Monomeric molten globule intermediate involved in the equilibriumHwei-Jen Lee1, Shang-Way Lu1and Gu-Gang Chang2 1 Department of Biochemistry, National Defense Medical Center, Taipei, Tai
Trang 1Monomeric molten globule intermediate involved in the equilibrium
Hwei-Jen Lee1, Shang-Way Lu1and Gu-Gang Chang2
1
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan;2Faculty of Life Sciences, National Yang-Ming University, Taipei, Taiwan
Duck d2-crystallin is a soluble tetrameric lens protein In
the presence of guanidinium hydrochloride (GdnHCl),it
undergoes stepwise dissociation and unfolding
Gel-filtra-tion chromatography and sedimentaGel-filtra-tion velocity analysis
has demonstrated the dissociation of the tetramer protein to
a monomeric intermediate with a dissociation constant of
0.34 lM3 Dimers were also detected during the dissociation
and refolding processes The sharp enhancement of 1-anilino
naphthalene-8-sulfonic acid (ANS) fluorescence at 1M
GdnHCl strongly suggested that the dissociated monomers
were in a molten globule state under these conditions The
similar binding affinity ( 60 lM) of ANS to protein in the
presence or absence of GdnHCl suggested the potential assembly of crystallins via hydrophobic interactions,which might also produce off-pathway aggregates in higher protein concentrations The dynamic quenching constant corres-ponding to GdnHCl concentration followed a multistate unfolding model implying that the solvent accessibility of tryptophans was a sensitive probe for analyzing d2-crystallin unfolding
Keywords: d-crystallin; lens protein; unfolding; dissociation; argininosuccinate lyase
d2-Crystallin,a highly concentrated yet soluble protein in
avian and reptile eye lens,acts as an important structural
protein for light refraction [1–3] Thermodynamic stability
for crystallins is essential in maintaining lens transparency
[4] Determining the mechanism of folding and assembly of
these proteins is important for understanding how they can
form stable transparent structures at high concentrations
The d2-crystallin in lens was recruited during evolution
from argininosuccinate lyase,an enzyme involved in
arginine biosynthesis derived from the urea cycle These
two proteins shared over 90% sequence homology and thus
have similar tertiary structures [5–7] d2-Crystallin has a high
helical content and constitutes a unique liquid-like region in
the center of the duck lens [8] The central 20-helix core
contributes to the major interactions between subunits,and
is crucial for subunit association The active site is located at
a boundary composed of three subunits [8–11]
Identifying and characterizing of possible conformational
states in the pathways leading to folding and unfolding are
important For d2-crystallin,characterization of the
inter-molecular association of the helix bundles and the partial
unfolded intermediate with exposed hydrophobic region
leading to polymerization remains to be elucidated [12]
Most of the established models of reversible unfolding
of proteins are based on experiments exploring the effect
of chemical denaturants or temperature These models, although providing useful information on the unfolding mechanism,are limited to small,monomeric proteins For multimeric proteins,detection of partially unfolded inter-mediate is always complicated by the dissociation step [13,14] Only in limited cases can dissociation and unfolding
be clearly distinguished [15–19] In previous studies we have demonstrated that duck d2-crystallin can be reversibly dissociated and unfolded by GdnHCl [15] The dissociation
of tetrameric d2-crystallin is accompanied by loss of argininosuccinate lyase activity at around 0.9M GdnHCl, which produces monomeric d2-crystallin as judged by gel-filtration chromatography [15] At higher GdnHCl concen-trations,the monomer unfolds via a partially unfolded intermediate before denaturation Structural information has revealed that tetrameric d2-crystallin possesses a double dimer structure [10] The dimeric form of d2-crystallin can be observed under acidic conditions [20]
In this report,we investigate the detailed dissociation process for d2-crystallin during chemical denaturation by GdnHCl Gel-filtration chromatography and analytical ultracentrifugation were used to identify the dissociation intermediate Unfolding of the dissociated monomers was investigated using 1-anilino-8-naphthalene sulfonate (ANS) binding,fluorescence studies and circular dichroism (CD)
Materials and methods
Materials Ultra-pure guanidine hydrochloride and acrylamide was purchased from Baker (Phillipsburg,NJ,USA) Tris (base), EDTA and NaCl were obtained from Merck AG
Correspondence to H.-J Lee,Department of Biochemistry,National
Defense Medical Center,no 161,Sec 6,Minchuan East Road,
Neihu 114,Taipei,Taiwan.
Fax: + 88 62 87923106,Tel.: + 88 62 87910832,
E-mail: hjlee@ndmctsgh.edu.tw
Abbreviations: ANS,1-anilinonaphthalene-8-sulfonic acid; GdnHCl,
guanidinium hydrochloride; CD,circular dichroism.
(Received 12 May 2003,revised 14 July 2003,
accepted 7 August 2003)
Trang 2(Darmstadt,Germany),and argininosuccinic acid
(diso-dium salt) from Sigma Chemical Co
1-Anilinonaphthalene-8-sulfonic acid (ANS) was obtained from Molecular Probe
(Eugene,OR,USA) All other chemicals were of analytical
grade and used without further purification
Purification of duck lens d2-crystallin
Duck d2-crystallin was purified as described previously
[6,15] except that 50 mMTris/HCl,0.5 mMEDTA,pH 7.5
was used to equilibrate the column and d2-crystallin was
eluted in the same buffer containing 0.12 M NaCl The
pooled d2-crystallin showed one major band upon SDS/
PAGE analysis The purity of the protein was further
analyzed by gel filtration chromatography using Superdex
200 HR (10/30) column equilibrated in 100 mMTris/HCl
buffer,pH 7.5 The relative area of the major peak
accounted for around 85% of the total protein Protein
concentrations were determined spectrophotometrically as
described previously [15]
Equilibrium unfolding and refolding studies
A GdnHCl stock solution (7M) was prepared in 50 mM
Tris/HCl buffer (pH 7.5),and the pH of the stock
solution was readjusted to pH 7.5 Equilibrium unfolding
and refolding experiments were performed according to
the methods described previously [15] The endogenous
argininosuccinate lyase activity of d2-crystallin was
moni-tored as a function of the appearance of fumarate at
240 nm Tryptophan fluorescence was measured by the
emission spectra at an excitation wavelength of 295 nm
Far-ultraviolet (200–250 nm) CD data were obtained in a Jasco 810 spectropolarimeter equipped with a thermo-statically controlled cell holder with a 10-mm path length cell
Fluorescence quenching measurements Fluorescence quenching experiments were performed by adding aliquots of stock acrylamide or KI solution into the GdnHCl denatured proteins The fluorescence emission spectra with excitation wavelength at 295 nm were moni-tored The concentrations of quencher added were less than 0.3M Sodium thiosulfate (0.1 mM) was added to the KI stock solution to prevent I–formation The inner filter effect due to the absorption of acrylamide or KI at 295 nm was corrected for by multiplying the fluorescence intensity by
10A/2,where A is the absorbance of the solution at 295 nm Fluorescence quenching data were fitted to a modified Stern–Volmer equation [21]:
F0=DF¼ 1=ðfaKSV½QÞ þ 1=fa where DF¼ F0– F,where KSVis the dynamic quenching constant and fawas the fractional maximum accessible protein fluorescence
ANS binding assay The exposed hydrophobic surfaces of the protein were assayed by incubating the GdnHCl-denatured protein in the dark with ANS (50 lM) for 4 h at 25C The fluorescence emission spectra of the protein solution at an excitation wavelength of 370 nm were monitored Appropriate blank
Fig 1 Gel-filtration profiles of the
equili-brium-unfolded duck d-crystallin in GdnHCl.
d-Crystallin 0.6 l M (A),or 2.4 l M (B),was in
equilibrium unfolded in 0–5 M GdnHCl The
M r markers (.) were (from left to right):
thyroglobulin (669 kDa),ferritin (440 kDa),
catalase (232 kDa),albumin (67 kDa),and
ovalbumin (43 kDa) The elution positions
corresponding to monomers (M),dimers (D),
tetramers (T),unfolding (U) and polymers (P)
forms are also labeled Refolding of the
unfolded duck d-crystallin (2.4 l M ) in
GdnHCl was examined by a 10-fold dilution
of equilibrium unfolded d-crystallins at 1,2
and 5 M GdnHCl to 0.1,0.2 and 0.5 M ,
respectively,and analysis by gel filtration
chromatography (dashed lines).
Trang 3spectra of ANS in the corresponding GdnHCl solutions
were subtracted from the observed values
Gel-filtration chromatography
Gel-filtration chromatography was performed with an
Amersham Biosciences A¨KTA FPLC system using a
Superdex 200 HR 10/30 column d2-Crystallin (0.6 and
2.4 lM,100 lL) equilibrated in buffer and 1M GdnHCl
was loaded onto the column pre-equilibrated with 50 mM
Tris/HCl buffer containing the same concentration of
GdnHCl,pH 7.5 Calibrated standards were measured
under the same conditions
The partition coefficient (Kav) of the eluted component
was calculated by the following equation:
Kav¼ ðVe V0Þ=ðVt V0Þ where, Veis the elution volume of the protein elution peak and Vtand V0 are the total volume and the void volume of the column,respectively
Analytical ultracentrifugation All analyses were performed at 20C using a Beckman Optima XL-A analytical ultracentrifuge and an An-60 Ti rotor For sedimentation velocity experiments,a sample volume of 0.45 mL was used,and the radial scans were recorded at 5-min intervals at rotor speed of 50 000 r.p.m for 2.5 h TheSEDFITsoftware was used for data analysis [22] Sedimentation equilibrium measurements were per-formed at two speeds,18 000 r.p.m and 20 000 r.p.m ORIGIN software was used for data analysis Protein partial specific volumes in GdnHCl solution were calcu-lated from amino acid composition [23] The solvent density was estimated as described [24] For each set of experiments,a single species model was used to estimate the apparent weight–average molecular mass (MW,app) of the protein The data were further fitted to a T–M association system A molar extinction coefficient of
Fig 2 Time-dependent unfolding of duck d 2 -crystallin analyzed by
gel-filtration chromatography (A) Traces a–c correspond to d 2 -crystallin
(2.4 l M ) incubated with 1 M GdnHCl for 0,10 and 60
min,respect-ively The M r markers (.) were (from left to right): thyroglobulin
(669 kDa),ferritin (440 kDa),catalase (232 kDa),albumin (67 kDa),
and ovalbumin (43 kDa) (B) Relative amount of monomers measured
from the peak height.
Fig 3 Continuous sedimentation coefficient (A) and molecular mass (B) distributions of duck d 2 -crystallin in GdnHCl d 2 -Crystallin (1.3 l M ) was equilibrated in 0 (d),0.6 (s),0.8 (m),1 (n) and 2 M (j) GdnHCl solution.
Trang 41.1· 105M )1Æcm)1 for d2-crystallin was used in
calcula-tion of dissociacalcula-tion constant [5] The fit quality of the
models was examined by the residuals and by
minimiza-tion of the fit variance
Results
Dissociation of tetrameric d2-crystallin in GdnHCl
A complex unfolding process is observed when tetrameric
duck d2-crystallin is equilibrated in GdnHCl solutions [15]
The protein appears to dissociate into monomers before
further unfolding occurs The detailed
dissociation/unfold-ing mechanism of the protein remains to be determined
In this study,we examined the Mr of the protein after
equilibration in various denaturing concentrations of
GndHCl Figure 1 shows the distribution of various species
under different GdnHCl at two different protein
concen-trations [0.6 lMfor (A) and 2.4 lMfor (B)] Dissociation of
the protein at 1M GdnHCl and the unfolding at higher
GdnHCl concentrations are observed The unfolded forms
easily polymerize and finally aggregated This process can
be reversed simply by dilution
The time course of the dissociation process of the protein
at 1MGdnHCl was also determined (Fig 2) Dissociation
takes place a few minutes after addition of 1M GdnHCl
(Fig 2A) More than one protein form was resolved,
including a form eluting earlier than the native tetramer and
a form of intermediate size between the tetramer and
monomer,which was ascribed to a dimeric form also
observed during refolding (Fig 1) Dissociation of the
tetrameric form reached equilibrium after 30 min (Fig 2B)
The peak that eluted earlier than native protein could represent a partially unfolded tetrameric form,which is probably a molten-globule state with exposed hydrophobic patches as detected by ANS binding (Fig 6A) This partially unfolded form is easily polymerized as demonstra-ted by chromatography with lower Kavand was dependent
on the protein concentration Upon dilution of the GdnHCl concentration aggregates redissolved (Fig 1)
Sedimentation experiments Dissociation of d2-crystallin in 1M GdnHCl was further examined by analytical ultracentrifugation Native tetra-mers apparently sedimented as a single species with a sedimentation coefficient (velocity of sedimentation divided
by the acceleration of the force field) of 9.14 s (Fig 3) Incubation of the protein in 0.2M or 0.4M GdnHCl resulted in a decrease in the s-value to 8.87 s and 8.51 s, respectively This result confirmed that the change in Kav observed by gel-filtration chromatography was due to protein unfolding Two components appeared at 0.6M GdnHCl (Fig 3) At 0.8MGdnHCl,two components were observed with sedimentation coefficients of 7.8 and 3.8 s, respectively,consistent with the species observed by gel-filtration chromatography (Fig 1) In 1M GdnHCl solution,only one component was detected with a sedi-mentation coefficient of 3.76 s (Fig 3) The predominant component detected by sedimentation coefficient distribu-tion had a molecular mass corresponded to that of a monomer However, Mr of the component with larger s-value at 0.8M GdnHCl was determined to about
130 kDa,which is consistent with it being a dissociated
Fig 4 Gel-filtration chromatography and
sedimentation equilibrium ultracentrifugation
analysis of duck d 2 -crystallin (0.6 l M )
equili-brated with 1 M (A,B) and 2 M (C,D) GdnHCl.
(A) and (C) Gel-filtration chromatography
analysis The M r markers used () were (from
left to right): thyroglobulin (669 kDa),ferritin
(440 kDa),catalase (232 kDa),albumin
(67 kDa),and ovalbumin (43 kDa) The
dashed line in (C) is the elution profile of
native d 2 -crystallin (B,D) Sedimentation
equilibrium analysis of the same samples s
(bottom panel) show absorbance at 280 nm.
The solid line indicates best fit to a
self-asso-ciating system The upper panel shows the
residuals for the best fit.
Trang 5dimer (Fig 3B) The dissociated monomers possessed
significant amount of secondary and tertiary structure as
judged by the CD and fluorescence changes (Fig 5A)
Under the same conditions,the Mr of the major
component was further analyzed by equilibrium
sedimen-tation As d2-crystallin is apparently polydispersed in a 1M
GdnHCl solution,a monomer–tetramer self-association
model was adopted in data analysis [25] An equilibrium
constant (Kd) of 0.34 lM3was obtained which predicted that
76% of the protein existed as monomers,consistent with the
results obtained from gel-filtration chromatography
ana-lyses (Fig 4A) Analysis of behavior at different protein
concentrations allowed the apparent molecular weight to be
estimated as 59 490 ± 210 Da by extrapolation The value
averaged from two rotation speeds has about a 15%
discrepancy compared to the calculated molecular weight of
monomeric d2-crystallin
Multistep unfolding of monomeric d2-crystallin
Further increases in GdnHCl concentration induced
unfold-ing of the dissociated monomers This unfoldunfold-ing process
was investigated by quenching of intrinsic protein
fluorescence by KI and acrylamide The solvent accessibility
of tryptophan residues in protein was subject to the conformational fluctuations KI is ionic in nature and can selectively quench exposed tryptophan residues,while the nonionic quencher acrylamide can nonselectively quench both buried and exposed tryptophan residues
The GdnHCl concentration-dependent changes of the dynamic quenching constant were apparently a multistate process as it did not conform to the smooth two-state transition observed for simple protein molecules (Fig 6) The dynamic quenching constants for the individual quencher interacting with d2-crystallin in native and 6M GdnHCl solution were 1.8 ± 0.1 and 9.4 ± 0.3 for acrylamide,and 0.4 ± 0.02 and 4.1 ± 0.07 for KI, respectively The fa value for KI was only 12% implying the low solvent accessibility of tryptophan residues of
d2-crystallin in the native form
ANS binding ANS is a sensitive probe commonly utilized to detect conformational changes of proteins especially in the molten globule state [26] Binding of ANS to the hydrophobic surface of proteins will lead to fluorescence enhancement as well as a blue shift in its emission maximum Native
d2-crystallin binds to ANS with maximum emission at
480 nm and slightly enhanced fluorescence intensity (Fig 5A) Denaturation of d2-crystallin results in complete loss of ANS binding The fluorescence intensity at 470 nm increases sharply at GdnHCl concentrations exceeding 0.5M and reaches a maximum at around 1M The fluorescence intensity gradually decreased as GdnHCl concentrations were further increased The unfolding curve using the ANS fluorescence probe suggested a multistate process,consistent with the results obtained by protein intrinsic fluorescence and CD The binding affinity (Kd)
of ANS to d2-crystallin in 0,0.84M and 2M GdnHCl solution was determined to be 53 ± 3.1,63 ± 3.6 and
72 ± 3.5 lM,respectively
Discussion
We have previously proposed a minimum model for the dissociation/unfolding of the tetrameric duck d2-crystallin
in the presence of GdnHCl The dissociation step involves dissociation of tetramer to monomers However,our present results demonstrate the formation of dimers during the dissociation step (Fig 1) These results are in agreement with the double dimer structure of the tetra-meric d2-crystallin The crystal structure of the protein indicated that dimers were bound together by the interaction of three helices,one of which is involved in tetramer formation [10]
The close contact interface between two tightly bound dimers of duck d2-crystallin structure is clearly shown by the surface model (Fig 6) The two subunits are structurally complementary to each other Area a of subunit A has a perfectly structural complementarily with area b from subunit B in a head-to-tail manner (high light in circle) (Fig 6B) The two b-sheets (label c) at subunit A and C stack together in a face-to-face manner forming another type of subunit association (Fig 6C) This association is not
Fig 5 Equilibrium unfolding of duck d-crystallin in GdnHCl (A)
Unfolding monitored by changes in tryptophan fluorescence at
340 nm (d),CD (s),or ANS fluorescence at 470 nm (m) All
experiments were measured at 25 C at 0.24 l M protein concentration.
(B) The dynamic quenching constant (K SV ) and the fractional
maxi-mum accessible protein fluorescence (f a ) verses GdnHCl The intrinsic
fluorescence of duck d-crystallin (0.24 l M ) in GdnHCl was quenched
by acrylamide (closed symbols) and KI (open symbols).
Trang 6as close and may be weaker than that shown in Fig 6B The
structural and biochemical data are compatible with a
T-D-M model for the dissociation process Dynamic exposure of
tryptophan residues in duck d2-crystallin revealed
conform-ational changes at different concentrations of GdnHCl
(Fig 5B) The results implied a more complicated process
for further unfolding of the dissociated monomer
Substantial tertiary structural changes occur in the presence of 1MGdnHCl but secondary structural changes are minimal The drastic increase of hydrophobic patches strongly suggests that there is a molten globule intermediate [26] This may be the reason why an off-pathway inter-mediate with an exposed hydrophobic core was detected under the present conditions (Fig 6) However,disturbance
Fig 6 Diagram of the surface of duck d-crystallin (1AUWÆpdb) (A) Each subunit of the tetrameric d-crystallin shows a surface with several convex and concave areas as indicated by a, b and c,which participate in subunit association Two neighboring subunits (A and B) associated in a tail-to-head manner to form a compact and tightly bound dimer (B) The top region of the structure (C) participates in another major region of subunit interaction (A and C) This figure was produced using VIEWERPRO (http://www.accelrys.com/viewer).
Trang 7of electrostatic interactions by GdnHCl may contribute to
the promotion of hydrophobic interactions [27] Incorrect
subunit interactions giving rise to aggregate formation
appears to be a competitive kinetic process with respect to
stable monomer [28] More aggregates accumulate at higher
protein concentration (Fig 3) Soluble polymerization is a
reversible process but aggregation at high protein
concen-tration is irreversible At 5M GdnHCl the aggregates are
dissolved,presumably due to the stronger ionic salt effect,
which disrupts interactions between protein molecules and
the polypeptide is in an unfolded state
Chakraborty et al [29] have reported similar GdnHCl
concentration-dependent reversible unfolding of
recombin-ant duck d2-crystallin based on CD experiments,and
proposed a tetramer-dimer-unfolded monomer mechanism
The total free energy change of the process was as high as
64.5 kcalÆmol)1 We have investigated this process with
multiple biophysical probes An elaborate model has been
derived (Scheme 1) based on the following observation:
First,our results revealed that the fluorescence signal was
more sensitive probe than CD for monitoring the
dissoci-ation transition Dissocidissoci-ation reflected a 50% intensity
decrease in tryptophan fluorescence and an 80%
enhance-ment in ANS fluorescence compared to only a 10%
decrease in ellipticity (Fig 5) A CD probe for oligomeric
protein unfolding may be relatively insensitive and miss
some important information
The shoulder at 0.6MGdnHCl in the ANS fluorescence
trace (Fig 5A) and Mr observed with sedimentation
velocity at 0.8 M GdnHCl could represent a dimeric
intermediate (Fig 3B) Thus,the proposed dimeric form
for human argininosuccinate lyase at 2.5 M GdnHCl
could be an artifact Our analytical ultracentrifugation
results indicated that d2-crystallin existed as monomer at
this concentration of GdnHCl (Fig 4) It is unlikely that
recombinant duck d2-crystallin has a grossly different
behavior from protein isolated from duck eyes We believe
that the mechanism shown in Scheme 1 more accurately
describes the dissociation/unfolding process of d2-crystallin
Another possible origin of the discrepancy between the
results of Sampaleanu et al [30] may be the denaturation
time used in these protocols We have performed
time-dependent denaturation of d2-crystallin experiments and
confirmed that the dimers separated upon mixing protein
with GdnHCl and their abundance decrease as incubation
time increased Monomer was the predominant species at
equilibrium (Figs 1 and 2) Sufficient incubation time is
therefore essential for allowing the denaturation reaction to
reach equilibrium [31]
Acknowledgements
We thank Matthew D Lloyd (University of Bath) for reading this
manuscript before publication and Yu-Chin Pon for technical
assistance This work was supported financially by the National
Science Council,Republic of China.
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