Correction of both NBD1 energetics and domain interface is required to restore ΔF508 CFTR folding and function.
ABSTRACT The folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of ΔF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508 CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.
- [show abstract] [hide abstract]
ABSTRACT: Misfolding accounts for the endoplasmic reticulum-associated degradation of mutant cystic fibrosis transmembrane conductance regulators (CFTRs), including deletion of Phe508 (DeltaF508) in the nucleotide-binding domain 1 (NBD1). To study the role of Phe508, the de novo folding and stability of NBD1, NBD2 and CFTR were compared in conjunction with mutagenesis of Phe508. DeltaF508 and amino acid replacements that prevented CFTR folding disrupted the NBD2 fold and its native interaction with NBD1. DeltaF508 caused limited alteration in NBD1 conformation. Whereas nonpolar and some aliphatic residues were permissive, charged residues and glycine compromised the post-translational folding and stability of NBD2 and CFTR. The results suggest that hydrophobic side chain interactions of Phe508 are required for vectorial folding of NBD2 and the domain-domain assembly of CFTR, representing a combined co- and post-translational folding mechanism that may be used by other multidomain membrane proteins.Nature Structural & Molecular Biology 02/2005; 12(1):17-25. · 11.90 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.Nature Structural & Molecular Biology 02/2005; 12(1):10-6. · 11.90 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.Nature Reviews Molecular Cell Biology 05/2007; 8(4):319-30. · 37.16 Impact Factor
Correction of Both NBD1 Energetics and
Domain Interface Is Required to Restore
DF508 CFTR Folding and Function
Wael M. Rabeh,1,2,4Florian Bossard,1Haijin Xu,1Tsukasa Okiyoneda,1Miklos Bagdany,1Cory M. Mulvihill,1Kai Du,1
Salvatore di Bernardo,1Yuhong Liu,3Lars Konermann,3Ariel Roldan,1and Gergely L. Lukacs1,2,*
1Department of Physiology
McGill University, Montre ´al, Quebec H3E 1Y6, Canada
3Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada
4Present address: Department of Sciences, New York University, P.O. Box 129188, Abu Dhabi, United Arab Emirates
The folding and misfolding mechanism of multido-
main proteins remains poorly understood. Although
thermodynamic instability of the first nucleotide-
binding domain (NBD1) of DF508 CFTR (cystic
partly accounts for the mutant channel degradation
in the endoplasmic reticulum and is considered as
a drug target in cystic fibrosis, the link between
NBD1 and CFTR misfolding remains unclear. Here,
we showthat DF508 destabilizesNBD1 both thermo-
dynamically and kinetically, but correction of either
defect alone is insufficient to restore DF508 CFTR
biogenesis. Instead, both DF508-NBD1 energetic
and the NBD1-MSD2 (membrane-spanning domain
2) interface stabilization are required for wild-type-
like folding, processing, and transport function,
suggesting a synergistic role of NBD1 energetics
and topology in CFTR-coupled domain assembly.
Identification of distinct structural deficiencies may
explain the limited success of DF508 CFTR corrector
molecules and suggests structure-based combina-
tion corrector therapies. These results may serve
as a framework for understanding the mechanism
of interface mutation in multidomain membrane
Cystic fibrosis transmembrane conductance regulator (CFTR) is
a multidomain, polytopic membrane protein that belongs to the
ATP-binding cassette (ABC) transporter C class superfamily
(Riordan, 2008). CFTR consists of two membrane-spanning
domains (MSD1, MSD2) with four cytosolic loops (CL1–4) and
three cytosolic domains: a regulatory (R) and two nucleotide-
binding domains (NBD1, NBD2). Deletion of F508 (DF508) in
the NBD1 is a cystic fibrosis (CF)-causing mutation found in at
least one allele of 90% of patients (http://www.genet.sickkids.
on.ca/cftr). This mutation diminishes the intrinsically low (20%–
40%) folding efficiency of CFTR to ?0.4% (Cheng et al., 1990;
Pedemonte et al., 2005) and results in ubiquitin (Ub)-dependent
endoplasmic reticulum (ER)-associated degradation, thereby
compromising the channel plasma membrane (PM) expression
(Riordan, 2008). Impaired gating kinetics and reduced metabolic
stability of the mutant further exacerbates the CFTR loss-of-
function phenotype (Dalemans et al., 1991; Sharma et al., 2004).
Low temperature, chemical chaperones, and second-site
suppressor mutations in the NBD1 or at the NBD1-MSD2 inter-
face can restore the PM functional expression of DF508 CFTR
up to 15% of wild-type (WT) CFTR (Aleksandrov et al., 2010;
Denning et al., 1992; He et al., 2010; Loo et al., 2009, 2010;
Sato et al., 1996; Teem et al., 1993, 1996; Thibodeau et al.,
2010). Comparable or poorer rescue efficiencies were achieved
by small molecule correctors identified by high-throughput
screens (HTSs) in vivo (Pedemonte et al., 2005; Robert et al.,
2010; Sampson et al., 2011; Van Goor et al., 2006, 2011) and
insilico (Kalid etal.,2010).Despite intensive efforts,theavailable
investigational corrector compound (VX-809) has similarly
modest efficiency (Clancy et al., 2011).
The original observation that DF508 NBD1 refolding is
impaired and the domain has marginally defective thermody-
namic stability (Qu and Thomas, 1996) is further elucidated by
the observation of localized structural perturbation of the flexible
surface loop of residues 509–511 in the crystal structures (Lewis
et al., 2005, 2010; Thibodeau et al., 2005). Although these find-
ings were consistent with the notion of formation of kinetically
trapped folding intermediate(s) (Thibodeau et al., 2005), it was
recognized that the DF508 CFTR misfolding coincides with a
global folding defect that affects the conformation of MSD1,
MSD2, and NBD2 (Cui et al., 2007; Du and Lukacs, 2009; Du
et al., 2005; Rosser et al., 2008). Based on the predicted
domain-swapped architecture of CFTR that manifests in the
invariability of contacts between coupling helices of the CLs
and NBDs from opposite halves in ABC exporters (Mornon
150 Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc.
et al., 2009; Serohijos et al., 2008a) (Figure 1A), engineered Cys
crosslinking confirmed that the F508 and its vicinity interface
with the CL4 and CL1 of MSD2 and MSD1, respectively (Fig-
ure 1A) (He et al., 2010; Serohijos et al., 2008a). These contacts,
similar to that of the NBD2-MSD1, are not detectable in nonna-
tive, core-glycosylated WT and DF508 CFTR (He et al., 2010;
Serohijos et al., 2008a), consistent with a possible role in domain
assembly. Jointly, these observations along with the observa-
tions on CFTR-interdependent domain folding and misfolding
helped to formulate the cooperative domain-folding model
that invokes energetic/kinetic conformational domain-domain
coupling as part of the CFTR co- and posttranslational folding
(Du and Lukacs, 2009).
More recent results established that the DF508 mutation
promotes the NBD1 thermal aggregation (Hoelen et al., 2010)
and compromises the thermodynamic stability of the NBD1
containing three solubilizing (S) mutations or deletion of the
regulatory insertion (DRI, residues 405–436) (Protasevich et al.,
2010; Wang et al., 2010). The DF508-NBD1 folding energetic
defect in the absence of second-site mutations and its contribu-
tion to DF508 CFTR global misfolding, however, are poorly
defined. We hypothesized that both NBD1 interface topology
and energetics are important determinants of CFTR domain
assembly. Our results show that either NBD1 energetics or the
NBD1-CL4 interface defect can instigate CFTR domain misas-
sembly. Conversely, genetic suppression of either energetic
or interface defects alone is insufficient to restore DF508
CFTR folding, processing, and function, whereas in combination
they result in mature protein with properties similar to those of
Thermodynamic Destabilization of Isolated NBD1
Variants by the DF508 Mutation
To determine the DF508-induced NBD1 energetic defect, full-
length WT and DF508 human NBD1 variants (amino acids 389–
678) with or without S and/or revertant (R) mutations were
purified from E. coli as described (Lewis et al., 2005). Both the
R mutations (G550E, R553Q, and R555K) and S mutations
theDF508CFTR folding andfunctional defect (Lewis etal.,2005;
Pissarra et al., 2008; Teem et al., 1993, 1996) and were assumed
to stabilize the domain either alone or in combinations (1S, 3S,
R, R1S, and R4S; see Figure 1B). The isolated NBD1s were
>90%–95% pure and monomeric (see Figures S1A and S1B
available online; data not shown). The secondary structure
composition and TNP-ATP-binding affinity of WT- and DF508-
NBD1 variants were comparable (Figures S1C and S1D; data
not shown) (Qu et al., 1997; Qu and Thomas, 1996; Stratford
et al., 2007).
The DF508 mutation reduced the apparent melting tempera-
ture (Tm) of the WT NBD1-1S from 41.8?C ± 0.2?C to 33.2?C ±
0.2?C (DTmz 8.6?C ± 0.2?C) (±standard error of the mean
[SEM]), based on ellipticity, as well as Trp fluorescence and
differential scanning fluorimetry (DSF) using different reporter
dyes (Figures 1C–1E, S1E, and S1F) (Niesen et al., 2007; Seni-
sterra et al., 2008). In the absence of second-site mutations,
the WT and DF508 NBD1-0S thermal denaturation propensity
was slightly increased (Tm?39.8?C ± 0.2?C and ?31.7?C ±
0.1?C, respectively) relative to NBD1-1S measured at 2.5 mM
ATP (Figures 1D and 1E). Because the protein yield was limited
(Figure S1G), only DSF scans could be performed on NBD1-0S.
The Tmdifference between the WT and DF508 NBD1 was
similar (6?C–8?C) for 0S, 1S, 3S, R, and R4S and in DRI (Protase-
vich et al., 2010), as well as at reduced ATP concentration
(Figures 1E, S1H, and S1I). Thermal unfolding preceded domain
aggregation for both WT and DF508-NBD1 (Extended Experi-
mental Procedures; Figure S1J). Assuming a reversible, two-
state folding mechanism with slow aggregation of the unfolded
form (Protasevich et al., 2010), we estimated the folding free
energy (DG0) based on the DSF data. Decreasing the tempera-
ture from 37?C to 20?C lowered the DG0of the DF508 and
WT NBD1-0S from +1.8 to ?4.2 kcal/mol and from ?1.0 to
?4.8 kcal/mol, respectively (Extended Results; Table S1).
tration (D0.5) of the WT NBD1 variants by ?0.8–1 M at 1 mM
ATP (Figure S2E). NBD1 fractional unfolding was calculated
by extrapolation of the CD data due to the oligomerization/
aggregation propensity of partially unfolded DF508-NBD1
(Figures 1F and S2A–S2D; Extended Results) (Strickland et al.,
1997; Wang et al., 2010). The estimated DG0between the
WT- and DF508-NBD1-1S and -3S was decreased by ?2.4 and
?1.4 kcal/mol, respectively, whereas the R1S and R4S mutation
stabilized the DF508 by ?1 and ?1.6 kcal/mol at 20?C (Fig-
ure 1G). Thermal unfolding analysis yielded comparable DG0
differences between WT and DF508-NBD1-0S, -1S, and -3S
(approximately ?1.7, ?1.6, and ?2.3 kcal/mol, respectively;
Extended Results; Table S1). Thus, thermal and chemical dena-
turation studies demonstrated the thermodynamic destabiliza-
tion of the DF508 NBD1 with a single or no S mutation at 37?C
Kinetic Destabilization of NBD1 by the DF508 Mutation
To determine the unfolding energy barrier between the native
and unfolded states, the rate of NBD1 unfolding was monitored
as a function of urea concentration by CD spectroscopy (Figures
2B, S2A, and S2B). The NBD1-1S initial unfolding kinetics
exhibited monoexponential behavior and was found to be
independent of protein concentration between 3 and 14 mM
(data not shown). The DF508-NBD1-1S extrapolated unfolding
rate in water (kuH20) was >30-fold (0.73 s?1) and ?17-fold
(0.04 s?1) faster than its WT counterpart at 20?C and
37?C, respectively (Figures 2C and 2D). The kuH20at 37?C was
determined by the extrapolation of kuH20obtained at 16?C–
30?C (Figure S2F). The unfolding activation energy (DGuz)
of the WT NBD1-1S was reduced in the DF508 from ?5.0 ±
0.11 to ?2.9 ± 0.04 kcal/mol (n = 6) at 20?C and from ?1.8
to ?0.2 kcal/mol at 37?C (Figure S2G; Table S1). In contrast
the R1S and R4S mutations partially restored kinetic stability of
the DF508 NBD1-1S by reducing the kuH20?3- to 4-fold
at 20?C (Figure 2C). Jointly, these results showed for the first
time that the DF508 reduces both thermodynamic and kinetic
components of the DF508-NBD1-1S energetic defect at
37?C, which could be reversed by second-site suppressor
Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc. 151
Figure 1. Folding Thermodynamics of Isolated NBD1 Variants
(A) Molecular models of CFTR closed state (Mornon et al., 2009). The hydrophobic cluster formed by F1068 and F1074 at the NBD1-CL4 interface by the
CL4-coupling helix (dark blue) and F508 (green) is indicated in the inset.
(B) R and S mutations are indicated in the crystal structure of the human NBD1 (PDB: 2BBO) (Lewis et al., 2010). Lower panel depicts the combination of second-
site S and R mutations used.
(C) Melting temperature difference (DTm) between WT- and DF508-NBD1-1S was measured using DSF in the presence of ANS, Nano Orange or Sypro Orange,
tryptophan fluorescence (Trp flu), and CD.
(D) Thermal unfolding scans of WT- and DF508-NBD1 in the absence and presence of 1S or 3S were acquired by DSF using Sypro-Orange.
(E) Summary of WT- and DF508-NBD1 Tmdeterminations by DSF as in (D).
(F) Isothermal urea denaturation curves of WT and DF508-NBD1 variants.
(G) The folding free energy (DG0) of NBD1 in water was estimated based on the isothermal urea denaturation curves at 20?C as in Extended Results.
Data are mean ± SEM. See also Figures S1 and S2.
152 Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc.
The increased conformational flexibility of the DF508-NBD1-
1S was verified by a modest but significant enhancement in
deuterium uptake, determined by hydrogen-deuterium ex-
change (HDX) mass spectrometry (MS) after 15 min incubation
at 24?C, but not at 0?C (Figure S3). Due to thermodynamic stabi-
lization, the extent of HDX for DF508-NBD1-1S was profoundly
reduced by the R1S mutation, consistent with the increased
backbone dynamics of the DF508-NBD1-3S, localized to resi-
dues 509–511 (Lewis et al., 2010).
The DF508 NBD1 Conformational Defect Is
Recognizable by Protein Quality Control
To ascertain that the DF508-NBD1 structural defect may serve
as a degradation signal in vivo, the metabolic turnover of
NBD1 fusion proteins was determined in prokaryotes and
eukaryotes. The degradation rate of the DF508-NBD1-1S was
?4-fold faster than the WT-NBD1-1S in E. coli at 37?C (data
not shown). To examine the effect of NBD1 conformational
stability on the ER export efficiency, NBD1s were tethered to
the C-terminally truncated CD4 (CD4T-NBD1) (Figure 3A). The
PM density of the chimeras was monitored by cell surface ELISA
as a surrogate measure of ER export efficiency at comparable
translational rates in COS7 cells at 37?C (Extended Results;
Figures S4A–S4C; Du and Lukacs, 2009). The DF508 mutation
decreased the PM expression of WT CD4T-NBD1 from 20% to
10% of CD4T, similar to that observed at the cellular expression
level (Figures 3B and 3C). The DF508 mutation decreased the
PM density of CD4T-NBD1 variants containing second-site
mutations, whereas conformational stabilization by the R4S
reversed the phenotype (Figure 3C). The low level of DF508
CD4T-NBD1-R1S expression could be attributed to the partially
normalized unfolding kinetics of the domain (Figure 2C). Stabili-
zation of WT NBD1 by second-site mutation, however, elevated
the chimera PM expression from 20% to 60% (Figure 3C). These
results in concert with the effect of F508E, F508R, F508G,
F508S, F508D, and F508N mutations revealed that the CD4T-
NBD1 PM density was proportional to the domain stability if
the NBD1 Tmwas >38?C (Figures 3D and S4D). Chimeras con-
taining NBD1s with Tm<38?C exhibited %10% expression of
CD4T in COS7 cells at 37?C (Figure 3D), consistent with the
domain instability and the consequential Ub-dependent ER
retention/degradation as shown below. These findings are remi-
niscent of the folding energy-dependent biosynthetic secretion
of transthyretin and BSEP (Sekijima et al., 2005) and suggest
that proteostatic mechanisms can recognize and eliminate
structurally damaged proteins with impaired folding energetics
not only in the lumen (Wiseman et al., 2007), but at the cyto-
plasmic surface of the ER as well.
Figure 2. Simplified Folding Free Energy Profile of WT and DF508
NBD1-1S and the Kinetic Stability Defect of the DF508 NBD1-1S
(A) It is assumed that the NBD1 follows a two-state folding equilibrium,
predominantly populated by the unfolded (U), native (N), or native-like
(N*) state without accumulation of the transition (T or T*) folding intermediate.
The folding free energy (DG0, blue numbers) at 20?C and 37?C was
approximated based on NBD1-1S chemical and thermal unfolding curves in
kcal/mol, respectively, as described in Extended Results. The unfolding
activation energy (DGuz, red numbers) was calculated from the kuH2O. The
folding activation energy (DGfz, black numbers) was calculated as the sum
of the DG0and the DGuz. The predicted folding energy changes are drawn
(B) Unfolding rate (ku) of WT- and DF508-NBD1 (1S and R1S) was measured
as a function of urea concentration by CD spectroscopy at 20?C. Data are
mean ± SEM.
(C) Unfolding rate constant of NBD1s in water (kuH2O) at 20?C was calculated
by extrapolation from experiments shown in (B). Data are mean ± SEM.
(D) The kuH2Oof WT and DF508 NBD1-1S at 37?C was calculated by extrap-
olation from kuH2Odetermined at 18?C–32?C as shown in Figure S2F.
See also Figures S2 and S3.
Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc. 153
Figure 3. The DF508 Imposed NBD1 Folding Defect Is Recognized In Vivo and In Vitro
(A) Schematic structure of truncated CD4 (CD4T) and chimeric variants.
(B) Immunoblot analysis of CD4T and CD4T-NBD1s expression. Equal amounts of lysates (50 mg) of transiently transfected COS7 cells grown at 26?C or 37?C
(C) The PM density of CD4T-NBD1 variants was determined by ELISA as percentage of CD4T and normalized for cellular protein.
(D) Correlation between the thermal stability of NBD1s and the PM density of CD4T-NBD1s. The Tmof NBD1s was determined by DSF and the PM density of the
chimeras by ELISA. Second-site mutations in combination with F508 or DF508 are indicated by filled (d) or empty (B) circles, respectively. F508 missense
mutations are colored.
as described in Experimental Procedures. NBD1 mobility shift was visualized by immunoblotting.
(F) Higher-molecular mass adducts as ubiquitinated NBD1 were quantified by densitometry and plotted as a function of denaturing temperature (left panel).
Correlation between the Tmvalues obtained by DSF and NBD1 unfolding temperature required for 50% ubiquitination (right panel). The data were fitted by linear
regression analysis (r = 0.89, p = 0.05).
Data are mean ± SEM. See also Figure S4.
154 Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc.
The in vitro ubiquitination propensities of WT and DF508
NBD1-1S, -3S, and -R1S were assessed following the native
NBD1 unfolding at 25?C–50?C for 5 min. Subsequent ubiq-
uitination was performed in the presence of E1 enzyme, UbcH5c
(an E2 Ub-conjugating enzyme), CHIP (C-terminal Hsp70-
interacting proteins, an E3 Ub-ligase), and ATP (Meacham
et al., 2001) for 30 min at 26?C in vitro. Ubiquitination was
monitored by the appearance of high molecular mass adducts
in anti-NBD1 immunoblots (Figure 3E). Half-maximal ubiq-
uitination was achieved at temperatures (T0.5) corresponding
to the Tmof NBD1-1S and -3S (Figure 3F). The unfolding tem-
perature required for 50% ubiquitination was increased propor-
tionally to the DF508-NBD1-R1S stability (Figure 3F). This result
indicates that the NBD1 conformational defect correlates
with the domain ubiquitination propensity that may signal ER
Figure 4. NBD1 Stabilization Increases the
Marginal Effect on the DF508 CFTR
(A) Folding efficiency of CFTR variants was
measured in stably transfected BHK cells. WT
CFTR variants were pulse labeled for 15 min, then
chased for 3 hr. DF508 CFTR variants were pulse
labeled for 15 min (chase 0) or for 150 min and
chased for 150 min (2.5 hr).
(B) WT (right panel) and DF508 (left panel) CFTR
folding efficiency was
percentage of pulse-labeled, core-glycosylated
conversion to complex-glycosylated form.
(C) Expression of HA-tagged CFTR variants was
detected in transiently transfected COS7 cells
by immunoblotting with anti-HA Ab. Probing of
Na+/K+-ATPase was used as a loading control.
(D) PM expression of WT (right panel) and DF508
(left panel) CFTR-3HA variants was measured in
transiently transfected COS7 cells by ELISA and
expressed as percentage of WT CFTR.
Data are mean ± SEM. See also Figure S5.
calculated as the
Conformational Stabilization of the
DF508-NBD1 Modestly Improves
DF508 CFTR Folding
As a next step, we assessed whether
DF508 CFTR global misfolding can be
solely attributed to impaired NBD1 ener-
was determined as a function of NBD1
thermal stability (Tm) by measuring the
conversion of radioactively pulse-labeled
core-glycosylated CFTR into complex-
glycosylated form in BHK cells (Figure 4A)
(Du et al., 2005). The 1S, 3S, R, or R1S
mutationsincreasedthe WT CFTRfolding
efficiency from ?27% up to ?52% (Fig-
ure 4B). The cellular and PM density
of WT CFTR was similarly increased, as
evidenced by immunoblotting and cell
surface ELISA, respectively, in BHK and
COS7 cells (Figures 4C and 4D). In con-
trast, stabilization of the DF508-NBD1 only marginally enhanced
the mutant folding efficiency (from 0.4% to 3.5%), expression,
and PM density (Figures 4A–4D). These and additional results
discussed in the following section show that both folding effi-
ciency and PM density of WT variants increased at ?37- and
?14-fold steeper slopes, respectively, than their DF508 or
F508X counterparts as a function of NBD1 Tm(Figures 5A and
5B) and suggest that NBD1 folding energetics can define the
WT but not DF508 CFTR domain-domain assembly. A possible
explanation for this phenomenon could be the permissive
effect of NBD1-CL4 interface stabilization by the F508 residue
for coupled domain folding, an assumption supported by the
(1) Although most missense mutations of the F508 (Q508,
S508, D508, and N508) largely preserved the NBD1
Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc. 155
Figure 5. NBD1 Energetic Stabilization Is Insufficient to Restore DF508 CFTR Folding, Expression, and Domain Assembly
(A) CorrelationbetweentheNBD1thermalstability(Tm)invitroand CFTRfolding efficiency invivo.Second-sitemutationsareindicatedinthebackground ofF508
(C) or DF508 (B). Missense mutations of F508 are depicted by colored squares (-), and F508N-3S and -R are indicated by solid triangle (:). CFTR folding
efficiency was determined as in Figure 4B. Data are mean ± SEM.
156 Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc.
thermal stability, monomeric state, secondary structure
composition, and TNP-ATP-affinity, they severely com-
promised CFTR folding (Figures 5A and S4D; data not
shown). Cys and Met substitutions of F508 retained WT
CFTR folding conceivably by preserving intra- and inter-
domain (CL4) side-chain interactions (Du et al., 2005).
(2) Although certain combinations of F508N and R or S
mutations conferred thermodynamic and kinetic stability
comparable with or exceeding that of the WT NBD1-1S
(e.g., F508N-R; DG0= ?5.1 ± 0.1 kcal/mol, Tmz47?C
and kuH20= 0.16 10?3sec?1; Figure S5), they failed to
restore WT-like folding and expression of the DF508
CFTR (Figures 5A and 5B). Similar to the 3S and R4S
mutations (Figures 5A and 5B), the stabilizing DRI muta-
tion (Protasevich et al., 2010) was unable to reinstate
WT-like folding and expression in DF508 CFTR (Figures
4B and 4D).
(3) To demonstrate that the hydrophobic cluster of the
NBD1-CL4 interface (Figure 1B) stabilizes the CFTR
native fold, the conformation of individual domains was
probed by limited trypsinolysis in isolated microsomes
(Du et al., 2005). The proteolytic fragmentation patterns
were visualized by immunoblotting using domain-specific
antibodies (Figure 5C). The NBD1-, NBD2-, MSD1-, and
MSD2-containing fragments, represented by 29–33, 29–
31, 35–37, and 53–76 kDa immunoreactive bands, have
been validated (Cui et al., 2007; Du et al., 2005) and are
indicated by dashed boxes in Figure 5C. Despite the
R1S-induced stabilization of the DF508-NBD1, the
conformational flexibility of MSD1-, MSD2-, and NBD2-
containing fragments could not be reversed, and it
remained proteolytically more sensitive than their WT
counterparts (Figure 5C). This observation implies that
correction of the NBD1 energetic defect is insufficient to
reinstate domain assembly. Remarkably, NBD1, MSD1,
and NBD2 were susceptible to comparable misfolding
upon disrupting the NBD1-CL4 interface by the R1070W
substitution in WT CFTR. This reduced the channel
folding efficiency by ?87% (Figures S6A and 6A–6C).
Conversely, stabilization of the NBD1-CL4 interface by
the V510D substitution (see bellow) increased the WT
CFTR folding efficiency by ?2-fold, supporting the critical
role of the NBD1-CL4 interface in the coupled domain
folding of CFTR (Figure 6B; see below).
Correction of Both DF508-NBD1 Energetics and
NBD1-CL4 Interaction Is Required to Synergistically
Restore DF508 CFTR Folding and Processing
Because CFTR biogenesis appears to be associated with the
formation of the NBD1-CL4 interface (He et al., 2010; Serohijos
et al., 2008a), we tested whether stabilization of the NBD1-CL4
interface alone or in combination with that of NBD1 was also
required to increase the DF508 CFTR folding efficiency.
R1070W or V510D substitutions at the interfaces restored the
proximity of the DF508 NBD1 and CL4 as shown by Cys
crosslinking. The NBD1-CL4 interface stabilization can be
accomplished by filling the cavity created by the DF508 with
the bulky hydrophobic side chain of R1070W or by salt bridge
formation between V510D in NBD1 and R1070 in CL4 (Fig-
ure S6B) (He et al., 2010; Loo et al., 2008, 2010; Thibodeau
et al., 2010).
R1070W, similar to V510D, alone modestly increased the
DF508 CFTR folding efficiency and cellular and PM expression
(Figures 6A–6E), in part confirming previous reports (He et al.,
2010; Thibodeau et al., 2010; Loo et al., 2010). In sharp contrast,
combining an interface mutation with NBD1 stabilization by 3S,
R, or R1S enhanced the DF508 CFTR folding efficiency by 25-
to 30-fold and led to 50%–80% of WT CFTR expression in
both COS7 and BHK cells (Figures 6A–6E and S7A). At the indi-
vidual domain level, the combination of R1S and R1070W, but
not R1S alone, largely restored domain assembly, as indicated
by the WT-like trypsin resistance of the MSD1, MSD2, and
NBD2 in DF508-CFTR-R1S-R1070W (Figure 5C). Similarly, the
DF508 CFTR folding and expression were synergistically
rescued by combining the DF508-NBD1 stabilizing mutation
DRI with R1070W or V510D (Figures 6F–6H and S7B), ruling
out nonspecific effects of second-site mutations. Direct ener-
getic stabilization of the DF508-NBD1-0S and -3S by the
V510D mutation was marginal (Figure S5).
The four-domain minimal folding unit of CFTR (CFTR-1218X)
that lacks the NBD2 mimics the cooperative domain folding
of WT CFTR (Du and Lukacs, 2009). Accordingly, substantially
increased folding efficiency of DF508-CFTR-1218X was only
achieved by synergistic stabilization of the NBD1-CL4 interface
(R1070W or V510D) and NBD1 (3S, -3R, or -R1S) (Figures 7A–
7C, and S7C).
The cooperative domain-folding model predicts that destabi-
lizing NBD1 or the NBD1-CL4 interface would limit the CFTR
folding efficiency as reported for several CF-causing mutations
in the CLs (Riordan, 2008). This was indeed the case. CF-
associated nonconserved amino acid substitutions (A559E or
R600G) that are solvent exposed and distant from the NBD1-
CL4 interface prevented the expression of soluble NBD1 in
E. coli and CFTR processing in mammalian cells (Figure S7D;
data not shown). Likewise, disrupting the NBD1-CL4 interface
by the R1070W mutation compromised both WT CFTR and
CFTR-1218X folding (Figures 6B, 6C, 6E, left panel, and 7C),
as well as the NBD1, NBD2, and MSD1 conformation, which
was partly rescued by stabilizing the NBD1 with 3S, R, or R1S
(Figure S6A). Together, these observations suggest that NBD1-
CL4 association results in mutually stabilized NBDs and MSDs.
This inference is supported by the partially regained protease
(B) PM density of CFTR was measured by cell surface ELISA and plotted as a function of the respective NBD1 Tm. Second-site mutations are indicated in the
background of F508 (C) or DF508 (B). Symbols are as in (A). Red and blue circles depict the combined effect of R1070W or V5010D interface and 3S, R, or R1S
stabilizing mutations on the DF508 CFTR expression. Data are mean ± SEM.
(C) Conformational stability of CFTR domains was determined by limited proteolysis. Isolated microsomes expressing CFTR variant were trypsin digested at 4?C
for 10 min and probed by domain-specific antibodies. Proteolytic fragments containing NBD1, MSD1, MSD2, or NBD2 are indicated by dashed boxes.
See also Figure S6.
Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc. 157
resistance of NBD1, MSD1, and NBD2 (Figure S6A) as well as
restored folding and processing upon NBD1 stabilization by
3S, R, R1S, or DRI in the CFTR-R1070W and CFTR-1218X-
R1070W (Figures 6B, 6C, 6E, and 7C).
Channel Gating in DF508 CFTR Is Corrected by
Interfacial and NBD1-Stabilizing Mutations
Synergistic correction of the DF508 CFTR channel-gating defect
could be observed by the interfacial and NBD1-stabilizing
Figure 6. Stabilization of NBD1 and NBD1-CL4 Interface Synergistically Rescues the DF508 CFTR Folding and Expression Defect
(A and B) The channel folding efficiency was determined by metabolic pulse-chase experiments in BHK cells as described in Figure 4B.
(C and D) Expression of CFTR variants was monitored by immunoblotting in transiently transfected COS7 cells.
(E) PM density of the CFTR-3HA variants was measured by cell surface ELISA.
Data are mean ± SEM. See also Figures S6 and S7.
158 Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc.
Figure 7. Combination of NBD1-Stabilizing and NBD1-CL4 Interface Mutations Rescues the DF508-CFTR-1218X Processing and the DF508
CFTR Channel-Gating Defect
(A–C) Folding efficiency (A), cellular (B), and PM expression (C) of the WT and DF508 CFTR-1218X containing NBD1 stabilizing and interface mutations.
(D and E) Correction of the DF508 CFTR gating defect by the combination of the 3S and R1070W mutations. Single-channel open probability (Po) of PKA-
phosphorylated channels was determined following the reconstitution of CFTR in phospholipid bilayer. The DF508 CFTR Pofold increase is indicated above the
bars. The processing defect of the DF508 CFTR variants was rescued at 26?C prior to microsome isolation. Data are mean ± SEM.
(F) Working model of CFTR domain folding and the effect of second-site suppressor mutations on DF508 CFTR coupled domain misassembly. For DF508 CFTR
the energetic and topological defect of DF508 NBD1 (D) compromises cooperative domain assembly by disrupting NBD1-MSD2, NBD1-MSD1, and MSD1-
MSD2 interfaces (He et al., 2008, 2010; Loo et al., 2008; Wang et al., 2007). The putative folding energy of individual domains is color coded. For DF508+R1S and
DF508+R1S+R1070W, although the DF508 NBD1 thermodynamic destabilization can largely be rescued by second-site suppressor mutations (e.g., R1S, 3S, or
DRI, indicated as D*), this failed to correct DF508 CFTR misassembly probably due to persistently impaired coupled domain folding. WT-like domain assembly
and stabilization of DF508 CFTR were achieved by a combination of NBD1 and NBD1-CL4 interface-stabilizing mutations (e.g., 3S and R1070W or V510D). M2*
and red line depict the R1070W mutation.
Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc. 159
mutation, measured by the open probability of reconstituted and
phosphorylated CFTR in a phospholipid planar bilayer (Fig-
ure 7D). The low temperature rescued DF508 CFTR open prob-
ability (Po= 0.11 ± 0.01, n = 6) was reduced to 30% of the WT
at ?60 mV holding potential. The 3S and R1070W mutations
increased the DF508 CFTR Poby 27% and 54%, respectively.
Combination of the suppressor mutations resulted in a Po
(0.28 ± 0.03, n = 8) that was 154% higher than the control (Fig-
ure 7E), indicating that DF508 CFTR functional rescue requires
the stabilization of both NBD1 energetics and the NBD1-CL4
The objective of this study was to elucidate the role of NBD1
and the structural consequences of the DF508 mutation in CFTR
multidomain assembly. At the isolated domain level, we showed
for the channel conformational maturation and function. The
DF508 impairs both structural prerequisites. In order to achieve
domain assembly and function close to that of the WT protein, it
is, therefore, essential that energetic and interface defects be
corrected simultaneously in the DF508 CFTR.
DF508 Destabilizes the NBD1 Both Thermodynamically
efficiency of CD4T-NBD1-DF508, as well as DF508 CFTR con-
taining 0S or 1S, demonstrate that the 1S mutation marginally
reverts the DF508-NBD1 structural defect and justified the use
of NBD1-1S as a surrogate domain for NBD1-0S. Thermal and
chemical denaturation scans revealed that the DF508 mutation
thermodynamically destabilizes the NBD1-0S and -1S at 37?C
(Figures 1D–1G), in line with recently published data using
variable numbers of second-site mutations (Hoelen et al.,
2010; Protasevich et al., 2010; Wang et al., 2010) but at variance
with earlier findings (Lewis et al., 2005; Qu and Thomas, 1996;
Thibodeau et al., 2005).
The thermodynamics of the NBD1-1S folding profile is pro-
foundly altered by the DF508 mutation. The attenuated (re)
folding kinetics of the DF508 NBD1 (Qu and Thomas, 1996;
Serohijos et al., 2008b; Thibodeau et al., 2005) could be partly
explained by the ?4-fold increased folding activation energy
(DGfz) of the isolated DF508 NBD1-1S at 37?C (Figure 2A). In
addition we discovered that DF508 NBD1-1S has ?10-fold
reduced unfolding activation energy (DGuz) and a ?17-fold faster
unfolding rate relative to its WT counterpart at 37?C (Figures 2A,
2D, and S2G). The combination of DF508 NBD1-1S rapid unfold-
ing (T1/2?1 s), reduced DG0, and impeded folding rate consti-
tutes the complex energetic folding defect and is primarily
responsible for DF508 CFTR (Denning et al., 1992), CD4T-
DF508-NBD1, and SUMO-DF508-NBD1-1S misprocessing, as
well as for the 2- to 10-fold enhanced NBD1 protease suscepti-
bility in DF508 CFTR (Cui et al., 2007; Du et al., 2005; Roy et al.,
2010; Thibodeau et al., 2010). Energetic stabilization of DF508-
NBD1 variants at reduced temperature (Figure 1E) also offers
a reasonable explanation for the native-like crystal structure of
DF508 NBD1-2S and -3S obtained at 4?C–20?C (Lewis et al.,
We demonstrated that multiple R and S mutations restore the
DF508 NBD1 conformational stability at 37?C (Figures 1 and 2)
but modestly increase the folding efficiency (Figure 4B), expres-
sion, and PM function of DF508 CFTR (Pissarra et al., 2008;
Teem et al., 1993). Although some of the R and S mutations
are solvent exposed or far from F508, they can stabilize NBD1
through long-range, intradomain interactions among the three
NBD1 subdomains with high contact order (Khushoo et al.,
2011), accounting for the structural basis of cooperative NBD1
folding and misfolding. Similar effects were observed in the
solvent-exposed W62G mutation of lysozyme that leads to un-
folding and fibril formation (Zhou et al., 2007).
Biophysical characterization also revealed that the WT NBD1-
manifests in the WT SUMO-NBD1and CD4T-NBD1 recognition
bythe protein quality control machinery in bacteria and mamma-
lian cells, respectively, and the domain ubiquitination in vitro.
Although interdomain interactions likely stabilize NBD1 confor-
mationally in WT CFTR, as suggested by the enhanced NBD1
protease susceptibility upon destabilizing the MSD1 or NBD2
by CF-causing point mutation (Cui et al., 2007; Du and Lukacs,
2009; Du et al., 2005; Xiong et al., 1997), or the NBD1-CL4 inter-
face by R1070W (Figure S6A), these stabilizing factors are
compromised due to coupled misfolding of MSD1, MSD2, and
NBD2 in DF508 CFTR and other mutants (Cui et al., 2007; Du
stabilizing NBD1 by second-site mutations is sufficient to
increase WT channel folding efficiency by ?2-fold (Figure S7E).
Stabilization of Both NBD1 Energetics and Domain
Interface Is a Prerequisite for DF508 CFTR Domain
At least three mechanisms may be responsible for multidomain
protein folding: (1) individual domains can fold and function
independently, (2) one or more domains can facilitate the folding
of the other(s) mutually and/or by acting as a folding template,
or (3) the domains(s) can stabilize the folded conformation by
interacting in its native state to prevent unfolding. The first
scenario has been ruled out (Cui et al., 2007; Du and Lukacs,
2009; Han et al., 2007). This work provides support for the
second and third mechanisms by correlating NBD1 energetics
and interface stabilization with CFTR folding efficiency and
(1) Here, we showed that both NBD1 and the NBD1-CL4
interface instability contribute to the intrinsically low-
folding efficiency of the WT CFTR in vivo (Riordan, 2008).
S, R, or DRI mutations increased the WT channel folding
efficiency (by 20%–80%) in proportion with NBD1 stabili-
zation, a tendency that is also observed in the back-
ground of NBD1-CL4 destabilization in R1070W-CFTR
(Figures 4B and 6B). Stabilization of the NBD1-CL4 inter-
face by salt bridge formation between V510D and R1070
also improved WT CFTR biogenesis (Figure 6B).
160 Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc.
Although protease susceptibility and FRET measure-
ments suggest that NBD1 undergoes cotranslational
folding (Du et al., 2005; Khushoo et al., 2011), it is likely
that NBD1 and interface stabilization can facilitate CFTR
folding by favorably influencing the folding thermody-
anism would be consistent with the lack of measurable
difference in the NBD1 protease susceptibility when
comparing WT and WT-3S CFTR (data not shown) and
ER-associated ubiquitination in the presence of corrector
molecules (Grove et al., 2009).
(2) Cysteine-crosslinking studies have demonstrated that
NBD1 S mutations (e.g., 3S) restored the NBD1-CL4
and NBD2-CL2 (MSD1) association (He et al., 2010).
Intriguingly, the proximity of selectively crosslinked inter-
face residues is insufficient to profoundly enhance DF508
CFTR and CFTR-1218X folding, presumably due to tran-
sient interactions of the domains (Figures 4B, 5C, and
6A–6C). Accordingly, pharmacological chaperones (e.g.,
RDR1, a substituted phenylhydrazone) and second-site
suppressor mutations used here (Figure 5A) and else-
where (He et al., 2010; Hoelen et al., 2010; Sampson
et al., 2011) failed to restore DF508 CFTR folding, despite
reverting DF508-NBD1 stability nearly to that of the WT
domain. The combination of NBD1 and NBD1-CL4 inter-
face stabilization, however, led to robust biochemical
and functional rescue, as well as the acquisition of
protease resistance in MSD1, MSD2, and NBD2 (Figures
5,6,and 7E).Thislastobservation suggeststhatstabiliza-
tion of MSD2 and MSD1 by NBD1 interactions is indis-
pensable for the conformational rescue of the NBD2 and
Based on the three-dimensional architecture and cooperative
folding model of CFTR as well as the energetic and topological
defect of the DF508 NBD1, it is reasonable to speculate that
the channel structure destabilization is initiated by the compro-
mised NBD1-CL1 interface (He et al., 2008). This view is consis-
tent with the increased metabolic turnover of the MSD1-NBD1-R
caused by DF508 (Rosser et al., 2008) (Figure 7F). MSD2 trans-
lation further exacerbates the tertiary structural defect due to the
disruption of the NBD1-CL4 hydrophobic cluster (He et al., 2010)
and consequential destabilization of the MSD2 and MSD1-
MSD2 packing (Cui et al., 2007; Du and Lukacs, 2009; Wang
et al., 2007). This in turn compromises the NBD2-CL2/CL3 and
NBD1-NBD2 interfaces, culminating in the global misfolding of
NBD2 and DF508 CFTR (Cui et al., 2007; Du et al., 2005) (Fig-
ure 7F). This multistep misfolding can be modulated by cytosolic
chaperones (Qu et al., 1997; Rosser et al., 2008; Wang et al.,
2006) and by strategically placed second-site suppressor muta-
tions that may also be mimicked by small molecule correctors.
Implications for Correcting CF and Other
Conformational Diseases Caused by Multidomain
Membrane Protein Misfolding
Conformational stabilization of mutant polypeptides with ener-
getically compromised but partially preserved ligand-binding
capacity has been achieved by pharmacological chaperones
to restore their biosynthetic processing (Bernier et al., 2004).
The combination of impaired energetics and domain interactions
rescue efficiency (<15%) of DF508 CFTR by individual corrector
molecules isolated in HTSs of chemical libraries (Clancy et al.,
2011; Van Goor et al., 2011; Wang et al., 2007). Our results
suggest a rational, structure-based screening strategy to isolate
combinations of corrector molecules that may preferentially
stabilize the mutant NBD1 and the NBD1-CL4 interface.
At a more general level, our results highlight the potential
consequence of destabilizing interface mutations in coopera-
tively folding multidomain membrane proteins and demonstrate
that folding of neighboring domains can be compromised by
interface mutations similar to a subset of multidomain soluble
polypeptides (Han et al., 2007). Extension of this paradigm to
multidomain PM proteins may serve as a framework in future
studies of folding diseases caused by mutations clustered at
domain interfaces, as in the case of the shaker CLC and
ABCC6 transporter (Feng et al., 2010; Fu ¨lo ¨p et al., 2009).
Cloning, Expression, and Purification of CFTR and NBD1 Proteins
Point mutations were introduced into WT and DF508 CFTR-3HA cDNA by
overlapping PCR mutagenesis as described before (Du et al., 2005). CD4T-
NBD1 expression constructs were generated by inserting PCR-amplified
NBD1 cDNAs into the XmaI/ApaI sites of pcDNA3-CD4T (Du and Lukacs,
The pET26b-derived expression vectors with N-terminal His6-SUMO
fusion tag containing the human WT or DF508 NBD1-3S and -R4S with the
crystallization domain boundaries were kindly provided by H. Lewis(Structural
GenomiX and CFFT) and C. Lima (Columbia University) and purified as
described (Lewis et al., 2005).
Circular Dichroism and Fluorescence Spectroscopy
Circular dichroism (CD) spectroscopy was performed using a Chirascan CD
spectrometer (Applied Photophysics, Leatherhead, UK). CD scans were
collected on proteins in 150 mM NaCl, 1 mM ATP, 3 mM MgCl2, and 50 mM
sodium phosphate buffer at pH 7.5 between 260 and 190 nm at 20?C or
25?C using a 0.2 mm path-length cuvette at ?14 mM NBD1 concentration.
CD measurements were acquired every 0.5 nm with 0.5 s as an integration
time and repeated three times with baseline correction. Thermal unfolding
scans were measured at 208 nm with a 0.5?C step size at 1?C/min ramp
rate with ± 0.1?C tolerance.
Tryptophan fluorescence of NBD1 (12 mM in a 1 mM ATP, 3 mM MgCl2,
150 mM NaCl, and 10 mM HEPES at pH 7.5) during thermal unfolding was
monitored using a Cary Eclipse spectrofluorometer (Varian, Palo Alto, CA,
USA) with a 3 3 3 mm micro-quartz cuvette. Fluorescence emission was
monitored at 340/350 nm at 290 nm excitation wavelengths at 300 nm/min
scan rate. The data were fitted to a Boltzmann sigmoid function to calculate
the Tmusing the Excel add-on package XLfit (IDBS, Bridgewater, NJ, USA)
as described (Senisterra et al., 2008).
DSF of NBD1 (7–12 mM) scans was obtained routinely in 150 mM NaCl, 20 mM
MgCl2, 10 mM HEPES, and 2.5 mM ATP at pH 7.5 in the presence of 23 Sypro
Orange or Nano Orange, using a Stratagene Mx3005p (Agilent Technologies,
La Jolla, CA, USA) qPCR instrument (Lo et al., 2004; Niesen et al., 2007). The
temperature ramp rate was 1?C/min. The data were fitted to a Boltzmann
Additional methodologies are included in the Extended Experimental
Cell 148, 150–163, January 20, 2012 ª2012 Elsevier Inc. 161
Supplemental Information includes Extended Results, Extended Experimental
Procedures, seven figures, and one table and can be found with this article
online at doi:10.1016/j.cell.2011.11.024.
We thank H. Lewis, C. Lima, J.R. Riordan, J. Young, E. Buck, and the Cystic
Fibrosis Foundation Therapeutics, Inc. for valuable reagents and feedbacks,
J. Young and D. Thomas for providing access to the Chirascan and qPCR
instruments, and N. Kartner, G. Veit, J. Szapor, D. Scicchitano, and F. Pianofor
forhelpful comments onthemanuscript. WethanktheCysticFibrosisFounda-
tion Therapeutics Inc, Cystic Fibrosis Canada, NIH-NIDDK (grant number
DK075302), Canadian Institute of Health Research, and the Canadian Founda-
tion for Innovation for operating and infrastructure support. W.M.R. was
partially supported by GRASP and a Canadian Cystic Fibrosis Foundation
postdoctoral fellowship. L.K. and G.L.L. are holders of Canada Research
Received: June 27, 2011
Revised: October 25, 2011
Accepted: November 3, 2011
Published: January 19, 2012
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