Deletion of Phe508 in the first nucleotide-binding domain
of the cystic fibrosis transmembrane conductance
regulator increases its affinity for the heat shock cognate
70 chaperone
Toby S. Scott-Ward
1,2
and Margarida D. Amaral
1,2
1 Universidade de Lisboa, Faculdade de Cie
ˆ
ncias de Lisboa, BioFIG, Centre for Biodiversity, Functional and integrative Genomics, Portugal
2 Centro de Gene
´
tica Humana, Instituto Nacional de Sau
´
de Dr. Ricardo Jorge, Lisboa, Portugal
Keywords
CFTR-interacting proteins; correctors;
mechanism of disease; small molecules;
surface plasmon resonance
Correspondence
M. D. Amaral, EMBL – European Molecular
Biology Laboratory, Meyerhofstrasse 1,
69117 Heidelberg, Germany
Fax: +49 6221 387 8306
Tel: +49 6221 387 8199
E-mail:
(Received 3 August 2009, revised 29
September 2009, accepted 1 October
2009)
doi:10.1111/j.1742-4658.2009.07421.x

action of small corrective molecules, demonstrating its potential to validate
additional therapeutic compounds for CF.
Structured digital abstract
l
MINT-7265886: mNBD1 (uniprotkb:P26361) binds (MI:0407)toHsc70 (uniprotkb:P19120)
by anti bait coimmunoprecipitation (
MI:0006)
Abbreviations
Ab, antibody; C4a, corrector 4a; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum;
ERQC, endoplasmic reticulum quality control; F508del, deletion of phenylalanine residue at position 508; h, human; Hsc70, heat shock
cognate 70; Hsp70, heat shock protein 70; I172, inhibitor CFTR
inh-172
; LA, apo-a-lactalbumin; m, murine; NBD1, first nucleotide-binding
domain; Red-LA, reduced apo-a-lactalbumin; SEM, standard error of the mean; SPR, surface plasmon resonance; V325, corrector VRT-325;
wt, wild-type.
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7097
Introduction
Cystic fibrosis (CF) is a life-threatening genetic disease
caused by malfunction of CF transmembrane conduc-
tance regulator (CFTR) [1], a Cl
)
channel that plays a
central role in transepithelial ion transport [2]. A single
amino acid deletion, of phenylalanine 508 (F508del), in
the first nucleotide-binding domain (NBD1) of CFTR
accounts for approximately 70% of CF chromosomes
worldwide [2]. This mutation prevents the correct fold-
ing of CFTR, thus causing its retention by the endo-
plasmic reticulum (ER) quality control (ERQC) as an
immature intermediate that is rapidly degraded by the

certainty (99%). Biochemical analyses of purified
NBD1 indicate that F508del reduces domain stability,
and hence promotes aggregation [14,15]. Molecular
dynamics modelling studies suggest that F508del-
NBD1 has more conformational freedom than the
wild-type (wt), thus exposing its hydrophobic interior
to the solution and impairing its interdomain contacts
[16,17]. Collectively, these data implicate NBD1 as the
most probable site for the interaction of CFTR with
Hsc70.
Several small molecules that improve CFTR folding,
biogenesis and function were recently identified in
high-throughput screens [18], such as corrector 4a
(C4a) and corrector VRT-325 (V325), which promote
trafficking of F508del-CFTR to the plasma membrane
[19,20]. However, the exact mechanism of action of
these compounds and their putative binding sites on
CFTR remain undefined.
Here, we used a novel approach, surface plasmon
resonance (SPR) [21], to quantify the interaction occur-
ring between F508del-CFTR and Hsc70, versus that of
the chaperone with wt-NBD1. Our data show that
F508del-mNBD1 binds Hsc70 with approximately five-
fold higher affinity than wt-mNBD1, and that both
ATP and ADP dramatically reduce NBD1–Hsc70
binding. Moreover, we also show that, in the presence
of a small molecule known to rescue the traffic of full-
length F508del-mCFTR to the plasma membrane, the
strength of the F508del-mNBD1–Hsc70 interaction is
reduced by $ 30%.

(right lane, both panels). The data in Fig. 1B show
that purified Hsc70 is present in immunoprecipitated
complexes of either wt-mNBD1 (lanes 2, 3 and 5) or
F508del-mNBD1 (lane 4) incubated in SPR flow
buffer. Moreover, the absence of Hsc70 in immunopre-
cipitates when BSA (Fig. 1B, lane 1) was used instead
of NBD1, or in the absence of L12B4 (lane 7), demon-
strates that the binding of Hsc70 does not occur with
all protein substrates. Increasing the concentration of
wt-hNBD1 by five-fold (Fig. 1B, lane 3) did not
noticeably increase the amount of NBD1 or Hsc70
recovered (compare with lane 2), suggesting that the
L12B4-coated beads were already saturated with
NBD1. Addition of MgATP (2 mm) markedly reduced
the binding of Hsc70 to wt-mNBD1 (Fig. 1B, lane 6),
consistent with the results of previous studies with
other Hsc70 substrates [8].
Analysis of stability of NBD1 under the SPR
interaction conditions
We used intrinsic tryptophan fluorescence spectroscopy
to assess the structure and stability of NBD1 under
assay conditions that would subsequently be used in
SPR interaction studies. A comparison of emission
spectra obtained before (0 min) and after (10 min)
incubation (Fig. 2A) reveals that there was only a
small decrease in the overall intensity of the fluores-
cence emitted from F508del-mNBD1 and no shift in
the peak wavelength. Furthermore, the intensity of
fluorescence emitted at 328 nm and 343 nm from
diluted F508del-mNBD1 (Fig. 2B) displayed a minor

Fluorescence (units)
343 nm
328 nm
F508del-mF508del-m
343
328
A
B
Fig. 2. Spectroscopic analyses show that NBD1 is stable under the
conditions used in SPR studies. (A) Fluorescence emission spectra
(300–400 nm) of F508del-mNBD1 (1 l
M) before (0 min) and after
(10 min) incubation in buffer at 25 °C (excitation wavelength of
295 nm). (B) Change in intrinsic tryptophan fluorescence emitted at
328 and 343 nm from F508del-mNBD1 (1 l
M) during incubation in
buffer (25 °C). Data are corrected for buffer fluorescence. Similar
results were obtained with other NBD1 preparations (F508del-
mNBD1, n = 3; wt-mNBD1, not shown, n = 2).
kDa P WB
47
83
25
16
Hsc70
37
50
75
25
kDa PWB

1
––
–
–
BSA (µ
M)
33 3 3 3 3
––
NBD1
A
B
Fig. 1. In vitro interaction of NBD1 with Hsc70 confirmed by immu-
noprecipitation (IP) under conditions used in SPR studies.
(A) SDS ⁄ PAGE (P) and western blot (WB) analyses of purified
F508del-mNBD1 (F508del-NBD1; 5 lg) and bovine Hsc70 (1 lg)
with L12B4 and SPA-815, respectively, to confirm their specificity
(see Experimental procedures). (B) In vitro analysis of the inter-
action of Hsc70 with NBD1 by immunoprecipitation under condi-
tions equivalent to those used for SPR. Protein G beads coated
with L12B4–NBD1 complexes were incubated with purified Hsc70
(1 l
M; 10 min at 25 °C) and analysed by western blot using either
SPA-815 or L12B4 (see Experimental procedures). Lanes: 1, BSA
(3 l
M; without NBD1); 2, wt-hNBD1 (1 lM); 3, wt-hNBD1 (5 lM); 4,
F508del-mNBD1 (1 l
M); 5, wt-mNBD1; 6, wt-mNBD1 with MgATP
(2 m
M); 7, wt-hNBD1 with beads (without L12B4); 8, L12B4-coated
beads only (without NBD1 and Hsc70). Similar results were

)1
; n = 3), with mini-
mal adsorption of Hsc70 onto BSA-coated surfaces
(< 0.2 pmolÆnmol
)1
). Hence, under these conditions,
the magnitude of Hsc70 binding to wt-mNBD1 was
substantially (approximately six-fold) lower than that
observed for L12B4.
Then, we tested the reverse situation by immobiliz-
ing Hsc70 on CM5 sensor chips (on-chip Hsc70), to
investigate its ability to bind folded or unfolded pro-
teins. The immobilized chaperone was found to
potently bind reduced apo-a-lactalbumin (Red-LA)
(Fig. 3C; 10 lm; 29.1 ± 3.8 pmolÆnmol
)1
; n = 3), an
Hsc70 substrate [13], whereas minimal binding was
detected with the nonreduced, folded form of the pro-
tein (LA) (10 lm; 1.1 ± 0.7 pmolÆnmol
)1
; n = 3). As
expected, virtually no binding of BSA (15 lm)to
Hsc70-coated surfaces could be detected (< 0.1 pmo-
lÆnmol
)1
; n = 20) under these conditions. These data
confirm that Hsc70 binding detected by SPR was only
significantly detected for unfolded protein substrates.
Interestingly, when Red-LA was applied in the pres-

–1
)
BSA
wt-h
wt-m
F508del-m
A
Binding (pmol·nmol
–1
)
BSA
wt-h
Applied: L12B4 Ab
Applied: Hsc70
0 500 1000
0
20
40
0 500 1000
0
20
40
D
BSA
Binding (pmol·nmol
–1
)
F508del-m
wt-h
wt-m

displayed minimal interaction with on-chip
BSA [
, (A) and (B)]. (C, D) The interaction
of applied Red-LA or LA (10 l
M) (C) and
wt-hNBD1, wt-mMBD1 or F508del-mNBD1
(0.5 l
M) (D) with on-chip Hsc70 (On-Chip
Hsc70; see Experimental procedures). BSA
(15 l
M) showed minimal interaction with
on-chip Hsc70 [–, (C) and (D), n = 15].
Periods of protein application (and associa-
tion) are indicated by the solid bars. After
protein application, dissociation was
measured by injecting flow buffer over the
protein-coated surface. Similar results were
obtained in additional experiments (n =3or
n = 4).
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7100 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS
(100 lm) dramatically reduced the binding of wt-
mNBD1 to Hsc70 (Fig. 1A; 3.7 ± 1.3 pmolÆnmol
)1
;
n = 3). These data show that we can use SPR to accu-
rately measure the specific binding of NBD1 to immobi-
lized Hsc70. The substantially higher binding of NBD1
to immobilized Hsc70 over that of Hsc70 chaperone
binding to immobilised NBD1 can be attributed to the

F508del-mNBD1 bound Hsc70 with a comparable
association rate, but had a five-fold lower dissociation
rate [k
a
, 4030 ± 655 m
)1
Æs
)1
; k
d
, (1.0 ± 0.2) ·
10
)5
s
)1
; n = 3). Using these constants, we calculated
that Hsc70 bound F508del-mNBD1 with five-fold
higher affinity than wt-mNBD1 [dissociation constant
(K
D
): F508del-mNBD1, 2.6 ± 0.5 nm; wt-mNBD1,
13.9 ± 0.8 nm; n = 3]. These data indicate that when
Phe508 is deleted, there is a significant increase in the
real-time affinity of mNBD1 for Hsc70 (P < 0.01).
Analysis of the dose–response data (Fig. 4D) revealed
that the maximum amount of F508del-mNBD1 bound
to Hsc70 at s aturating concentration appears to be lower
than that of wt-mNBD1 (B
max
app

25
50
75
100
0
50
100
0 1000 2000 0 1000 2000
0
50
100
Time (s)
Binding (pmol·nmol
–1
)
NBD1 (µM)
Binding (pmol·nmol
–1
)Binding (pmol·nmol
–1
)
Time (s)
[NBD1] (µ
M)
wt-m
F508del-m
0.02
0.1
0.2
0.05

M) shown on a
highly expanded scale. Binding was
normalized independently for each dissocia-
tion curve to an initial maximum value (100)
at the start of each dissociation phase
(920 s). First-order fits (
) to the data
denote relative dissociation rates. (D) The
change in amount of NBD1 bound by
on-chip Hsc70 at equilibrium with increasing
concentrations of wt-mNBD1 (d) and
F508del-mNBD1 (s; mean ± SEM; n = 3).
Other details as in legend to Fig. 3.
T. S. Scott-Ward and M. D. Amaral Interaction of Hsc70 with F508del-NBD1
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7101
wt-mNBD1 and F508del-mNBD1 (0.5 lm) to Hsc70.
In contrast, the introduction of a control molecule,
NADP (500 lm), did not significantly reduce the bind-
ing of either wt-mNBD1 or F508del-mNBD1 (0.5 lm)
to immobilized Hsc70 (Fig. 5C; wt-mNBD1, P = 0.45;
F508del-mNBD1, P = 0.46; n = 3). Interestingly, the
binding of wt-mNBD1 and F508del-mNBD1 to Hsc70
was also reduced by the addition of ADP (500 lm),
although, at this concentration, the degree of inhibi-
tion was less than for ATP (P = 0.01; n = 3). Fig-
ure 5C, displaying a comparison of the summarized
data for wt-mNBD1 and F508del-mNBD1, shows that
the interaction of wt-mNBD1 with Hsc70 was most
potently inhibited by increasing ATP concentration
(IC

25
50
0
500 1000
0
25
50
[Compound] (µ
M)
Time (s)
Binding (pmol·nmol
–1
)
C4a
Con
Binding (pmol·nmol
–1
)
Binding (pmol·nmol
–1
)
I172
Incubation
Incubation
+ ATP
C4a
V325
I172
*
*

23
203
Binding (pmol·nmol
–1
)
Binding (pmol·nmol
–1
)
Binding (pmol·nmol
–1
)
Time (s) Time (s) [Adenine nucleotide] (µM)
wt-m
F508del-m
3
ATP (µ
M)
53
503
103
23
203
3
53
wt-m F508del-m
ATP
ADP
NADP
ABC
Fig. 5. Adenine nucleotides reduce the binding of NBD1 to on-chip Hsc70. The interaction of (A) wt-mNBD1 (0.5 lM) and (B) F508del-

Fig. 6B indicates that the magnitude of inhibition
($ 30%) was comparable to that observed with acute
compound application. However, following preincuba-
tion of NBD1 with C4a or V325 at increased MgATP
concentration (106 lm), we observed no effect of these
corrector compounds on the NBD1–Hsc70 interaction
(Fig. 6C; P = 0.74–0.95; n = 3).
Discussion
Various components of the ERQC recognize aberrant
conformations of secretory proteins and target them
for proteasomal degradation so as to avoid clogging of
the secretory pathway. In the case of the CFTR protein
bearing F508del, the major CF-causing mutation, it has
been shown that the molecular chaperone Hsc70 plays
a major role in this disposal mechanism [11,12]. In the
present study, we have used an SPR approach to inves-
tigate how deleting F508 from NBD1 of mCFTR alters
the interaction of the domain with the molecular chap-
erone Hsc70, and whether small molecule correctors of
CFTR folding can affect this critical interaction.
Both wt-NBD1 and F508del-NBD1 bind Hsc70
Our data indicate that Hsc70 can interact with both
wt-NBD1 and F508del-NBD1, in agreement with pre-
vious studies [12] showing that both wt-CFTR and
F508del-CFTR can associate with this chaperone via
NBD1. The strength of NBD1–Hsc70 binding determined
here by SPR is substantially higher (low nanomolar K
D
)
than the majority of previous non-SPR assessments of

NBD1 to annexin A5, and that deleting Phe508 had
no effect on the annexin A5–NBD1 interaction. Inter-
estingly, they demonstrated that CPX, a potential
NBD1 small molecule ligand [33], inhibited binding.
Overall, the data suggest that Hsc70 interacts with
NBD1 at alternative sites to annexin A5 and with
different characteristics.
The enhanced binding of Hsc70 to wt-hNBD1 rela-
tive to wt-mNBD1 that we find here may be due to
variations in amino acids between these forms of
NBD1 that help to stabilize this domain against inter-
action with Hsc70. Interestingly, when some of the
variant residues in NBD1 of F508del-mCFTR are
substituted for residues at corresponding positions in
F508del-hCFTR, they act as revertants of the folding
and trafficking defects [14]. This is the case for Thr539
(Ile539 in humans), a so-called revertant of F508del-
hCFTR [34], and also Ser429 (Phe429 in humans),
recently shown to contribute to rescue of the traffick-
ing defect of F508del-hCFTR [35]. The presence of
these ‘profolding’ residues in mNBD1 is a probable
explanation for the recently reported attenuated
processing ⁄ trafficking defect of F508del-mCFTR in
comparison with F508del-hCFTR [36].
F508del increases the affinity of CFTR NBD1 for
Hsc70
Our data demonstrate that deleting Phe508 from
mNBD1 increases five-fold the affinity of the domain
for the Hsc70 chaperone. However, our data also indi-
cate that Hsc70 binds $ 30% more wt-mNBD1 than

association of Hsc70 with F508del-CFTR relative to
that with wt-CFTR [11], which appears to constitute
the first ERQC checkpoint occurring in vivo [9,12].
Our data reported here thus suggest that the absence
of Phe508 increases the accessibility of Hsc70 to one
or more binding sites on NBD1.
Binding sites on NBD1 promoting Hsc70
interaction
A critical issue in this field is the location of the func-
tionally important Hsc70-binding site(s) on NBD1; in
particular, whether removal of Phe508 creates a novel
Hsc70-binding site in NBD1. Although it is known
that Hsc70 and Hsp70 bind short, hydrophobic peptide
pockets exposed on substrate proteins, the exact pri-
mary sequence of these peptides is variable [6,30].
Accordingly, the NBD1 proteins employed in this
study (Thr389–Gly673) contain many of these short,
hydrophobic sequences, including the region around
Phe508, constituting potential Hsc70-binding sites.
Analysis of hNBD1 and mNBD1 sequences with the
limbo program, which predicts likely binding sites of
the Hsp70 ⁄ Hsc70 homologue, DnaK, identified three
novel regions (Ser466–Leu472, Leu568–Pro574, and
Asp614–Gln621), all three of which are distant from
Phe508. Qu and Thomas [14] localized a putative
Hsc70-binding site to Gly545–Ala561 [13], a hydropho-
bic pocket that is partially exposed in the crystal struc-
ture of NBD1 and that is also distant from Phe508.
However, the residue limits of the NBD1 used in this
study (Gly404–Ser589) were different from those used

tion–dissociation profile, suggesting that it did not
alter the kinetics of the interaction. Second, ADP,
rather than enhancing NBD1–Hsc70 binding, as pre-
dicted for an Hsc70-mediated effect, substantially
reduced binding. Third, the IC
50
values for ATP inhi-
bition of wt-mNBD1 and F508del-mNBD1 binding to
Hsc70 ($ 20 and $ 110 lm, respectively, this study)
are comparable to the previously reported apparent
dissociation constants for ATP and wt-hNBD1 and
F508del-hNBD1 ($ 90 lm [42]) but substantially
higher than the dissociation constant for ATP and
Hsc70, which is in the order of $ 0.7 lm [43]. Finally,
ATP caused a reduction of only $ 20% in the Hsc70
binding of denatured lactalbumin, itself not predicted
to interact with ATP, whereas Hsc70 binding to
wt-mNBD1 was reduced by $ 80%. Our data also
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7104 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS
show that the ATP concentration required to inhibit
the mNBD1–Hsc70 interaction is dramatically
increased by deleting Phe508. Less optimal ATP bind-
ing to F508del-NBD1 would be consistent with the
gating defect of F508del-CFTR, characterized by long
interburst intervals [44], according to the current
model of CFTR channel gating [45]. However, regard-
ing the isolated domain, ATP binds wt-NBD1 and
F508del-NBD1 with equivalent affinity, arguing
against such an effect. However, it remains plausible

plausible explanation, nevertheless, is that C4a binds
directly to a site on monomeric NBD1 that stabilizes
homodimerization, similarly to the ATP effect (see
above). This could likewise occlude the Hsc70-binding
site(s) and thus reduce Hsc70 binding. Strikingly, we
observed a significantly reduced effect of C4a at high
ATP concentrations.
Our data demonstrate that SPR provides a powerful
approach to quantifying the Hsc70–NBD1 interaction
and the impact of F508del (or other NBD1 mutations)
and corrector compounds on this folding-sensitive
association. In particular, our data show that ATP dra-
matically reduces the ability of mNBD1 to bind Hsc70,
and indicate that it is mostly this ability of ATP to
displace Hsc70 from NBD1 binding that is impaired by
F508del. C4a significantly inhibits the F508del-mNBD1
interaction with Hsp70, suggesting direct binding. As
this effect is abolished at high ATP concentrations, ATP
and C4a may compete for the same F508del-mNBD1
binding site ⁄ surface. We conclude that this SPR
approach constitutes a useful assay for the determina-
tion of whether and how correctors affect the Hsc70–
F508del-NBD1 interaction, which will improve our
understanding of the mechanism of action of small
molecules with therapeutic potential for CF, a critical
step in bringing them to the clinical setting. Future stud-
ies should focus on the quantification of the effect of
these correctors on the NBD1–Hsc70 interaction in the
presence of other relevant CFTR domains (e.g. intra-
cellular cytoplasmic loop 4 and ⁄ or nucleotide-binding

materials were obtained from GE Healthcare (Milwaukee,
WI, USA). All other chemicals, proteins (BSA and bovine
apo-a-lactalbumin) and reagents were purchased from
Sigma Aldrich (St Louis, MO, USA) or BDH (Poole, UK),
and were of research grade or higher (‡ 99% purity).
T. S. Scott-Ward and M. D. Amaral Interaction of Hsc70 with F508del-NBD1
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7105
Biochemical analysis of purified NBD1 and Hsc70
Coimmunoprecipitation of purified NBD1 (1 lm, unless
otherwise stated) and Hsc70 (1 lm) in SPR flow buffer
[150 mm KCl, 2 mm MgCl
2
, 0.1% (v ⁄ v) Triton X-100,
0.1% (v ⁄ v) dimethylsulfoxide, 1 mm b-mercaptoethanol,
20 mm Hepes, pH 7.0] was performed using L12B4, as pre-
viously described [39]. SDS ⁄ PAGE and western blot using
SPA-815 (Hsc70) and L12B4 (NBD1) were also performed
as previously described [39]. Protein quantification was
performed with a modified Lowry method.
Spectroscopic analysis of NBD1
Wild-type and F508del-mNBD1 (1 lm) were diluted in
NaCl ⁄ P
i
(150 mm NaCl, 20 mm Na
2
PO
4
,1mm b-mercap-
toethanol, pH 7.4) and, following excitation at 295 nm, the
emitted intrinsic tryptophan fluorescence was measured at

immediately.
The effect of small molecules (C4a, V325, I172; 10 mm,
100% dimethylsulfoxide) was determined by diluting these
compounds to different concentrations (as indicated in
the figure legends) in flow buffer containing BSA
(0.2 mgÆmL
)1
), and applying them either immediately (acute
effect) or after 30 min of incubation at 16 °C (incubated
effect). The final dimethylsulfoxide concentration was
adjusted to 0.5%. Protein-coated CM5 chips were used for
2 weeks, or until nonspecific binding increased (‡ 5%). All
experiments were performed in parallel with an inactivated,
or blank, flow cell not coated with protein.
Data analysis
All SPR sensograms were corrected for buffer-induced
refractive index changes at an uncoated reference surface,
analysed using biaevaluation software (biaeval; v. 3.2;
GE Healthcare), and displayed in sigmaplot (v. 10; Systat,
San Jose, CA, USA) as pmoles of interacting protein (e.g.
NBD1) bound per nmole of immobilized protein (e.g.
Hsc70). Molar concentrations of the proteins were calcu-
lated from their measured concentration (mgÆmL
)1
), using
their molecular masses as determined from amino acid com-
position (wt-NBD1, 31 976 Da; F508del-NBD1, 31 829 Da;
wt-hNBD1, 31 969 Da; bovine Hsc70, 71 241 Da).
The kinetics of interaction (association, k
a

pmolÆnmol
)1
) was determined from kinetic analysis and
plotted against [mNBD1] (sigmaplot). The [MgATP]
required to inhibit binding by 50% [IC
50
, lm; Eqn (2)] was
determined by plotting the amount of mNBD1 bound at
320 s against [MgATP]
B
eq
¼
B
max
app
½NBD1
K
D
app
þ½NBD1
ð1Þ
B ¼ B
min
þ
B
max
À B
min
1 þ 10
ðIC

value for each [NBD1]; (b) these
values were averaged; and (c) the averaged values were
used to calculate a single K
D
(k
d
⁄ k
a
). The mean K
D
was
then determined by averaging K
D
values from repeat dose–
response experiments (n = 3). For mean K
D
app
and B
max
app
values, the K
D
app
and B
max
app
were determined directly
for each dose–response dataset as described, and the
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7106 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS

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