulate lipid acyl chain selection. Gly88Cys functions as a dedicated myristoyltransferase at pH 7, but at pH 8, it
introduces a destabilizing thiolate anion that extinguishes the exciton and broadens selection to include C15 acyl chains. Cys methylation with
methyl p-nitrobenzenesulfonate (MNBS) restores the exciton at pH 8 and renders PagP selective for C13 acyl chains. (C) Synthetic scheme for a
suite of alkyl p-nitrobenzenesulfonates used to inscribe the perimeter of the PagP hydrocarbon ruler by site-specific chemical alkylation.
˚
ruler (Figure 1A,B) (15). Recently, a 1.4 A resolution X-ray
structure of PagP crystallized from a mixture of sodium dodecyl
sulfate (SDS) and 2-methyl-2,4-pentanediol (MPD) has revealed
a crenel gating mechanism for the transit of the phospholipid
between the hydrocarbon ruler and the outer membrane external
leaflet (13).
in length by a solitary methylene unit (15, 21). Mutation of Gly88
lining the hydrocarbon ruler floor can modulate lipid acyl chain
selection (21). Appropriate amino acid substitutions shorten the
selected acyl chain by a degree predictable from the expected rise
in the hydrocarbon ruler floor. In prior investigations of PagP
hydrocarbon ruler mutants, where Gly88Ala, Gly88Cys-S-methyl,
and Gly88Met were constructed, acyl chain selection was
predictably shifted toward C15, C13, and C12 acyl chains,
PagP is exquisitely selective for a 16-carbon palmitate chain
because its hydrocarbon ruler excludes lipid acyl chains differing

9048 Biochemistry, Vol. 49, No. 42, 2010
Khan et al.
respectively, with the expected unitary methylene resolution (21).
These mutants display an aromatic exciton couplet during far-
ultraviolet circular dichroism (far-UV CD) and a thermal unfold-
ing temperature (Tu) of 88 °C that is characteristic of wild-type
PagP (21). In this LDAO detergent system, PagP precipitates when
unfolded above 80 °C (21). The thermal melting profiles are thus at
equilibrium only when a perturbation shifts the transition to
temperatures below 80 °C (22).
Table 1: Electrospray Ionization Mass Spectrometry of Modified PagP
Mutants
exact mass (Da)
experimental mass (Da)
wild typea
20175.49
20249.64
20221.58
[40441.14]
20235.61
20249.64
20263.67
20277.77
20291.73
20305.76
20308.77
20085.34
20141.46
20131.43
[40260.84]
20159.49
20157.46
20345.04
20409.99
20531.04
20174.53 ( 0.4
20249.41 ( 0.7
20220.15 ( 0.6
[40439.32 ( 1.0]
20234.53 ( 0.5
20251.10 ( 1.9
20263.50 ( 1.4
20277.20 ( 1.9
20291.78 ( 1.8
20305.71 ( 1.8
20309.51 ( 1.9
20084.31 ( 2.4
20141.71 ( 1.9
20129.81 ( 1.0
[40258.63 ( 2.4]
20159.01 ( 1.9
20155.40 ( 3.5
20344.01 ( 3.6
20409.35 ( 1.9
20529.31 ( 2.2
Gly88Meta
Gly88Cysa
[S-S dimer]
G88C-S-methyla
G88C-S-ethyl
Interestingly, the Gly88Cys mutant behaves as a dedicated
myristoyltransferase at pH 7, but at pH 8, a buried thiolate anion
extinguishes the exciton and destabilizes the enzyme so it selects
both C14 and C15 acyl chains. Gly88Ser and Gly88Thr PagP
mutants were also found to behave as myristoyltransferases
having good stability, but only the Gly88Cys mutant at pH 7
was highly selective for myristate both in vitro and in vivo (22).
Introduction of charged and polar groups more distally within
the hydrocarbon ruler does not extinguish the exciton, but these
substitutions do destabilize the enzyme and compromise the
resolution of acyl chain selection (22). Branched uncharged polar
groups and unbranched charged groups each support substantial
low-resolution enzymatic activity in PagP Gly88 mutants, but
groups that are both charged and branched are by comparison
enzymatically inactive. Hydrocarbon ruler mutants with optimal
stability and acyl chain resolution have so far been obtained
primarily with unbranched aliphatic Gly88 substitutions, but the
full extent of the hydrocarbon ruler remains to be elucidated.
Despite the widespread importance of integral membrane
enzymes of lipid metabolism in signal transduction and mem-
brane biogenesis processes (23, 24), their molecular mechanisms
for lipid substrate interrogation remain poorly understood
because many such enzymes have proven recalcitrant toward
extraction from the membrane environment by a functional
detergent (25). In contrast, PagP is a heat-stable 161-amino acid
β-barrel enzyme, which intrinsically lacks Cys residues, and can
reversibly unfold and refold in a defined detergent micellar
enzymatic assay system (21). Methyl p-nitrobenzenesulfonate
(MNBS) was previously used for site-specific chemical methyla-
tion of Gly88Cys PagP, and the S-methylated enzyme maintained
stability and methylene unit acyl chain resolution (21). We have
now prepared customized alkylating reagents to fully explore
PagP hydrocarbon ruler acyl chain selectivity (Figure 1B,C). Like
a piston inscribing the perimeter of a cylinder, we have mapped
out the occluded surface of the hydrocarbon ruler and, simulta-
neously, evinced the nature of localized physical forces at work in
determining its specificity.
G88C-S-n-propyl
G88C-S-n-butyl
G88C-S-n-pentyl
G88C-S-n-hexyl
G88C-S-n-aminopentyl
wild type [Nle]
Gly88Met [Nle]
Gly88Cys [Nle]
[S-S dimer] [Nle]
G88C-S-ethyl [Nle]
Met72Leu
Met72Leu [SeM]
wild type [SeM]
Gly88Met [SeM]
aThe wild-type E. coli PagP protein used in this study lacks the
N-terminal signal peptide and contains a C-terminal His6 tag. The amino
acid sequence with Gly88 underlined and the five methionine residues in
italics: MNADEWMTTFRENIAQTWQQPEHYDLYIAITWHARFA-
YDKEKTDRYNERPWGGGFGLSRWDEKGNWHGLYAMAFKDS-
WNKWEPIAGYGWESTWRPLADENFHLGLGFTAGVTARDNW-
NYIPLPVLLPLASVGYGPVTFQMTYIPGTYNNGNVYFAWMRFQ-
FLEHHHHHH. A disulfide-linked dimeric species of the Gly88Cys mutant
is observed by this ESI-MS procedure, which was previously developed to
validate the G88C-S-methyl mutant (21).
mass spectrometer. The spectra were reconstructed using the
MassLynx 4.0 MaxEnt 1 module. Folded proteins were concen-
trated through Ni2þ affinity chromatography and dialyzed
against 10 mM Tris-HCl (pH 8.0) and 0.1% LDAO to remove
imidazole and β-mercaptoethanol (βME). All protein stock
solutions were maintained at a concentration of 2 mg/mL
determined using the Edelhoch method (27) or bicinchoninic
acid assay (28). Purified PagP and its mutant derivatives all
display masses accurately matching theoretical predictions
(Table 1). Site-directed mutagenesis was performed as described
previously (21) to create Met72Leu PagP using the primers
M72L-F (50-GGCATGGCCTGTATGCCCTGGCATTTAA-
GGACTCGTGG-30) and M72L-R (50-CCACGAGTCCTTA-
AATGCCAGGGCATACAGGCCATGCC-30). Three-dimen-
sional portraits of PagP or the hydrocarbon ruler were rendered
with PyMol (29) using an energy-minimized coordinate file, as
described previously (21), and PDB entries 1THQ (15) and
3GP6 (13).
Preparation of Alkyl 4-Nitrobenzenesulfonates. Alkyl
4-nitrobenzenesulfonates were prepared by adaptation of a
published method (Figure 1C) (30). The representative procedure
was used for n-pentyl 4-nitrobenzenesulfonate. Metallic sodium
(0.19 g, 8.4 mmol, freshly cut and washed with hexanes to remove
mineral oil) was added to n-pentyl alcohol (5 mL) and the mixture
allowed to react until gas evolution ceased. The resulting solution
of sodium n-pentoxide was added in a dropwise manner to a
solution of 4-nitrobenzenesulfonyl chloride (2.0 g, 8.4 mmol) in
diethyl ether (50 mL) and the mixture stirred at room tempera-
ture under a nitrogen atmosphere. After 3 h, the reaction mixture
was added to H2O (100 mL) and the product was extracted with
CHCl3 (2 ꢀ 100 mL). The combined organics were washed with
EXPERIMENTAL PROCEDURES
Protein Expression and Purification. Cells were grown in
LB medium at 37 °C with ampicillin at 100 μg/mL or tetracycline
at 12.5 μg/mL as appropriate (26). The expression and purifica-
tion of PagP and its mutant derivatives were performed as
previously described (21). The procedure depends on isopropyl
β-D-thiogalactopyranoside (IPTG) induction in E. coli BL21-
(DE3) from the pETCrcAHΔS vector, which removes the native
signal peptide and introduces a C-terminal hexahistidine tag (14).
Protein samples precipitated from water prior to detergent
folding were dissolved in a 50:50 solution of 1% formic acid
and acetonitrile just prior to electrospray ionization mass spec-
trometry (ESI-MS). The sample concentration was maintained
at 1 ng/μL and the sample injected directly onto a Waters/
Micromass Q-TOF Ultima Global quadrupole time-of-flight

Article
Biochemistry, Vol. 49, No. 42, 2010 9049
water (50 mL), saturated aqueous NaHCO3 (100 mL), and water
(50 mL) again prior to being dried over Na2SO4. The solvent was
removed on a rotary evaporator and the crude product purified
by column chromatography (SiO2, 20% EtOAc in hexanes) to
yield the pure product as a yellow solid (1.65 g, 72% yield): mp
gradually cooled to 37 °C. Under a N2 barrier, 0.5 mL of 5.2 mM
ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or n-aminopentyl
4-nitrobenzenesulfonate (2-fold molar excess of the βME) dis-
solved in acetonitrile was added to the reaction mixture. The
reactions were conducted at 37 °C for 2 h with ethyl, 4 h with
n-propyl, 8 h with n-butyl, and overnight with n-pentyl, n-hexyl,
and n-aminopentyl 4-nitrobenzenesulfonate. Reactions were
quenched by addition of 5 μL of 14 M βME. The reaction
mixture was dialyzed exhaustively against distilled water, and the
precipitated proteins were collected. A small amount of the
precipitate was stored for the purposes of ESI-MS as described
above. The remainder was dissolved in 10 mM Tris-HCl (pH 8.0)
and 6 M Gdn-HCl for protein refolding as described pre-
viously (21).
1
55-56 °C; H NMR (300 MHz, CDCl3) δ 8.41 (d, 2H, J =
8.8 Hz), 8.12 (d, 2H, J = 8.8 Hz), 4.14 (t, 2H, J = 6.5 Hz), 1.69
(m, 2H), 1.30 (m, 4H), 0.87 (t, 3H, J = 6.5 Hz); 13C NMR (75
MHz, CDCl3) δ 140.5, 127.5, 122.8, 113.0, 70.4, 26.9, 25.7, 20.3, 12.1.
The more elaborate n-aminopentyl 4-nitrobenzenesulfonate
(Figure 3A) was prepared in two steps. A mixture of Boc-
protected 5-amino-n-pentan-1-ol (0.25 g, 1.2 mmol) and dry
CH2Cl2 (3 mL) was placed under a nitrogen atmosphere at
0 °C, and a solution of 4-nitrobenzenesulfonyl chloride (0.32 g,
1.2 mmol) dissolved in dry CH2Cl2 (3 mL) was added dropwise
via syringe over the course of 15 min. The mixture was stirred at
0 °C for 2 h and allowed to warm to ambient temperature for
24 h. The reaction was quenched and the mixture subsequently
washed three times with 1 M HCl (5 mL) and once with saturated
aqueous NaCl (5 mL). The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The crude yellow solid was
dissolved in minimal EtOAc and purified by flash chromatogra-
phy (SiO2, 20 to 40% EtOAc in hexanes) to afford the corre-
sponding Boc-protected 4-nitrobenzenesulfonate derivative as a
Norleucine and Selenomethionine Substitution. To incor-
porate norleucine (Nle) or selenomethionine (SeM) into PagP
and its mutants, the appropriate expression plasmids were
transformed into methionine auxotrophic cell line E. coli B834-
(DE3). Nle incorporation was based on the procedure described
by Budisa (32), except that we adapted media designed for SeM
labeling. Four stock solutions were prepared. First, a stock
solution of 50ꢀ amino acid mix was prepared by adding to
500 mL of autoclaved water 1 g each of the standard L-amino
acids, excluding cystine, trans-proline, tyrosine, glutamine, and
methionine. The solution was passed through a 0.22 μm filter,
and 40 mL aliquots were stored at -80 °C. Second, a 1000ꢀ trace
metal stock solution was prepared to yield a final concentration
in culture media of 10 μM for each metal: 12.4 g of
(NH4)6(Mo7)24 4H2O, 2.4 g of CoCl2 6H2O, 2.5 g of CuSO4
1
pale yellow solid (0.28 g, 60% yield): mp 82-88 °C; H NMR
(300 MHz, CDCl3) δ 8.38 (d, 2H, J = 9.1 Hz), 8.08 (d, 2H, J =
9.1 Hz), 4.49 (s, 1H), 4.11 (t, 2H, J = 6.5 Hz), 3.05 (q, 2H, J =
6.6 Hz), 1.65 (quintet, 2H, J = 6.6 Hz), 1.48-1.30 (m, 13H); 13C
NMR (75 MHz, CDCl3) δ 156.1, 150.9, 142.1, 129.3, 124.6, 79.4,
71.7, 40.3, 29.6, 28.7, 28.5, 22.1; HRMS m/z 411.1201 [(M þ
Na)þ calcd 411.1194]. A solution of this Boc-protected inter-
mediate (0.05 g, 0.12 mmol) in MeOH (10 mL) was treated with
HCl (1 mL of a 4 M solution in dioxane). The mixture was stirred
at ambient temperature for 1 h, and all solvents were evaporated
in vacuo. The crude yellow product was triturated in CH2Cl2,
filtered, and washed with CH2Cl2 to afford the n-aminopentyl
4-nitrobenzenesulfonate product (Figure 3A) as its HCl salt (pale
3
3
3
5H2O, 0.62 g of H3BO3, 2.0 g of MnCl2 4H2O, and 1.4 g of ZnCl2
3
were added to 0.1% HCl in a final volume of 1.0 L and
autoclaved. Third, a buffer solution was prepared by dissolving
4.0 g of (NH4)2SO4, 18.0 g of KH2PO4, 42.0 g of K2HPO4, and
2.0 g of Na3-citrate in 800 mL of water and autoclaved. Finally,
we prepared the TyB “Tyrosine and nucleotide bases” mix by
dissolving 0.16 g of tyrosine and 2.0 g each of adenine, guanosine,
thymine, and uracil in 2.0 L of autoclaved water by heating the
sample to 50 °C with stirring and then autoclaving. After the
samples had been cooled to room temperature, the following
were added to the TyB mix in order: 80 mL of the 50ꢀ amino acid
stock, 8 mL of 20 mg/mL glutamine (filter-sterilized), 80 mL of
50% glycerol (autoclaved), 5 mL of the 1000ꢀ trace metal mix,
4 mL of 0.5 M CaCl2 (filter-sterilized), 4 mL of MgSO4 (filter-
sterilized), 1.6 mL of 5 mg/mL thiamine (filter-sterilized),
1000 mL of autoclaved water, and 800 mL of the buffer solution.
The TyB mix was stored at 4 °C until it was used. A solution of
Nle was also prepared via addition of 0.33 g to 10 mL of water
for a final medium concentration of 5 mM at pH 2.31 and filter-
sterilized.
1
yellow solid, 0.024 g, 60% yield): mp 104-108 °C; H NMR
(500 MHz, CD3OD) δ 8.48 (d, 2H, J = 8.8 Hz), 8.17 (d, 2H, J =
8.8 Hz), 4.19 (t, 2H, J = 6.2 Hz), 2.90 (t, 2H, J = 7.6 Hz), 1.75
(quintet, 2H, J = 7.6 Hz), 1.65 (quintet, 2H, J = 7.6 Hz), 1.46
(quintet, 2H, J = 7.5 Hz); 13C NMR (125 MHz, CD3OD) δ
151.2, 141.7, 129.3, 124.5, 71.1, 39.3, 28.3, 26.7, 25.3; HRMS m/z
289.0858 [(M þ H - Cl)þ calcd 289.0863].
Site-Specific Chemical Alkylation of Gly88Cys PagP.
MNBS is commercially available, and we have previously
adapted the S-methylation procedure developed by Heinrick-
son (31) for the covalent modification of Gly88Cys PagP (21). In
the work presented here, the customized alkylating reagents
described above were utilized to incorporate ethyl, n-propyl,
n-butyl, n-pentyl, n-hexyl, and n-aminopentyl groups into the
Gly88Cys PagP mutant. The reactions were conducted in capped
glass tubes. To achieve buffer concentrations of 6 M guanidine
hydrochloride (Gdn-HCl), 0.25 M Tris-HCl (pH 8.6), 3.3 mM
disodium ethylenediaminetetraacetic acid (EDTA), and 25%
acetonitrile (v/v), 25 mg of Gly88Cys PagP was dissolved in a
volume of 3.75 mL of 8 M Gdn-HCl, 0.34 M Tris-HCl (pH 8.6),
and 4.4 mM EDTA and the volume adjusted to 5 mL with 1.25
mL of 100% acetonitrile. The solutions were flushed with N2 for
1 min to create an anoxic barrier, and 50 μL of 260 mM βME was
added (10-50-fold molar excess of the protein). The tubes were
tightly sealed and placed in a 50 °C bath for 1.5 h and then
For Nle substitution, wild-type, Gly88Met, and Gly88Cys
PagP strains were grown in 5 mL of LB medium at 37 °C
overnight, and 1 mL of each overnight culture was inoculated
into 1 L of prewarmed minimal medium described above to
which a limited amount of Met (0.05 mM) had been added. After
the OD reached 0.6, the Met-exhausted culture was supplemen-
ted with 5 mM Nle and incubated for 10 min before induction
with 1 mM IPTG. Cells were induced for 4 h at 37 °C before being
harvested. As a control, we also grew these mutants in the same
medium supplemented with 5 mM Met. The protein was purified
as described previously (21).
For SeM substitution, 10 mL overnight cultures of wild-type
and Gly88Met PagP strains were prepared in minimal medium as

9050 Biochemistry, Vol. 49, No. 42, 2010
Khan et al.
described above, but supplemented with 0.3 mM Met. After 16 h,
the cultures were harvested at 7000 rpm for 10 min. The pellet was
washed twice with 10 mL of minimal medium without Met and
then resuspended in 20 mL of minimal medium supplemented
with 0.05 mM Met. The suspension was then added to 980 mL of
minimal medium supplemented with 0.05 mM Met. After an OD
of 0.7 had been reached, 10 mL of a 300 mM filter-sterilized SeM
solution was added to the Met-depleted culture to give a final
SeM concentration of 3 mM. After incubation for 10 min, the
culture was induced with 1 mM filter-sterilized IPTG and grown
for 4 h before the cells were harvested. The protein was purified as
described previously (21), except that solutions were maintained
in a reduced state by addition of 20 mM βME. No oxidation
of SeM to SeM selenoxide in PagP was evident by ESI-MS
(Table 1).
CD Spectroscopy. Samples to be analyzed by far-UV CD
were maintained at a concentration of 15 μM in 10 mM Tris-HCl
(pH 8.0) and 0.1% LDAO using a cuvette with a path length of
1 mm. An Aviv 215 spectrophotometer was linked to a Merlin
Series M25 Peltier device for temperature control. For each
sample, three accumulations were averaged at a data pitch of
1 nm and a scanning speed of 10 nm/min. The temperature was
maintained at 25 °C, and data sets were obtained from 200 to
260 nm for far-UV CD. Thermal denaturation profiles were
obtained by heating the samples from 20 to 100 °C at 218 nm with
a temperature slope of 2 °C/min and a response time of 16 s.
Preparation of [32P]Kdo2-Lipid A. To prepare radiolabeled
described (21). Synthetic sn-1,2-diacylphosphatidylcholine
(PtdCho) with unbranched saturated acyl chains of defined
composition, and Kdo2-lipid A, were obtained from Avanti
Polar Lipids (Alabaster, AL). The assays were conducted in
0.5 mL microcentrifuge tubes, in which sufficient Kdo2-lipid A
was added to achieve a concentration of 10 μM in a final assay
volume of 25 μL. To this sample was added a trace amount of
[32P]Kdo2-lipid A, yielding a density of 200 cpm/μL. Sufficient
PtdCho was added to attain a final concentration of 1 mM. These
constituents were dried under a gentle N2 stream and subse-
quently dissolved in 22.5 μL of a reaction cocktail containing
0.1 M Tris-HCl (pH 8.0), 10 mM EDTA, and 0.25% dodecyl
maltoside (DDM). The reaction was initiated via addition of
sufficient PagP, serially diluted in DDM to remove inhibitory
LDAO, to achieve a linear reaction profile. All reactions were
conducted at 30 °C and were stopped by directly spotting 4 μL of
the reaction mixture to the origin of a Silica Gel 60 TLC plate.
The TLC plate was resolved in a CHCl3/pyridine/88% formic
acid/H2O system (50:50:16:5, v/v) within a tightly sealed glass
tank. The constituents of the tank were allowed to equilibrate for
a period of 3 h prior to being exposed to the TLC plate. After the
plate was dried, it was exposed to a Molecular Dynamics
PhosphorImager screen overnight to visualize the reaction. The
product was quantified using ImageQuant. Detergent exchange
acyltransferase assays for wild-type and Gly88Cys-S-n-butyl
PagP were conducted by dilution of the protein samples in
reaction buffers containing either 0.25% DDM, 0.1% Cyclofos
7, or 0.1% LDAO [all detergents were obtained from Anatrace
(Maumee, OH)]. Wild-type PagP was assayed using di16:0
PtdCho, and Gly88Cys-S-n-butyl PagP was assayed using
di10:0 PtdCho. The inhibitory effects of LDAO upon PagP were
determined via addition of increasing concentrations of LDAO
to the assay.
lipid A bearing two units of 3-deoxy-D-manno-oct-2-ulosonic acid
(Kdo2-lipid A), a 10 mL culture of E. coli WBB06 was grown
overnight as described previously (33). Briefly, a 1:100 dilution
was made in 5 mL of LB containing 25 μCi of [32P]ortho-
phosphate (Perkin-Elmer) and grown at 37 °C for 4-5 h. After
being harvested, the pellet was washed with phosphate-buffered
saline (PBS) (pH 7.4) (26) and harvested again. The cells were
suspended in a CHCl3/MeOH/PBS (pH 7.4) mixture (1:2:0.8,
v/v) and allowed to sit at room temperature for 1 h with slight
agitation. The tube was centrifuged at 7000 rpm and room
temperature for 10 min, and the supernatant was collected.
Appropriate amounts of CHCl3 and H2O were added to form
a two-phase Bligh-Dyer solution [CHCl3/MeOH/H2O mixture
(2:2:1.8, v/v)] (34), and the lower phase was extracted. The lower
phase was dried under N2, and the dried lipids were resuspended
in a CHCl3/MeOH/H2O mixture (2:3:1, v/v) (solvent A) and
applied to a DE-52 cellulose column. We prepared the resin by
washing dry DE-52 resin with 2.4 M NH4-acetate three times and
then washing the resin with distilled water. The resin was then
washed twice with a MeOH/2.4 M NH4-acetate mixture (8:2, v/v)
and then twice with a CHCl3/MeOH/2.4 M NH4-acetate mixture
(2:3:1, v/v). The resin was washed several times with solvent A to
remove all the salt. This resin was packed in a glass column to
which the sample was applied. The column was washed with
2 column volumes of solvent A and then eluted with serial 5-8
column volumes of solvent A containing 60, 120, 240, 360, and
480 mM NH4-acetate as the aqueous component. Ten microliters
of each fraction was visualized by thin layer chromatography
(TLC) in the CHCl3/pyridine/88% formic acid/H2O solvent
system (50:50:16:5, v/v) to identify the fractions containing
Kdo2-lipid A with a monophosphate moiety at position 1. The
TLC plates were visualized using a PhosphorImager (Molecular
Dynamics).
RESULTS AND DISCUSSION
PagP and its mutants were produced in E. coli BL21(DE3)
using IPTG-inducible PagP expression plasmid pETCrcAHΔS
(14). The expressed proteins possess a C-terminal hexahistidine
tag and lack the N-terminal signal peptide to target PagP for
secretion to the outer membrane. The proteins are expressed as
insoluble aggregates, which can be dissolved in Gdn-HCl, puri-
fied by Ni2þ-ion affinity chromatography, and folded by dilution
into the detergent LDAO (21). Site-specific chemical alkylation
of Gly88Cys PagP was performed by an established procedure
(Figure 1B) (21, 31) and confirmed by ESI-MS of the purified
proteins (Table 1). Alkyl p-nitrobenzenesulfonates were prepared
from the corresponding sodium alkoxides and 4-nitrobenzene-
sulfonyl chloride as described in Experimental Procedures
(Figure 1C). PagP phospholipid:lipid A palmitoyltransferase
activity is monitored in vitro using a defined detergent micellar
enzymatic assay with TLC separation of radioactive lipid pro-
ducts (21). The inhibitory LDAO detergent used during PagP
folding is exchanged by dilution into DDM to support enzymatic
activity. Wild-type PagP is highly selective for a palmitoyl group at
the sn-1 position in a glycerophospholipid but largely unspecific
for the polar headgroup (1). We employ the palmitoyl donor
di-16:0-PtdCho and the palmitoyl acceptor [32P]Kdo2-lipid A.
When challenged with a spectrum of donor acyl chain lengths,
wild-type PagP functions as a dedicated palmitoyltransferase
whereas Gly88 PagP mutants can utilize shorter acyl chains.
Analysis of Gly88Cys-S-alkyl Mutants. We examined
the hydrocarbon ruler profiles for the Gly88Cys-S-alkyl PagP
Acyltransferase Assays. The hydrocarbon ruler assays
were performed using a TLC-based approach as previously

Article
Biochemistry, Vol. 49, No. 42, 2010 9051
F
IGURE 2: Modulating the PagP hydrocarbon ruler by site-specific chemical alkylation. (A) PagP specific activity was monitored with a suite of
PtdCho’s having defined saturated acyl chain compositions varying from C14 to C8. Panels from top to bottom represent the activity profiles for
Gly88Cys PagP modified with S-ethyl, S-n-propyl, S-n-butyl, S-n-pentyl, and S-n-hexyl alkyl groups, respectively. (B) Far-UV CD identifies the
β-barrel and exciton signatures characteristic of wild-type PagP. (C) Thermal melts at 218 nm reveal exciton loss near 40 °C followed by β-barrel
unfolding above 80 °C. (D) LDAO inhibition profiles for wild-type PagP and the Gly88Cys-S-alkyl substitutions using their preferred acyl chain
donors. (E) PagP specificactivities for wild-type and Gly88Cys-S-n-butyl PagP were assayed using C16 and C10 acyl chain donors, respectively, in
three different detergent systems. ND means not detected.
derivatives using a suite of diacyl PtdCho’s varying in saturated
acyl chain length from C14 to C8 (Figure 2A). Gly88Cys-S-ethyl,
S-n-propyl, and S-n-butyl PagP were all highly specific for C12,
C11, and C10 acyl chains, respectively. As expected, Gly88Cys-S-
n-pentyl PagP utilized a C9 acyl chain optimally, but with only
half the specific activity compared to that of the wild type and the
shorter alkyl substitutions when using their optimal substrates.
Gly88Cys-S-n-hexyl PagP was by comparison inactive (Figure 2A).
Far-UV CD analysis of the folded S-alkylated proteins clearly
identified the positive 232 nm ellipticity derived from the intact
aromatic exciton (Figure 2B) (21). The Tu of 88 °C for wild-type
PagP β-barrel unfolding is elevated by ∼2-4 °C among the
spectrum of Gly88Cys-S-alkyl derivatives (Figure 2C). Addition-
ally, the 218 nm component of the exciton is observed by its loss
above 40 °C, consistent with the known thermal sensitivity of the
exciton (Figure 2C) (21). We had previously observed an ∼2 °C
increase in thermal stability for Gly88Cys-S-methyl PagP
(21), and that finding is recapitulated here (Figure 2C).

9052 Biochemistry, Vol. 49, No. 42, 2010
Khan et al.
F
IGURE 3: Effect of the incorporation of ammonium ion within the hydrocarbon ruler. (A) PagP specific activity was measured using defined
PtdCho’s varying in saturated acyl chain length from C16 to C8 for the Gly88Lys and Gly88Cys-S-n-aminopentyl enzymes. Also shown is a
superposition of bound LDAO and SDS molecules identified in the hydrocarbon ruler of two PagP crystal structures (PDB entries 1THQ and
3GP6). The numbering represents the positions that correspond to the carbon atoms of the palmitoyl group thought to occupy this position in the
Michaelis complex. The two detergents are aligned with the side chains of Gly88Lys and Gly88Cys-S-n-aminopentyl PagP together with the
aminoalkylating reagent used to generate the latter substitution. Far-UV CD (B) and thermal melts at 218 nm (C) for wild-type PagP compared
with those of Gly88Lys and Gly88Cys-S-n-aminopentyl PagP. ND means not detected.
Although S-alkylation appears to stabilize PagP by occluding
the hydrocarbon ruler cavity (Figure 2C), such stabilization was
not observed in Gly88Met PagP (21).
Cyclofos-7 remain the only detergents known to support PagP
activity without interfering as unspecific substrates or as compe-
titive inhibitors (15, 16).
The observed donor chain length limit of C10 (Figure 2A)
happens to coincide with the PtdCho transition from displaying
bilayer to micellar properties in water (35). If shorter acyl chains
had been supported, a substrate that also functioned as a
detergent might have replaced DDM in the in vitro PagP assay
system. DDM thus remains an intrinsic component of the assay,
but its detergent properties disrupt bilayer phospholipid phase
behavior (25). The C10 limit might better reflect the minimal
occluded surface area between the donor acyl chain and hydro-
carbon ruler required for PagP to selectively engage its substrate.
We thus tested inhibition by the detergent LDAO, which also
gradually became ineffective in the DDM micellar assay as the
selected acyl chains were shortened to C10 (Figure 2D). Inhibi-
tion at 10 mM LDAO corresponds to a mole fraction of two
LDAO molecules to one DDM molecule. Although LDAO
inhibition in the mixed micelles was gradually lost, we could
not replace DDM to assay the Gly88Cys-S-n-butyl PagP deri-
vative in LDAO alone (Figure 2E). Either the LDAO at a high
mole fraction can still inhibit competitively, or else the physical
properties of the LDAO micelles themselves are somehow
incompatible with Gly88Cys-S-n-butyl PagP activity. The hydro-
carbon ruler floor was shown previously to be solvent accessible
when DDM supported the palmitoyltransferase reaction, but
solvent exchange and enzymatic activity were both blocked when
the enzyme was folded in LDAO micelles (22). DDM and
Effect of Incorporation of Ammonium Ion within the
˚
Hydrocarbon Ruler. A recent 1.4 A resolution X-ray structure
of PagP crystallized from SDS/MPD has revealed a single SDS
detergent molecule buried within the hydrocarbon ruler (13). The
nine terminal carbons of the SDS molecule are tightly held and
take a zigzagging electron density pattern in contrast to the
tubular pattern observed closer to the headgroup. In the terminal
eight carbons of the acyl chain, the SDS in the pocket overlaps
˚
very well with the LDAO molecule at 1.9 A resolution (15), but
the paths diverge closer to the headgroup region (Figure 3A). If
the bound detergent molecules correspond to a palmitoyl group
in the Michaelis complex, then the overlapping detergent electron
density might define the fully occluded surface of the hydro-
carbon ruler. Alternatively, the SDS molecule might better
indicate the full hydrocarbon ruler because it extends further
than the LDAO molecule before diverging. We have previously
shown that Gly88Lys introduces a structural destabilization into
PagP (Tu = 74 °C) that broadens the spectrum of selected acyl
chains, but with the preferred substrate being the expected
C11 (22). We wondered if providing an aminoalkyl substitution
might extend acyl chain selection beyond the limit of C10
observed with our Gly88Cys-S-alkyl substitutions (Figure 2A).
By incorporating an n-aminopentyl group using the corre-
sponding aminoalkylating reagent (see Experimental Proce-
dures), we tested for acyl chain selection out to C8 but found

Article
Biochemistry, Vol. 49, No. 42, 2010 9053
F
IGURE 4: Role of side chain flexibility and localized thioether-aromatic dispersion attraction in engineering a dedicated PagP lauroyltransfer-
ase. (A) PagP specific activity was measured using defined PtdCho’s varying in saturated acyl chain length from C16 to C10. The unnatural amino
acid Nle was substituted for Met by misaminoacylation of tRNAMet. Panels from top to bottom represent the activity profiles for Gly88Met,
Gly88Met [Nle], and Gly88Cys-S-ethyl [Nle], respectively. For comparison, the behavior of Gly88Cys-S-ethyl PagP is shown in Figure 2. (B)
Far-UVCD and (C) thermal melts at 218 nm revealthat substituting the five methionines in wild-type and Gly88CysPagP (six inGly88MetPagP)
with Nle does not compromise structure or stability. (D) Schematic representation of side chain conformational preferences for Nle (left) and Met
(right). Viewed in Newman projection down the central χ3 torsion axis, the penultimate atom is rotated to emphasize the enthalpic preference of
Nle for maintaining the terminalmethyl group inthe extendedanti conformation, whereas Met isslightlyfavored toadopt one ofthe two available
gauche conformers.
the Gly88Cys-S-n-aminopentyl PagP to be enzymatically inactive
by comparison with Gly88Lys PagP (Figure 3A). The incorpora-
tion of ammonium groups is most easily discerned by compar-
isons between two different pairs of nearly isosteric partners:
Gly88Nle with Gly88Lys and Gly88Cys-S-n-hexyl with
Gly88Cys-S-n-aminopentyl. Far-UV CD (Figure 3B) showed no
major structural perturbation, while thermal melts (Figure 3C)
showed that both NH3þ-containing partners were destabilized by
the same amount relative to their CH3-terminated counterparts.
Unlike the significant differences observed between S-ethylcysteine
and Met (see below), these data show no dependence on the location
of the ammonium ion in the folded state. This suggests that the large
desolvation energy penalties that must be paid to bury an ammo-
nium ion in the core of a protein are the dominant effects for this set
of compounds.
most likely specifies diminishing binding forces between van der
Waals-contacted groups localized within a hydrophobic micro-
environment. In the absence of water or electrostatic perturba-
tions, London dispersion forces appear to be a dominant source
of intermolecular attraction, especially when nonpolar surface
contacts are maximized without steric repulsion (36). Localized
dispersive attractions should be paramount for the highly
polarizable thioether sulfur atom, which is more electron-dense
than typical hydrocarbon moieties (37). If a thioether-containing
side chain fits snugly within the hydrocarbon ruler, van der
Waals-contacted dispersive attractions with neighboring aro-
matic side chains should be more pertinent compared to inter-
actions with aliphatic or hydroxylated side chains that lack
conjugated resonance-stabilized π-electron systems (38, 39).
Interestingly, Gly88Cys-S-ethyl PagP behaves as a dedicated
lauroyltransferase (Figure 2A), but acyl chain selection by its
regioisomer Gly88Met PagP is imperfect by comparison (21)
(Figure 4A). The main observed differences are the partial
Role of Side Chain Flexibility and Localized Thioether-
Aromatic Dispersion Attraction in Engineering a Dedi-
cated PagP Lauroyltransferase. The C10 acyl chain transition

9054 Biochemistry, Vol. 49, No. 42, 2010
Khan et al.
selection, thus creating another dedicated lauroyltransferase, but
without increasing its Tu (Figure 4A,C).
We interpret these findings in terms of the preferred con-
formational equilibria for the n-butyl side chain of Nle versus
the thioether side chains of Met and S-ethylcysteine, combined
with localized thioether-aromatic dispersion attraction solely
for S-ethylcysteine. As cogently articulated by Gellman (36), the
Nle side chain displays an enthalpic preference for the extended
anti conformation, whereas Met slightly favors one of the two
available gauche conformers (Figure 4D). We calculated identical
gas-phase bond rotation barriers and conformational energies for
the side chains of S-ethylcysteine and Met, indicating that
differences in their acyl chain selectivity and thermal stability
must be explained by interactions these side chains experience
within the folded structure of PagP. Although the inherent
conformational flexibility of Met is matched by that of
S-ethylcysteine, only the latter substitution displays a stabilized
Tu of 92 °C, which appears to lock the side chain in the extended
anti conformation as is evident in its superb C12 acyl chain
selectivity. Reflecting the thioether sulfur atom to the opposite
wall of the hydrocarbon ruler in Gly88Met PagP causes the local
environment to support only the wild-type Tu of 88 °C. Without
any corresponding stabilization, the Met thioether side chain is
now free to explore its wider available conformational space and
thus opens the hydrocarbon ruler for partial utilization of C13
acyl chains. The same reasoning can be applied to Gly88Cys-
S-methyl PagP, which has the same terminal methyl group
flexibility as Met, but is stabilized by an apparent thioether-
aromatic dispersion attraction (Tu = 90 °C), thus locking the side
chain in its extended conformation to yield superb selection of
C13 acyl chains (21). This localized stabilization might also
explain the excellent acyl chain selectivity of Gly88Cys-S-n-
propyl and Gly88Cys-S-n-butyl PagP (Figure 2A,C). Gly88Met
[Nle] PagP is a dedicated lauroyltransferase only because it
intrinsically prefers the extended anti conformer, but without
the thioether sulfur atom, it does not display the stabilized Tu
(Figure 4C).
The hydrocarbon ruler microenvironments surrounding the
bound detergent methylene groups, which correspond with the
thioether sulfur atoms in Gly88Met PagP (position 14) or
Gly88Cys-S-ethyl PagP (position 15), lend support to a role for
localized thioether-aromatic dispersion attraction only for the
latter substitution (Figure 5A). Whereas the thioether sulfur
atom of Gly88Cys-S-ethyl PagP is likely cradled in its extended
anti conformation at position 15 by the aromatic side chains
of Tyr70, Trp156, and Tyr26, the same atom in Met at position
14 is likely exposed to the phenolic oxygen atom of Tyr70
together with the hydroxylated Thr108 and aliphatic Leu128.
These distinctly different microenvironments might explain the
observed stabilization of the same thioether sulfur atom only
when it is localized at the deeper of the two hydrocarbon ruler
positions (38, 39).
SeM Substitution Is Highly Destabilizing to PagP. To
further explore the role of dispersion forces within the PagP
hydrocarbon ruler, we replaced each Met residue (Figure 5B)
with SeM, which introduces a larger and more electron dense
selenium atom in place of sulfur. SeM is routinely used for
phasing in X-ray crystallography, but its utility appears to be
underrepresented among integral membrane protein struc-
tures (40). PagP [SeM] did not crystallize in our original structure
determination, but mixing the labeled and native proteins did
yield crystals suitable for phasing (15). Instability associated with
F
IGURE 5: Views of the hydrocarbon ruler and solvation shell for
PagP in SDS/MPD. (A) Pseudo-2-fold symmetric views of the
hydrocarbon ruler-bound SDS molecule (PDB entry 3GP6) empha-
size the functional groups surrounding thioether positions 15
(Gly88Cys-S-alkyl) and 14 (Gly88Met), thus revealing distinctly
different local environments for the sulfur atom. (B) Solvation of
˚
PagP in SDS/MPD at 1.4 A resolution reveals the belt of high hydro-
phobic surface potential. Met residues 0, 6, 72, 140, and 157 are
shown as sticks, as are embrasure Pro residues 28 and 50. The buried
SDS is shown in space-filling format, whereas the remaining
5 SDS, 56 water, 10 MPD, 6 sulfate, and 2 lithium molecules are
shown in dots format.
utilization of a C13 acyl chain by Gly88Met PagP and the Tu that
is increased from 88 °C, for wild-type and Gly88Met PagP, to
92 °C, for Gly88Cys-S-ethyl PagP (Figures 2C and 4B). To
dissect the possible contribution of localized thioether-aromatic
dispersion attraction from side chain conformational prefer-
ences, we substituted Met residues with the unnatural amino
acid Nle through misaminoacylation of tRNAMet (32) (see
Experimental Procedures). Substituting the five methionines in
wild-type and Gly88Cys PagP (six in Gly88Met PagP) with Nle
does not compromise structure and stability as ascertained by far-
UV CD and thermal melts (Figure 4B,C). However, Nle sub-
stitution of Gly88Met PagP corrects the imperfect acyl chain

Article
Biochemistry, Vol. 49, No. 42, 2010 9055
F
IGURE 6: SeM substitution is highly destabilizing to PagP. Far-UV CD (A) and thermal melts at 218 nm (B) reveal that substituting methionines
in wild-type and Gly88Met PagP with SeM compromises stability. PagP specific activity was measured using defined PtdCho’s varying in
saturated acyl chain length from C17 to C10. Wild-type (C) and Gly88Met (D) PagP were monitored with and without SeM substitution. ND
means not detected.
SeM labeling of the β-barrel membrane protein FadL has also
been reported (41). SeM-labeled wild-type and Gly88Met PagP
were prepared in the presence of βME, and no oxidation of SeM
to SeM selenoxide was evident by ESI-MS (Table 1). Although
the exciton and β-barrel structure of PagP are evident in far-UV
CD, we were surprised by the extent of the destabilization
associated with SeM substitution in the corresponding thermal
melts (Figure 6A,B). The greatest instability previously asso-
ciated with an active PagP mutant is that of Gly88Cys PagP at
pH 8, which loses its exciton and has its Tu reduced by 22 °C
because of a buried thiolate anion (21) (see also Figure 4B,C). By
comparison, SeM-substituted wild-type and Gly88Met PagP
each display the exciton, but their Tu values are reduced by
34 °C (Figure 6B). PagP with Nle and PagP without Nle were
virtually indistinguishable in terms of stability (Figure 4C), and
wild-type acyl chain selection (Figure 6C) was not distinctly
different for the Nle-substituted protein (not shown). However,
SeM substitution of the five intrinsic Met residues drastically
broadened acyl chain selection and reduced the specific activity
(Figure 6C). The introduction of a sixth SeM residue at position
88 did not further destabilize the protein (Figure 6B), but the
residual enzymatic activity was extinguished (Figure 6D). We
conclude that substituting Met88 with SeM compromises lipid
acyl chain selection without any apparent PagP destabilization.
Of the five Met residues in wild-type PagP, only Met72 is
buried and lines the hydrocarbon ruler wall (Figures 1B and 5).
Two conformers of this thioether side chain are apparent in the
SDS/MPD structure (13), and the corresponding selenide might
be envisaged to influence lipid acyl chain selection from this
position through disruption of neighboring van der Waals
packing interactions (Figure 6C). We purified Met72Leu PagP,
both with and without SeM substitution (Table 1), and found
Met72 to be entirely responsible for the perturbation observed in
the wild-type enzyme upon SeM substitution (Figure 7). No
destabilization was associated with SeM substitution of the four
surface-exposed SeM residues. Met6 and the non-native Met0 are
each located in the N-terminal helix, whereas the C-terminal
Met140 and Met157 are both exposed on the β-barrel surface
(Figure 5B). These four surface residues all map onto the belt of
high hydrophobic surface potential marking the transmembrane
˚
domain in the 1.4 A resolution SDS/MPD structure (13, 42). SeM
residues buried within the hydrophobic core or exposed to
solvent on the surface of soluble globular proteins are usually
reasonably well tolerated (32, 40, 43). Our findings point to a
strongly destabilizing influence of SeM at position 72 in PagP.
The Lennard-Jones potential describes a narrow optimal distance
range between neutral molecules for the ideal interaction energy
to balance weak van der Waals attraction with stronger steric
repulsion (44). We thus suspect that the larger van der Waals
radius and/or bond lengths associated with the selenide com-
pared to the sulfide at residue 72 are somehow incompatible
with local packing arrangements necessary for optimal PagP
stabilization (45).
Concluding Remarks. The thioether-aromatic dispersion
attraction described here can also be regarded specifically as a
methionine-π attraction (38), or more generally as a sulfide-
π attraction (39). In an aqueous environment, the energetic stabi-
lization of the methionine-π attraction is on the order of 0.3-
0.5 kcal/mol (38). Similarly, conformational energies at equilib-
rium in the gas phase indicate that the Nle side chain enthalpically
favors the anti conformer (Figure 4D) by 0.8 kcal/mol (36, 46). In
our LDAO detergent system, these energetics translate into a Tu
increase of 2-4 °C for the S-alkylated Gly88Cys PagP deriva-
tives. Coulombic repulsions are typically stronger than London
dispersion attractions (44), hence the 14 and 22 °C decreases in Tu
observed with the buried ammonium (Figure 3C) (22) and

9056 Biochemistry, Vol. 49, No. 42, 2010
Khan et al.
the primary factor controlling its monomer-dimer equilibrium
(49, 50). Our use of alkylnitrobenzenesulfonates to inscribe
the PagP hydrocarbon ruler perimeter has allowed us to titrate
the substrate affinity to the point where PagP can no longer
function, all without changing the fold of the protein or
dramatically altering the dispersion attractions governing lipid
binding.
ACKNOWLEDGMENT
We thank Philip Hultin (University of Manitoba, Winnipeg,
MB), Jian Payandeh (University of Washington, Seattle, WA),
Alan Davidson (University of Toronto, Toronto, ON), and
ꢀ
Murray Junop, Alba Guarne, Ricky Cheng, and Richard Epand
(McMaster University) for helpful advice and discussions.
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F
IGURE 7: SeM destabilization is attributed to Met72. Far-UV CD
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(A) and thermal melts at 218 nm (B) reveal that substituting
methionines in Met72Leu PagP with SeM no longer compromises
stability. PagP specific activity was measured using defined PtdCho’s
varying in saturated acyl chain length from C17 to C10. (C) Me-
t72Leu PagP was monitored with and without SeM substitution.
thiolate ions (Figure 4C) (22), respectively. The observed desta-
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13. Cuesta-Seijo, J. A., Neale, C., Khan, M. A., Moktar, J., Tran, C. D.,
ꢁ
ꢀ
Bishop, R. E., Pomes, R., and Prive, G. G. (2010) PagP crystallized
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The conclusion that the thioether bond cannot be regarded as
simply equivalent to its corresponding methylene unit was made
previously in a study using aminoalkylbromides to modify an
introduced Cys residue in RNase A (47). Iodoalkanes have also
been reported previously to modify an introduced Cys residue in
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phospholipase OMPLA was more recently subjected to covalent
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