effects in fusion proteins containing an intrinsicallydisordered and a globular protein Ilaria Sambi1, Pietro Gatti-Lafranconi1*, Sonia Longhi2and Marina Lotti1 1 Dipartimento di Biotecn
Trang 1effects in fusion proteins containing an intrinsically
disordered and a globular protein
Ilaria Sambi1, Pietro Gatti-Lafranconi1*, Sonia Longhi2and Marina Lotti1
1 Dipartimento di Biotecnologie e Bioscienze, Universita` di Milano-Bicocca, Italy
2 Architecture et Fonction des Macromole´cules Biologiques, Universite´ Aix-Marseille I et II, France
Introduction
Until very recently, one of the pillars of protein science
has been the so-called structure–function paradigm,
which posits the formation of a unique 3D structure as
the prerequisite for biological function [1] However,
during the last decade, numerous proteins have been
described that fail to adopt a stable tertiary structure
under physiological conditions and yet display biologi-cal activity [2] This condition, defined as intrinsic disorder, has been found to be widespread in func-tional proteins Importantly, disordered regions are often required for biological activity, indicating that the lack of stable secondary and tertiary structure is a
Keywords
conformation; fusion proteins; intrinsically
disordered proteins; stability; viral proteins
Correspondence
M Lotti, Dipartimento di Biotecnologie e
Bioscienze, Universita` di Milano-Bicocca,
Piazza della Scienza 2, 20126 Milano, Italy
Fax: +3902 6448 3569
Tel: +3902 6448 3527
E-mail: marina.lotti@unimib.it or
S Longhi, Architecture et Fonction des
Macromolecules Biologiques (AFMB), UMR
6098 CNRS et Universite´s d’Aix-Marseille I
et II, 163, Avenue de Luminy, Case 932,
13288 Marseille, Cedex 09, France
Fax: +33 (0) 4 91 26 67 20
Tel: +33 (0) 4 91 82 55 80
E-mail: sonia.longhi@afmb.univ-mrs.fr
*Present address
Biochemistry Department, University of
Cambridge, UK
(Received 30 June 2010, revised 10 August
2010, accepted 23 August 2010)
doi:10.1111/j.1742-4658.2010.07825.x
Intrinsically disordered proteins (IDPs) are functional proteins either fully
or partly lacking stable secondary and tertiary structure under physiologi-cal conditions that are involved in important biologiphysiologi-cal functions, such as regulation and signalling in eukaryotes, prokaryotes and viruses The func-tion of many IDPs relies upon interacfunc-tions with partner proteins, often accompanied by conformational changes and disorder-to-order transitions
in the unstructured partner To investigate how disordered and ordered regions interact when fused to one to another within the same protein, we covalently linked the green fluorescent protein to three different, well char-acterized IDPs and analyzed the conformational properties of the fusion proteins using various biochemical and biophysical approaches We observed that the overall structure, compactness and stability of the chime-ric proteins all differ from what could have been anticipated from the structural features of their isolated components and that they vary as a function of the fused IDP
Abbreviations
GFP, green fluorescent protein; IDP, intrinsically disordered protein.
Trang 2resource rather than a defect According to this novel
perspective, the straightforward quest for structural
features engaged in a function is moving towards a
dynamic view in which function arises from
conforma-tional freedom Fully or partly nonstructured proteins
are generally referred to as intrinsically disordered
(IDPs) or intrinsically unstructured proteins, or also as
natively unfolded proteins, a term that emphasizes the
fact that, to fulfil their tasks within the cell, these
poly-peptides rely on existing as a dynamic ensemble of
dif-ferent conformations [3–5] The intrinsic flexibility of
IDPs indeed provides a clue with respect to their broad
biological functions and high occurrence among
pro-teins with signalling and regulatory roles [4–6]
Consis-tent with their central position in biological networks,
many disordered proteins are tightly regulated through
the control of their synthesis and degradation and by
post-translational modifications (e.g phosphorylation)
[7] Because of its functional relevance, disorder is
widespread in nature, as shown by computational
anal-yses at the genomic level, which indicate that more
than half of all eukaryotic proteins contain
unstruc-tured regions (> 50 residues) and 25–30% of them are
mostly disordered [8] According to their pivotal role
in signalling and regulation, IDPs are involved in
sev-eral different pathologies [9], such as cancer [10],
as well as cardiovascular [11] and neurodegenerative
diseases [9,12]
Unstructured protein regions often undergo
disor-der-to-order transitions upon interaction with their
partners, as well as upon post-translational
modifica-tions [13,14] Although the structural effects of
inter-molecular associations have been thoroughly
investigated [15–17], the mutual influence that ordered
and disordered regions exert on each other when they
are embedded in the same protein has received less
attention This is the case for proteins in which more
compact regions co-exist with fully or mostly unfolded
ones, such as, for example, in KNR4, a 505 residue
yeast protein involved in the coordination of cell wall
synthesis and cell growth [18], the nucleoprotein and
phosphoprotein from measles and Sendai viruses
[19,20] and the Rhabdoviridae phosphoprotein [21]
Although the isolated disordered domains of these
lat-ter proteins have been studied in depth, comprehensive
data on the full-length polypeptides are lacking, with
the data available so far only suggesting that
unstruc-tured regions maintain this feature in the context of
the entire proteins [19–21] However, evidence that
dis-ordered regions may impact on linked globular
domains arises from work performed in a different
context In particular, studies by Bae et al [22] focused
on the prediction of rotational tumbling times of
proteins containing disordered segments, and high-lighted the effects of the unordered regions on the properties of covalently linked globular domains (in this case on the tumbling of the rigid part), with the extent of the perturbation being proportional to the length of the disordered region
With the aim of investigating the reciprocal confor-mational effect of covalently linked structured and unstructured protein regions, we fused green fluores-cent protein (GFP) with disordered fragments of dif-ferent origin and compactness and investigated the properties of these fusion proteins using biochemical and biophysical methods GFP is a globular protein with a stable fold and known 3D structure [23] Its flu-orophore provides a specific marker to monitor struc-tural changes in GFP only As disordered moieties, we used the unstructured regions of two measles virus proteins (NTAIL and PNT) and the whole Saccharo-myces cerevisiae SIC1 protein Although they are all IDPs, these proteins have different structural features and a different extent of disorder NTAIL is the C-ter-minal domain (residues 401–525) of the viral nucleo-protein that is exposed at the surface of the nucleocapsid [24] Disorder confers a high structural plasticity to NTAIL, thus allowing the establishment
of interactions with various partners [25–29] PNT is the unstructured N-terminal region of the P protein of the viral RNA polymerase complex [30,31] SIC1 is a
284 residue inhibitor of the cyclin-dependent yeast pro-tein kinase whose conformation in isolation has been described recently [32,33]
We report that differences in the intrinsic properties
of the IDP (length, a-helix propensity, compactness) result in fusions with different conformational proper-ties that are not accounted for by the features of their components in isolation
Results and Discussion
Fusion proteins are produced in soluble form though at levels lower than the constituent proteins
Plasmids for the expression of fusion proteins were designed to encode proteins bearing a histidine tag for immobilized metal-affinity chromatography purifica-tion All chimeras consist of the IDP (NTAIL, PNT or SIC1), a linker of 14 residues containing the TEV pro-tease cleavage sequence (Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Ser) and the GFP, in that order (Fig 1) The lengths of the resulting fusion proteins are: 386 resi-dues for NTAIL-GFP (43.2 kDa), 490 resiresi-dues for PNT-GFP (53.4 kDa) and 545 residues for SIC1-GFP
Trang 3(61.7 kDa) Isolated IDPs and GFP were expressed
from similar constructs and in the same host cells
The expression protocol was optimized to obtain
similar amounts of all proteins and to minimize both
the formation of inclusion bodies and spontaneous
proteolysis Indeed, it has been observed that IDPs are
prone to undergo proteolytic degradation during
puri-fication even upon addition of protease inhibitors to
cell extracts [5] The culture conditions found to satisfy
all these requirements were: transformed Escherichia
coli BL21 [DE3] cells were grown at 37C until D600
of 0.4–0.5 was reached, then induced with 100 lm of
isopropyl thio-b-d-galactoside at 37C for 2 or 6 h,
depending on whether single or fusion proteins were to
be expressed, respectively Under the above conditions,
all proteins were found to be mainly soluble and
prote-olytic events were negligible (Fig 2) Despite repeated
attempts (data not shown), we could not improve the
expression level of SIC1-GFP, which systematically
remained very poor Notably, all the fusion proteins
were fluorescent, thus suggesting that the GFP moiety
adopts a native-like conformation
Conformational properties of the fusion proteins vary as a function of the unstructured moiety NTAIL, PNT and SIC1 have been previously shown
to belong to the family of IDPs on the basis of their biochemical and biophysical properties [30,32,34] Accordingly, their far-UV CD spectra recorded at
20C show the distinctive IDP profile, characterized
by a large negative peak at 200 nm The ellipticity val-ues observed at 200 and 222 nm are consistent with the existence of some residual helical structure By con-trast, the GFP spectrum is typical of a structured pro-tein with predominant b-strand content, as indicated
by the well-defined positive peak at 195 nm and the broad negative peak with a minimum at 218 nm (Fig 3) Spectra of the fusion proteins combine fea-tures of ordered and unordered components Although minima corresponding to helical structures (a well-defined inflection point at 203–207 nm and a less pro-nounced inflection point at 220–222 nm) are clearly observed, the negative ellipticity values at 200 nm, together with the low ellipticity in the range 185–
195 nm, suggest the presence of unordered structures (Fig 3) Notably, these spectroscopic hallmarks of dis-order are particularly pronounced for the NTAIL and SIC1 fusion proteins (Fig 3A, C), whereas PNT-GFP exhibits a less disordered nature (Fig 3B)
To highlight possible mutual effects of disordered and ordered moieties within the fusion proteins, we calculated the theoretical average spectra of equimolar IDP and GFP mixtures by averaging the spectra of the individual IDP and GFP proteins Note that each average spectrum describes what would be expected in case the two components do not affect each other’s conformation We then compared the average spectra with the experimental measured spectra of equimolar IDP + GFP mixtures The CD spectra of NTAIL + GFP (Fig 3A) and PNT + GFP (Fig 3B) mixtures superimpose quite well onto their respective calculated theoretical average spectra, indicating that, when the two separated components are mixed, they do not undergo any significant structural rearrangement The spectra of both NTAIL-GFP and PNT-GFP fusion proteins are clearly different from those of the mix-tures either calculated or measured, suggesting that structural rearrangements are induced in the fusion by the forced close proximity of the two proteins In par-ticular, NTAIL-GFP and PNT-GFP spectra indicate a lower and higher extent of order with respect to the average spectra, respectively By contrast, the spectrum
of the SIC1-GFP fusion protein superimposes onto the calculated average spectrum, suggesting that the two domains do not impact on each other’s conformation
Fig 1 Schematic representation of IDP-GFP constructs From the
N- to C-terminus, each fusion protein contains the hexahistidine tag
(H 6 ), the IDP (NTAIL, PNT or SIC1), a TEV cleavage sequence (TEV)
and the GFP.
Fig 2 Expression and purification of fused polypeptides and
indi-vidual proteins M, molecular weight markers; TF, total protein
frac-tion; SOL, soluble protein fracfrac-tion; proteins purified by immobilized
metal affinity chromatography (IMAC).
Trang 4when they are covalently linked More difficult to
explain are the rearrangements observed in the
SIC1 + GFP mixture (Fig 3C), which suggest that
structural rearrangements only take place when the
two proteins exist as individual moieties in solution
This observation might be accounted for by the
inter-action depending on orientation factors, with the two
moieties exhibiting a considerably reduced conforma-tional freedom if covalently linked
In view of obtaining further insight into the struc-ture of the fusion proteins, we estimated the content of a-helices, b-strands, b-turns and unordered regions by the cdsstr deconvolution method (Fig 4) Although this type of analysis does not yield secondary structure content values that are in perfect agreement with those derived from structural data obtained by other meth-ods, it is assumed to be applicable and trustworthy if its aim is a comparison of the secondary structure con-tent of a restricted set of spectra obtained under the same conditions, as in our case [35]
At 20C, NTAIL, PNT and SIC1 share a high tent of unordered stretches (50–59%) and a low con-tent in a-helices and b-strands (2–3% and 22–29%, respectively), whereas GFP is rich in b-strands (46%) and exhibits a low content in unordered regions (20%), in agreement with previous data available for these proteins [23,30,32,34] Interesting differences arise from a comparison of the secondary structure content
of each protein in isolation with that of fusion pro-teins We observed that the linkage with GFP does not alter the b-a-turn-unordered ratio typical of the unstructured moiety when the IDP is NTAIL or SIC1, whereas, in the fusion PNT-GFP, the structured part appears to prevail, raising the percentage of the differ-ent secondary structures to a value close to that of GFP alone (Fig 4) Comparison between the measured and the averaged secondary structure contents (see Materials and methods) clearly shows that the second-ary structure composition of the fusion proteins devi-ates from the mean of the single contributions (Fig 5) Although this analysis does not allow an assessment of whether the observed deviations in the structural con-tent reflect structural transitions taking place in only one of the two moieties or rather reflect structural rearrangements distributed over the whole polypeptide,
we can speculate that the increase in order in PNT-GFP likely reflects a gain of structure within PNT That PNT possesses an inherent propensity to undergo
a disorder-to-order transition has already been reported, with this gain of structure concerning the first 50 residues [30] Conversely, the less ordered nat-ure of the NTAIL-GFP fusion protein with respect to the mean of the secondary structure contents of the two components could be ascribed either to partial unfolding of GFP or to loss of residual structure by NTAIL, with the transiently populated a-helical regions of the latter [26,34,36–39] adopting preferen-tially an extended (e.g disordered) conformation when linked to GFP
A
B
C
Fig 3 Far-UV CD spectra The CD spectrum of each of the fusion
proteins is compared with that of individual proteins, with the
theo-retical average spectrum and with the spectrum of equimolar
pro-tein mixtures (A) NTAIL-GFP; (B) PNT-GFP; (C) SIC1-GFP Spectra
were recorded in 10 m M sodium phosphate (pH 7.5) at 20 C.
Trang 5Notably, the analysis of the GFP sequence alone
and of GFP bound to any of the three IDPs using the
anchor server, which predicts binding sites within
dis-ordered regions [14,40], shows that the GFP
N-termi-nal part becomes more disordered (Fig S1) This can
account for the increased disorder measured by far-UV
CD on the NTAIL-GFP fusion protein compared to
the NTAIL + GFP mixture According to anchor,
NTAIL contains possible binding regions either
(Fig S1) In this case, however, the experimental data suggest that these binding regions are not compatible with GFP binding anchor predicts numerous possible binding regions within PNT (Fig S1) As a result
of order increasing in PNT-GFP compared to PNT + GFP, one (or more) of the binding regions probably effectively binds to GFP, albeit not to a great extent, because this interaction is not measured in the PNT + GFP mixture Finally, the predicted SIC1 binding regions (Fig S1) may interact with GFP, thus accounting for the increased order in the SIC1 + GFP mixture compared to the theoretical average As a result of steric restrictions, the most likely candidate for GFP binding is the 183–194 stretch (or the weaker ones between 214–229 and 253–259) predicted by anchor
Regardless of the distribution within the fusion pro-tein of such folding and unfolding events, we can clearly state that, in the presence of the same globular domain (GFP), the overall structure of the fusion pro-tein varies as a function of the unstructured moiety The conformational stability of the chimeras was investigated by recording variations in the mean resi-due ellipticity at 195 nm when heating the protein samples from 20 to 100C (Fig S2) We observed that, although isolated GFP undergoes a cooperative unfolding transition between 70 and 90 C, all IDPs display almost constant negative mean residue elliptic-ity values, consistent with the absence of cooperative unfolding that typifies unstructured proteins, and a moderate increase of ellipticity at the highest tempera-tures, consistent with the process of temperature-induced folding common to several IDPs [41] Heat induced transitions recorded for the fusion proteins were intermediate between these two scenarios Only for NTAIL-GFP was a defined transition visible in the range 75–85C range, whereas PNT-GFP and SIC1-GFP did not display a classical two-state confor-mational transition We also monitored the mean resi-due ellipticity at 195 nm during recooling to 20 C, and recorded the CD spectra of the cooled solutions
in the whole range (260–185 nm) to assess the revers-ibility of unfolding (data not shown) GFP denatur-ation was found to be only partly reversible, with the sample exhibiting some helical structure but not the native b-strand content, whereas the CD spectra of the IDPs acquired before and after the heating⁄ recool-ing process were fully superimposable Unfoldrecool-ing of fusion proteins was not fully reversible and showed a trend very similar to that of isolated GFP, suggesting that the GFP moiety retains an inability to recover its native conformation when covalently linked to an IDP
Fig 4 Secondary structure content of IDPs, GFP and fusion
pro-teins a-helix, b-strands, turns and unordered regions percentages
were calculated using CDSSTR
Fig 5 Deviation from the theoretical secondary structure average
composition Differences are calculated for each fusion and each
kind of structure by comparing the percentages derived from
exper-imental spectra with the theoretical average compositions as
obtained by averaging the secondary structure content of each
indi-vidual IDP and GFP.
Trang 6Compactness of fusion proteins depends on the
disordered moiety
In further experiments, we investigated the effect of
the disordered domain on the electrophoretic mobility
of the fusion proteins using SDS⁄ PAGE migration
analysis (Table 1) IDPs are known to migrate slower
in SDS⁄ PAGE than globular proteins with the same
molecular mass as a result of their relative enrichment
in acidic residues [5] The apparent Mr of the isolated
IDPs as observed in SDS⁄ PAGE was larger than
expected (Table 1), whereas, for GFP, the expected
and observed values were very close Because all fusion
proteins exhibited an apparent molecular mass
(Mr App) significantly higher than expected, we
con-cluded that the presence of a covalently linked IDP is
sufficient to affect GFP migration and that the extent
of this modification is correlated with the specific
disordered component, as suggested by the observed
differences in the Mr App⁄ Mr thratio
We next addressed the impact of the disordered
moi-ety onto the overall compactness of the fusion proteins
by size exclusion chromatography Because these studies
are quite demanding in terms of protein amounts, we
only focused onto those proteins (NTAIL-GFP and
PNT-GFP) that could be produced and purified in
suffi-cient quantity (Table 1) In gel filtration experiments,
the elution volume of a given protein can be directly
cor-related with the protein apparent molecular mass by
interpolation with a calibration curve in which elution
volumes of several globular proteins of known size are
correlated with their molecular masses [42] The
hydro-dynamic radius of a protein (Stokes radius, Robs
S ) can then be deduced from its apparent molecular mass and
compared with the theoretical Stokes radius expected
for either the native (RsN) or the fully denatured (RsU)
form of a protein of the same size (for details, see
Materials and methods) As expected, the GFP hydro-dynamic behaviour reflected the properties of a globular protein, with a Robs⁄ RsN ratio very close to the unit, whereas ‘aberrant’ elution profiles in gel filtration experiments were systematically observed for all IDPs Previous data showed that NTAIL behaves in gel filtra-tion as a protein of 36 kDa, whereas its expected mass is
15 kDa [34] The corresponding Robs
S was 27 A˚, a value closer to the radius expected for a fully denatured state (RsU= 35 A˚) compared to globular protein (RsN=
19 A˚) [34] In the present study, PNT (expected mass of
25 kDa) was found to elute with an apparent molecular mass of 115 kDa, in agreement with previous studies [30] This very high value of the apparent molecular mass corresponds to an observed Stokes radius of 41 A˚, which is closer to the value expected for the fully un-ordered (RsU= 46 A˚) than the globular (RsN= 23 A˚) form The apparent molecular weight of SIC1 was reported to be 50 kDa instead of 33 kDa, and the inferred Stokes radius was 30 A˚, with the expected RsU and RsNbeing 53 and 25 A˚, respectively [32]
The apparent molecular masses of both NTAIL-GFP and PNT-NTAIL-GFP were higher than calculated from their amino acid sequence (96 kDa instead of 43 kDa and 73 kDa instead of 53 kDa, respectively) The extent of the discrepancy however was not the same The calculated Robs
S and comparison with the relative
RsN and RsU showed that the two fusion proteins have distinctive hydrodynamic behaviours that could not be anticipated from the characteristics of flexi-bility of their unstructured component The Robs
S of the NTAIL-GFP fusion protein (38 A˚) is closer to the
RsN(28 A˚) than to the RsU(61 A˚), whereas the Robs
S of PNT-GFP (35 A˚) is closer to the RsN(31 A˚) than to the RsU(69 A˚) (Table 1)
Thus, the hydrodynamic values of NTAIL-GFP and PNT-GFP proteins do not reflect the sum of the
Table 1 Apparent Mrof IDPs-GFP, IDPs and GFP derived from SDS ⁄ PAGE and gel filtration Hydrodynamic radii were inferred from the apparent molecular mass according to Uversky [46] ND, not determined.
Mr th
(kDa)
SDS ⁄ PAGE Gel filtration
Mr App (kDa) Mr App⁄ M r th
Mr App (kDa) Mr App⁄ M r th
RsN (A ˚ )
RsU (A ˚ )
R obs S
(A ˚ ) R obs
S ⁄ R sN R obs
S ⁄ R sU
Reference for gel filtration
present study
Trang 7behaviour of their single components because isolated
NTAIL is less extended than isolated PNT The
struc-tural disorder and flexibility typical of isolated NTAIL
is maintained and appears to increase in the fusion,
resulting in a Stokes radius that is even higher than
expected from the mean of the NTAIL and GFP radii
By contrast, the high flexibility of PNT is not reflected
in PNT-GFP, suggesting that structural modifications
of PNT occur in the fusion protein, in agreement with
the CD data Because isolated NTAIL is less
unor-dered than isolated PNT but their GFP fusions show
opposite behaviours, these experiments further indicate
that specific, rather than generic, interactions occur
CD spectra in the near ultraviolet region (250–
350 nm), also known as the aromatic region, reflect the
symmetry of the aromatic amino acid environment
and, consequently, characterize the protein tertiary
structure Proteins with rigid tertiary structure are
typi-cally characterized by intense near-UV CD spectra,
with unique fine structure, which is reflective of the
unique asymmetric environment of individual aromatic
residues Conversely, IDPs are characterized by low
intensity near-UV CD spectra with low complexity
[41] Accordingly, the near-UV CD spectrum of GFP
shows a very pronounced peak at 280 nm, whereas the
spectra of the IDPs are very flat, with no such a clear
peak being detectable (Fig 6A) Notably, the spectra
of GFP linked to a disordered moiety are much
smoother, with the decrease in the intensity of the
peak being IDP-dependent Indeed, the spectrum of
the PNT-GFP fusion protein reflects a higher extent of
order than that of GFP fused to NTAIL or to SIC1
(Fig 6A), in agreement with the data inferred from
both far-UV CD spectroscopy and size exclusion
chro-matography analyses In the same vein, the visible CD
spectra (Fig 6B) of GFP alone, as well as of GFP
fusion proteins, show a very pronounced negative peak
at 517 nm, with an intensity in the the order: GFP >
PNT-GFP > NTAIL-GFP > SIC1-GFP This peak
reflects the asymmetric and therefore rigid environment
of the green chromophore The gradual reduction in
the intensity of the peak in the fusion proteins is
indic-ative of progressive loss of ordered structure as PNT,
NTAIL or SIC1 are added (Fig 6B)
In conclusion, near and visible CD data are in good
agreement with the data provided by far-UV and
size-exclusion chromatography studies and, taken together,
they converge to show that PNT-GFP is the most
compact and ordered fusion protein, whereas
SIC1-GFP is the most disordered one
All the results obtained in the present study so far
point to reciprocal and different effects of the two
moie-ties of the fusion However, they still do not unravel
whether one of the two domains is more affected in its conformation; in other words, whether order prevails
on disorder or vice versa In an attempt to assign these effects to a specific domain, we analyzed changes in GFP fluorescence and resistance to proteolysis of the fusion proteins
GFP stability is not affected by fusion with the disordered domain, whereas IDPs are only marginally protected from proteolysis by the linked GFP
The presence of a natural chromophore in the globular part of the fusion provides a sensitive probe for assess-ing possible conformational changes The fluorescence emission spectra of GFP, NTAIL-GFP and PNT-GFP shared the typical features of the GFP chromophore, with a well-defined peak at 527 nm and a shoulder at
A
B
Fig 6 Near-UV and visible CD spectra for tertiary structure analy-sis CD spectra acquired in the near-UV (A) and in the visible (B) wavelength range Spectra were recorded in 10 m M sodium phos-phate (pH 7.5) at 20 C.
Trang 8570 nm [43] The GFP and PNT-GFP emission peaks
at 527 nm were almost superimposable, whereas the
fluorescence intensity of the corresponding peak in
NTAIL-GFP was lower However, on the basis of the
higher scattering peak of NTAIL-GFP at 475 nm,
such a decrease can likely be attributed to partial
pre-cipitation of the protein rather than to conformational
changes (Fig 7) When denaturation experiments were
performed, both fusion proteins displayed transitions
in the emitted fluorescence in the temperature range
75–80C, which is similar to those observed with the
isolated GFP (data not shown) Such temperature
values are slightly lower than the ones obtained by CD
analysis, as expected for a technique that specifically
targets the active site instead than averaging the whole
protein secondary structure content The above
observa-tion rules out any effect by the covalently linked IDP on
GFP stability, at least in the protein regions around the
chromophore or critical for its stabilization, and is in
agreement with the results of the spectroscopic analyses
described above, where the GFP moiety, both alone and
IDP-linked, proved unable to recover its native
confor-mation after thermal unfolding
Globular proteins are rather resistant to proteases,
whereas the extended structure of IDPs makes them
prone to proteolytic attacks [4,5,41,44,45] For this
rea-son, and in view of understanding which domain is
affected by structural rearrangements, we assessed
whether the presence of GFP is able to protect the
unstructured part of the fusion or, in contrast, GFP
becomes more protease-accessible when linked to an
IDP In Fig 8, we show a time-course analysis of a
limited tryptic digestion of NTAIL-GFP and
PNT-GFP, as well as of their isolated IDP moieties In these
experiments, GFP was very resistant to degradation even with enzyme : substrate molar ratios as high as
1 : 40 and an incubation time of up to 1 h (data not shown), whereas both IDPs started to degrade after
1 min of tryptic digestion, and were no longer detect-able after 5 min (Fig 8) A significant degradation of NTAIL was already apparent in the absence of the enzyme (t0 in Fig 8A), consistent with a high prote-ase-susceptibility of this protein Interestingly, a fragment of the same apparent molecular mass (approximately 17 kDa) was also observed in the sam-ple containing NTAIL-GFP before the addition of trypsin (see t0 in Fig 8A), suggesting that fusion with GFP would not protect this specific proteolytic site within NTAIL As shown in Fig 8A, after 20 min of incubation, full-length NTAIL-GFP disappeared and a band of the same molecular mass of GFP became
Fig 7 Fluorescence emission spectra of NTAIL-GFP, PNT-GFP and
GFP at 20 C Proteins were excited at 474 nm and spectra were
recorded in the range 465–620 nm in 10 m M sodium phosphate
(pH 7.5) at 20 C.
A
B
Fig 8 Limited proteolyis of NTAIL-GFP, PNT-GFP and their compo-nent proteins Two micrograms of purified proteins were incubated with trypsin at a 1 : 400 enzyme : substrate molar ratio for 1, 2, 5,
10, 20 and 60 min Samples were separated on 16% SDS ⁄ PAGE and stained by Coomassie (A) NTAIL-GFP, NTAIL and GFP (B) PNT-GFP, PNT and GFP M, molecular weight markers; 0¢, untreated samples The ‘framed’ bands were further analyzed by
MS (Fig S3).
Trang 9detectable (Fig 8A) In the course of incubation, a
fragment with an apparent Mr of 32 kDa (i.e bigger
than GFP) accumulated and persisted also after 1 h of
digestion (Fig 8A, box a)
Isolated PNT migrated as a unique band in the
con-trol sample but underwent fast degradation upon
tryp-tic treatment with disappearance of the full-length
polypeptide as early as after 5 min of incubation
Pro-teolysis of PNT-GFP proceeded with the formation of
a relatively stable fragment with an apparent mass of
31 kDa (Fig 8B, box b), in addition to that
corre-sponding to GFP alone, already after 1 min of
incuba-tion (Fig 8B)
Persistent protein fragments (see ‘framed’ bands a
and b in Fig 8) were processed by tryptic in-gel
diges-tion and the resulting peptides were analyzed by
MS⁄ MS (Fig S3) The Mr, as determined by MS, of
band a from NTAIL-GFP was 31.6 kDa and that of
band b from PNT-GFP was 30.8 kDa Sequencing
showed that these protease-resistant fragments
encom-pass the trypsin cutting sites at position 128 in
NTAIL-GFP and at positions 226, 235 and 236 of
PNT-GFP, respectively These results indicate that the
complete proteolytic digestion of both IDPs requires
longer incubation when they are fused with GFP, with
a proteolytic fragment still containing part of the IDP
being detectable after as long as 60 min of incubation
in both cases (Fig 8) That the GFP sensitivity
towards proteolysis was not affected by its linkage to
an unstructured part was checked in two additional
experiments Western blotting analysis of a
time-course digestion (up to 20 min) of NTAIL-GFP and
PNT-GFP ruled out the presence of fragments
react-ing with anti-GFP antibodies smaller than full-length
GFP (Fig S4) Moreover, the fragment of
approxi-mately 20 kDa produced from PNT-GFP after 20 min
of tryptic digestion (Fig 8B, band c) was found to
span the same amino acid sequence as the band of the
same size that was detectable after 2 min of digestion
of PNT alone (Fig 8B, band d) This protein
fragment encompasses a region of PNT upstream that
is in band b (Fig S3), thus ruling out the possibility
that it could correspond to a GFP proteolytic
frag-ment On the basis of these two lines of experimental
evidence, we concluded that the proteolytic resistance
of GFP is not affected by the presence of the fused
IDP
In both fusion proteins, proteolysis occurs within
the unstructured part only and proceeds from the
N-terminus towards the GFP moiety, resulting in the
generation of a trypsin-resistant fragment that contains
GFP after 1 h of incubation The persistence of GFP
in these fragments, besides highlighting its resistance
towards proteolysis, also suggests protection of the C-terminal region of the IDP by the GFP moiety However, we cannot exclude the possibility that pro-tection only arises from steric hindrance (i.e from a reduced substrate accessibility to trypsin), rather than being the result of local structural rearrangement within the IDP
How do ordered and disordered parts affect each other?
In conclusion, we have observed that different IDPs fused to the same globular protein result in polypep-tides with distinctive secondary structure content and compactness that are not merely the average of their two components The finding that their overall struc-ture and compactness are not consistent with those that are predicted on the basis of the behaviour of the isolated IDP was intriguing Indeed, although PNT alone is more flexible than NTAIL, PNT-GFP is by far more structured and compact that the NTAIL fusion This observation may suggest that linkage with GFP confers the two IDPs with folding propensities that differ from those of the isolated NTAIL and PNT proteins Nonetheless, our attempts to highlight spe-cific structural rearrangements within either the IDP or the GFP moiety that could account for the specific conformational features observed were hindered by the complex nature of proteins Association with a disor-dered moiety left GFP almost unchanged, whereas the IDP was marginally stabilized towards proteolysis However, despite the higher compactness of the PNT-GFP fusion protein with respect to NTAIL-PNT-GFP, no higher proteolytic resistance of PNT-GFP could be detected It could be speculated that the high flexibility
of IDPs prevents the formation of stable interactions, causing delocalized structural rearrangements that failed to manifest in the experiments conducted in the present study
Materials and methods
Construction of expression plasmids The NTAIL-GFP, PNT-GFP and SIC1-GFP constructs were obtained in two steps: the single IDP-encoding sequences were cloned in the pET22 plasmid (Novagen, Madison, WI, USA) and then the GFP encoding sequence was inserted downstream The cloning strategy was differ-ent in each case and is described below
The DNA fragment encoding NTAIL with a hexahisti-dine tag fused to its N-terminus was obtained by PCR from the pDest14⁄ NTAILHN plasmid [36] To remove the NcoI
Trang 10restriction site at position 321, amplification was carried
out in two separated reactions yielding products N1 (from
nucleotides 1–330) and N2(from nucleotides 331–396) N1
was amplified using a forward primer (FWN1: 5¢-TACCG
TTAACATCGATATGCATCATCATCATCATCATAC-3¢),
designed to introduce a ClaI restriction site at nucleotide
position –6 and a reverse primer (REVN1: 5¢-CCTGCC
ATTGCTTGCAGCC-3¢) that introduced a silent mutation
at nucleotide position 321 resulting in the suppression of
the NcoI site Product N2was amplified with a forward
pri-mer (FWN2: 5¢-GGCTGCAAGCAATGGCAGG-3¢) that
introduced the same silent mutation as above at nucleotide
position 321 and a reverse primer (REVN2: 5¢-ATCGCC
ATGGTCCCGGGCATATGGGATCCCTGGAAGTACA
GGTTTTCGTCTAGAAGATTTCTGTC-3¢) designed to
remove the NTAIL stop codon and to introduce a fragment
encoding a TEV cleavage sequence and a NcoI restriction
site at position +38 after the end of the NTAIL sequence
N1 and N2were mixed, digested with DpnI to remove the
methylated DNA template, and used as the template in a
PCR reaction with primers FWN1and REVN2to yield the
complete NTAIL amplification product
The DNA fragment encoding PNT with an N-terminal
hexahistidine tag was obtained by PCR using the
pET21a⁄ PNT-H6plasmid [30] as the template The forward
primer (5¢-TACCGTTAACATCGATATGCATCATCATC
ATCATCATGC-3¢) was designed to insert a ClaI
restric-tion site at nucleotide posirestric-tion )6, whereas the reverse
primer (5¢-ATCGCCATGGTCCCGGGCATATGGGATC
CCTGGAAGTACAGGTTTTCCTTTTTAATGGGTGTC
CC-3¢) was designed to remove the stop codon and to
introduce a DNA fragment encoding a TEV cleavage
sequence and a NcoI restriction site at position +38 after
the end of the PNT sequence
The DNA sequence encoding SIC1 with a hexahistidine
tag fused to its N-terminus was obtained by PCR from the
plasmid pET21⁄ SIC1 [32] with a forward primer (5¢-TAC
CTGGCCAATGAATATGCATCATCATCATCATCATA
CTCCGTCGACCCCACC-3¢) designed to introduce a
hexahistidine tag and a ClaI restriction site at nucleotide
position –6 and a reverse primer (5¢-ATCGCCATGGTC
CCGGGCATATGGGATCCCTGGAAGTACAGGTTTT
CGCCATGCTCTTGATCCC-3¢) designed to remove the
stop codon and to introduce a DNA fragment encoding a
TEV cleavage sequence and a NcoI restriction site at
posi-tion +38 after the end of the SIC1 gene fragment
PCR reactions contained 2.5 mm dNTPs, 5 lm of each
primer, 10 ng of plasmid DNA and 5 U of Triple Master
DNA Polymerase (Eppendorf, Hamburg, Germany) The
amplification program was: after a first denaturation step
at 94C for 5 min, 25 cycles of 20 s at 94 C, 20 s at 50 C
and 2 min at 72C were performed, followed by a final
elongation step of 10 min at 72C
All PCR products (NTAIL, PNT and SIC1) were
digested with DpnI and purified by precipitation with
ethanol, restricted with ClaI and NcoI, checked by agarose (0.8%, w⁄ v) gel electrophoresis and purified from the gel (QIAquick Gel Extraction Kit; Qiagen, Valencia, CA, USA) The pET22 vector was digested with NdeI, filled-in with the Klenow fragment of the E coli DNA polymerase I (New England Biolabs, Beverly, MA, USA) to produce blunt ends, and finally cleaved with NcoI In this way, the sequence pelB, allowing targeting to the periplasm of pro-teins expressed from pET22, was removed The digested PCR products and pET22 were ligated with T4 Ligase (New England Biolabs) The final constructs are referred to
as pET⁄ NTAIL, pET ⁄ PNT and pET ⁄ SIC1
The GFP gene (cloned from a pET19b⁄ GFP plasmid) was then inserted downstream NTAIL, PNT and SIC1 at the NcoI and ScaI sites to obtain pET⁄ NTAIL-GFP, pET⁄ PNT-GFP and pET ⁄ SIC1-GFP These constructs encode for fusion proteins bearing a 14 residues linker containing the TEV cleavage sequence between the two components The final constructs were transformed into the E coli DH5a strain (Novagen) and the sequence of their ORFs was checked by DNA sequencing on both strands
Expression and purification of fusion proteins The E coli BL21[DE3] strain (Novagen) was used as the host for heterologous expression Transformed cells were grown overnight at 37C in low-salt LB medium contain-ing 100 mgÆL)1 ampicillin, diluted 1 : 50 in 200 mL of the same broth and incubated at 37C until until D600of 0.4– 0.5 was reached Induction was performed by adding
100 lm isopropyl thio-b-d-galactoside Cells were then grown at 37C for either 2 h when expressing single IDPs
or for 6 h when expressing fusions and GFP
Cells were collected by centrifugation and resuspended
in 2 mL of lysis buffer (50 mm sodium phosphate, pH 8.0,
300 mm NaCl, 5 mm imidazole) containing the protease inhibitors cocktail P8465 (Sigma-Aldrich, St Louis, MO, USA) After 20 min of incubation on ice, cells were dis-rupted by sonication (four cycles of 10 s each at 50% power output) Cell extracts were centrifuged for 30 min
at 10 000 g at 4C and the His-tagged proteins recovered
as soluble proteins from the supernatant They were then purified by immobilized metal-affinity chromatography on
a Ni2+-nitrilotriacetic acid resin (Qiagen) The clarified lysate was added to a pre-packed resin suspension (2 mL
of resin per 200 mL of culture), eluted by gravity and then reloaded four or five times on the column After washing with 50 mm sodium phosphate (pH 8.0), 300 mm NaCl buffer containing increasing concentrations of imid-azole (25–50 mm), proteins were eluted with 50 mm sodium phosphate (pH 8.0), 300 mm NaCl and 250 mm imidazole When required, buffer exchange was performed
by gel filtration on PD-10 columns (GE Healthcare, Mil-waukee, WI, USA) and samples were concentrated with a