Brønsted analysis of the refolding and unfolding rates of a family of mutants with a variety of side chain substitutions for P37 and P38 reveals that the two prolines, which are located
Trang 1Context-dependent effects of proline residues on the stability
and folding pathway of ubiquitin
Maria D Crespo, Geoffrey W Platt, Roger Bofill and Mark S Searle
School of Chemistry, Centre for Biomolecular Sciences, University Park, Nottingham, UK
Substitution of trans-proline at three positions in ubiquitin
(residues 19, 37 and 38) produces significant
context-dependent effects on protein stability (both stabilizing
and destabilizing) that reflect changes to a combination
of parameters including backbone flexibility, hydrophobic
interactions, solvent accessibility to polar groups and
intrinsic backbone conformational preferences Kinetic
analysis of the wild-type yeast protein reveals a predominant
fast-folding phase which conforms to an apparent
two-state folding model Temperature-dependent studies of the
refolding rate reveal thermodynamic details of the nature of
the transition state for folding consistent with hydrophobic
collapse providing the overall driving force Brønsted
analysis of the refolding and unfolding rates of a family of
mutants with a variety of side chain substitutions for P37 and P38 reveals that the two prolines, which are located in a surface loop adjacent to the C terminus of the main a-helix (residues 24–33), are not significantly structured in the transition state for folding and appear to be consolidated into the native structure only late in the folding process We draw a similar conclusion regarding position 19 in the loop connecting the N-terminal b-hairpin to the main a-helix The proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways
Keywords: folding kinetics; NMR structural analysis; proline mutations; protein folding pathway; protein stability
Proline is unique amongst the natural amino acid residues;
the five-membered ring significantly reduces the flexibility of
the polypeptide chain by restricting rotation around the
N-Ca bond to a relatively small region of conformational
space This factor, coupled with the lack of an amide NH
hydrogen bond donor means that proline is not readily
accommodated into regular (a-helical or b-sheet) protein
secondary structure It is, however, more abundant in
connecting loops playing a specific role in b-turn sequences
[1,2], and as a helix capping residue or as a helix terminator
[3–5] Prolines confer pre-organization and rigidity in the
context of small peptide protease inhibitors [6,7], a concept
that has been widely used in biomolecular and
supramole-cular design to overcome the potential energetic cost of loss
of conformational entropy when dynamic molecules
asso-ciate, or when a flexible polypeptide chain folds In the
context of protein folding, the observation that cis and trans
forms of the Xaa-Pro peptide bond are nearly isoenergetic
[8], and separated by a significant activation barrier, can
lead to slow-folding kinetic phases due to the population of
the non-native cis-form in the unfolded state, where the rate
limiting step is the isomerization of the Xaa-Pro peptide
bond [9–12] Heterogeneity in the unfolded state due to slow
isomerization reactions potentially complicates the kinetic
elucidation of folding pathways and the ability to identify partially folded intermediate states or parallel folding pathways [13–21] However, the observation of a wide variation in the amplitude of slow folding phases associated with prolyl isomerization (in many cases less than expected
on the basis of frequency of occurrence in the primary amino acid sequence) suggests that not all non-native cis prolines result in slow folding phases, and that cis–trans isomerization in some structural contexts need not be rate limiting [22–29] More recent studies demonstrate that nonprolyl cis-peptide bonds also contribute to the hetero-geneous pool of unfolded molecules [18,30] Although individual cis-peptide bonds contribute little to the popu-lation ( 0.15–0.5%) in the unfolded protein, their large number generates a significant proportion of slow folding molecules [18,30,31]
We report on the effects of proline on the stability and folding kinetics of ubiquitin, a small model system of 76 residues that is uncomplicated by disulphide bonds and bound cofactors [32] Ubiquitin has been the subject of a number of investigations regarding its folding mechanism Early studies had suggested that the protein populates an intermediate state identified on the basis of deviations of kinetic data from linearity in the refolding arm of chevron plots at low denaturant concentrations [33] More recent studies [13,14,34,35] report apparent two-state kinetics under similar conditions, suggesting that the roll-over effect
in the refolding kinetics may be a consequence of either transient aggregation that is exacerbated by the stabilizing effects of inorganic salts [15,35], or due to data fitting at rates near the instrumental limits where interference from slower phases can decrease apparent folding rates resulting
Correspondence to M S Searle, School of Chemistry, Centre for
Biomolecular Sciences, University Park, Nottingham NG7 2RD, UK.
Tel.: +44 115 9513567, E-mail: mark.searle@nottingham.ac.uk
Abbreviations: TSE, transition state ensemble; GdmCl, guanidinium
chloride.
(Received 8 July 2004, accepted 30 September 2004)
Trang 2in chevron rollover effects [13,14] HX labelling studies and
stopped-flow CD similarly found no evidence for an early
intermediate in the first 2 ms of folding [36]
We show that proline substitutions in yeast ubiquitin
at positions 19, 37 and 38 produce context-dependent
effects on stability with removal of proline at specific
sites having the effect of either significantly increasing
stability (P38A) or destabilizing the protein (P19S and
P37A) A full kinetic analysis of the major fast folding
phase of wild-type yeast ubiquitin (WT*) and of a
number of nondisruptive single-point Ala mutants and
several double mutants, using F-value analysis and
Brụnsted plots, shows that the transition state ensemble
(TSE) is tolerant to proline substitutions at positions 19,
37 and 38, and that these residues are not well structured
in the transition state for folding
Materials and methods
Protein expression
A pKK223-3 plasmid construct containing the yeast
ubiquitin gene was used to express the wild-type protein in
Escherichia colistrain BL21(DE3) under the control of the
isopropyl thio-b-D-galactoside (IPTG)-inducible tac
pro-moter The F45W mutant gene was cloned by overlap PCR
methodology using the wild-type yeast ubiquitin gene in
pKK223-3 (Pharmacia Biotech) as a template The mutated
cassette was inserted between the EcoRI and HindIII
restriction sites of pKK223-3, and the mutation confirmed
by DNA sequencing Competent E coli cells were
trans-formed with this construct Expression and purification
were as described for the wild-type yielding typically
10Ờ15 mgẳL)1of ubiquitin, as previously described [34]
NMR structural analysis
All NMR experiments were performed on a Bruker
Avance600 spectrometer TOCSY and NOESY
experi-ments were used as previously described [34] on 1-mM
protein samples at pH 5.5 Spectra were referenced to
internal trimethylsilylpropionate Data were processed and
assigned using BrukerXWINNMR andANSIGsoftware [37]
Structural models were visualized usingMOLMOL[38]
Equilibrium stability measurements
Protein stability was determined by fluorescence
measure-ments on 1.5 lM solutions of protein in 25 mM acetate
buffer at pH 5.0 and 298 K The change in fluorescence at
358 nm was monitored as a function of guanidinium
chloride (GdmCl) concentration The linear extrapolation
method was used [39Ờ42] assuming that the stability varies
with the concentration of denaturant [D], according to the
expression DGDỬ DGeq+ m [D], where DGD is the
stability at a given [D], m is the constant of proportionality,
and DGeq is the stability in water alone The fraction of
folded protein Ffis derived from fluorescence measurements
according to FfỬ (fD) fU)/(fN) fU), where fD is the
measured fluorescence at a given [D] and fU and fNare
the limiting values for the unfolded and native states,
respectively The mid-point of the unfolding transition
[D]50%for each mutant was determined by nonlinear least squares fitting to the expression:
FfỬ expơmđơD ơD50%ỡ=RT=đ1 ợ expơmđơD
The equilibrium stability DGeq was determined from the expression DGeqỬ Ờm[D]50%, where m for a set of mutants
is assumed constant (10.9 ổ 0.23 kJẳmol)1ẳM )1) [34,43] This approach is justified by the NMR analysis which shows that all of the mutants fold to a native-like structure with only minor localized chemical shift perturbations Thus, mutations are not producing significant changes in the hydrophobic surface area buried, justifying the use of the same m-value for stability measurements Additional cor-rections were used to allow for a small linear denaturant dependence of the fluorescence of both the folded and the unfolded state [39]
Kinetics experiments Fluorescence-detected kinetic unfolding and refolding measurements were performed using an Applied Photo-physics Pi-star 180 spectrophotometer Temperature was regulated using a Neslab RTE-300 circulating program-mable water bath All kinetics experiments were per-formed in 25 mM acetate buffer pH 5.0 at 298 K Refolding experiments were performed by 1 : 10 dilution
of unfolded protein (15 lM in 7M GdmCl) into buffered solutions of different GdmCl concentrations yielding a final protein concentration of 1.36 lM For unfolding experiments, a buffered solution of native protein was unfolded by a 1 : 10 dilution to yield final concentrations
of GdmCl near or above the midpoint of the equilibrium unfolding transition (concentrations of GdmCl in the range 3.7Ờ7.3M) Kinetic measurements for both unfold-ing and refoldunfold-ing reactions were averaged four to six times at each GdmCl concentration In all cases, the GdmCl concentration was determined using a refracto-meter [40]
Analysis of kinetic data The kinetic traces were analysed using a multiexponential fitting procedure (two or three components) The kinetic data were analysed assuming an apparent two-state model using standard equations described in detail by others [41,43,44] The observed rate constant kobs is the sum of the folding and unfolding rates, kobsỬ kfold+
kunfold where kobs is dependent on [D] according to the expression:
lnkobsỬ lnơkunfoldexpđmunfoldơD=RTỡ
ợ kfoldexpđmfoldơD=RTỡ đ2ỡ The dependence of lnkobson [D] gives extrapolated values for kunfoldand kfoldin water alone, together with the slopes
of the folding and unfolding components munfoldand mfold The temperature dependence of the refolding rate was examined at a denaturant concentration of 0.4MGdmCl and 1.81 lMprotein and the data fitted according to the following expressions [30]:
Trang 3lnkobs¼ lnko DGz =RT ð3Þ
where kois the temperature independent pre-exponential
factor ( 108), and the temperature dependence of the
activation free energy DG is given by:
DGz ¼ DHz þ DCpz ðT 298Þ
T½DSz þ DCpz lnðT=298Þ ð4Þ with DH, DCp and DS representing the change in
activation enthalpy, heat capacity and entropy of formation
of the TSE for folding (U-) Reported errors reflect the
quality of the nonlinear least squares fit to the experimental
data
Results
Context-dependent effects of proline substitutions
on protein stability
We have used the F45W mutant of yeast ubiquitin as our
wild-type protein (WT*) for mutational and biophysical
studies The partially buried indole side chain (Fig 1)
undergoes a significant (fourfold) quenching of fluorescence
on folding but has previously been shown to have only a
relatively small effect on the stability (DDG 1 kJÆmol)1)
and structure of human ubiquitin [45] Our own structural
analysis of F45W mutants of the yeast protein confirms this
We have explored the context-dependent effects of proline
on ubiquitin stability by introducing a number of
substitu-tions at posisubstitu-tions P37 and P38 The equilibrium stability of
the mutants was determined from the change in fluorescence
at 358 nm as a function of GdmCl concentration The data
show that in each case the fraction unfolded fits well to a
two-state transition with the observation of a range of
mid-point denaturant concentrations, [D]50%values, indicating
significant context-dependent effects of the mutations on
protein stability (Fig 2; Table 1) The P37A mutation
produces a large shift in the transition mid-point for
denaturation from 2.62MGdmCl (WT*) to 2.18MGdmCl
This equates to a reduction in stability of 4.5 ± 0.6 kJÆ mol)1 In contrast, the P38A mutation results in a significant increase in stability of)4.6 ± 0.6 kJÆmol)1 The A37A38 double mutant is slightly less stable than WT* (1.1 ± 0.6 kJÆ mol)1), showing that the contributions from P37A and P38A are approximately additive
We also examined the effects of substituting a proline residue at position 19 in the loop region connecting the N-terminal b-hairpin to the main a-helix (Fig 1) Proline is highly conserved at this site in many species; however,
in yeast ubiquitin residue 19 is serine The mutation S19P produces a significant increase in stability of )5.3 ± 0.7 kJÆmol)1 Thus, the P19S, P37A and P38A mutations produce contrasting effects that do not appear to simply relate to entropic factors concerning changes in backbone flexibility
Structural analysis of the proline mutants by NMR NMR structural analysis was used to establish whether the substitutions of P37 and P38 are substantially perturbing the conformation and dynamics in this region of the protein,
or more specifically, whether structural effects are transmit-ted to the C terminus of the adjacent main a-helix (residues 24–33) We have completed an NMR backbone assignment
of WT* for comparison with P37A, P38A and the A37A38 double mutant and have examined chemical shift perturba-tions and the pattern of NOEs in the vicinity of the mutation sites Deviations of Ha signals from random coil chemical shifts provide a sensitive probe of local tions to secondary structure [46,47] We find that perturba-tions are largely confined to the residues immediately adjacent to the mutation site, in particular Ile36 (Fig 3) In the case of the P37A mutant, some small (< 0.1 p.p.m.) longer range effects are observed involving residues on the Pro37 face of the main a-helix (namely, Asp24, Ser28 and Gln31) The characteristic pattern of NH–NH sequential NOEs enables us to map the extent of structure formation within the main a-helix (residues 24–33), and examine the integrity of the helix C-capping motif and of the short helix (residues 38–40) In ubiquitin, the C-capping motif involves
a hydrogen bond between Gly35 NH and the backbone carbonyl of Gln31 This interaction positions Ile36 to form hydrophobic contacts to Ile30 and results in strong NH-NH sequential NOEs between Gln34« Gly35 « Ile36 Fur-ther, the NH signal of Ile36 is > 1 p.p.m upfield shifted by these interactions These NOEs are clearly evident in the NOESY data for WT*, P37A, P38A and A37A38 Further, Ile36 NH has the characteristic upfield shift that confirms that the C terminus of the helix and the C-capping motif are not disrupted by the proline mutations Extending the analysis to the short helix (residues 38–40), the strong sequential NH–NH NOEs from D39 through to Q41 are preserved in all mutants The P38A mutation appears to extend the helical turn by one residue with Ala38 having a
3JNH–Ha value < 6 Hz with evidence of i,i+3 NOEs to Gln41 NOE contacts from Ala38 protons to the side chains
of Lys27 and Gln31 in the main a-helix are also evident and confirm that the Ala38 methyl group occupies the same hydrophobic pocket as the side chain of Pro38 Modelling the structure with Ala substitutions imposed on the backbone conformation of WT* shows that the pattern of
P37
P38
P19
W45
Fig 1 Ribbon structure modelled on the X-ray structure of human
ubiquitin [32] The position and orientation of the side chains of Pro19,
Pro37 and Pro38 are highlighted along with the F45W mutation
(drawn using MOLMOL [38]) The sequences of human and yeast
ubiquitin differ at the following positions: P19S, E24D and A28S.
Trang 4NOEs is entirely consistent with native-like /,w angles Thus, we conclude that the Pro to Ala substitutions are not significantly perturbing the backbone conformation and dynamics of the protein around the mutation sites and in the adjacent a-helix Analogous NMR studies of the S19P mutant (data not shown) also establish that chemical shift perturbations are entirely localized to the mutation site and immediately flanking residues
Kinetic analysis of ubiquitin folding The folding kinetics of WT* have been analysed from refolding and unfolding stopped-flow experiments in GdmCl at 298 K and pH 5.0 in 25 mM acetate buffer The refolding traces for WT* in the range 0–2.5MGdmCl are best analysed in terms of a multiexponential fit reflecting
at least three resolved folding phases The fast phase, which accounts for 87% of the amplitude of the fluorescence change, has an extrapolated folding rate in water of 303 s)1, while several minor slower folding phases are also evi-dent with extrapolated rate constants k2¼ 34 s)1 and
Fig 2 Equilibrium denaturation curves for
yeast ubiquitin (WT*) and various mutants.
Fraction unfolded is plotted against
concen-tration of GdmCl at pH 5.0 in 25 m M acetate
buffer at 298 K and was monitored by
tryptophan fluorescence Stability data are
shown in Table 1.
Table 1 Equilibrium stability data for ubiquitin mutants (pH 5.0,
25 m M acetate buffer, 298 K) determined by GdmCl denaturation
monitored by changes in tryptophan fluorescence.
Mutant
m eqa
(kJÆmol)1Æ M )1 ) [D] 50%
b
DG eqc (kJÆmol)1)
a
Errors in m eq are less than ± 0.35.bDenaturant concentration
at the mid-point of the folding/unfolding transition; fitting errors
are less than ± 0.008 c Equilibrium stability determined from
the [D] 50% value assuming a mean m-value (± SE) of 10.9 ±
0.23 kJÆmol)1Æ M )1
Fig 3 Ha chemical shift analysis of residues
22–46 of the yeast ubiquitin mutants P37A,
P38A and the double mutant A37A38 These
residues span the main a-helical region
(resi-dues 21–35) N terminal to the X37 and X38
mutation sites, and the sequence of the short
helix (residues 38–40) and fourth strand of
b-sheet (residues 42–46) on the C-terminal side
of the mutation sites (Fig 1) Differences in
chemical shifts with respect to random coil
values [46,47] are plotted against sequence
position.
Trang 5k3¼ 0.14 s)1, and relative amplitudes of 11% and 2%,
respectively The k2 and k3 processes, also identified for
human ubiquitin [13,33], have previously been attributed to
slow rate-limiting cis–trans prolyl isomerization reactions
However, we have shown using double-jump (interrupted
unfolding) experiments (data not shown) that k2is a direct
refolding event whose amplitude is unaffected by the
equilibration time of the double-jump experiment In an
isomerization-limited process, the population of the
non-native cis-isomer would be expected to build up only slowly
in the unfolded state (rate constant < 2 s)1[30]) While k2
does not show these characteristics, the slowest phase (k3) is
consistent with a cis–trans rate-limiting event, showing a
significant reduction in amplitude at short aging times
We concentrate here on the major fast folding phase
which yields a chevron plot with both the folding and
unfolding arms varying linearly with the concentration of
denaturant Linearity is clearly observed when either
GdmCl or urea are used as denaturants (Fig 4A) The
kinetic stability calculated from the folding and unfolding
rate constants are in good agreement with those estimated
from the equilibrium denaturation measurements Further,
as can be seen in Fig 4A, the linear refolding and
unfolding arms of the chevron plots in GdmCl and urea
extrapolate to very similar lnkobs values at [D]¼ 0, and
give closely similar stability estimates, consistent with
two-state folding under these different conditions
Addition-ally, we see no evidence for a burst-phase in fluorescence
amplitude in the refolding experiment at low denaturant
concentrations (Fig 5A) Only when refolding
experi-ments are conducted in moderate concentrations of
stabilizing salts, such as 0.4M Na2SO4, do we see any
evidence for deviations from a two-state model Under
these conditions rollover effects are now apparent in the
refolding data at low denaturant concentrations (Fig 4B),
together with burst-phase changes in the fluorescence
intensity (Fig 5B) [33,35] We conclude that the data
collected for yeast ubiquitin at protein concentrations
< 2 lM are adequately described in terms of a two-state
folding model in concurrence with recent detailed studies
of human ubiquitin [13,14,35]
Kinetic experiments on the Pro mutants reveal that the
changes in protein stability associated with the Pro
substi-tutions are largely manifested in effects on the unfolding
rather than refolding kinetics (Table 2) The chevron plot
analysis shown in Fig 6 reveals little change in the m-values
for either the refolding or unfolding phases, indicating that
the TSE is not significantly perturbed by the mutations, nor
do we see any evidence for deviation from the two-state
folding model using the criteria described above
Tolerance to substitutions at the P37P38 site
Kinetic studies with other systems, aimed at probing the
nature of the TSE for folding, have focused primarily on
nondisruptive Ala or Gly substitutions, arguing that more
sterically demanding substitutions have the potential to
shift the position of the TSE along the folding pathway or
even stabilize intermediate states [48,49] We have
exam-ined the robustness of the TSE for folding in the current
context by also introducing more polar or sterically more
diverse mutations in place of P37 and P38 We have
considered three double mutants with a combination of polar, nonpolar and b-branched side chains: SQ, QL and VV, in addition to the Ala substitutions already described
Equilibrium denaturation experiments monitored by fluorescence show that these double mutations have a modest destabilizing effect of < 5 kJÆmol)1 (Fig 2; Table 1), suggesting that their location close to the surface
Fig 4 Chevron plot analysis of the logarithm of the refolding and unfolding rates vs concentration of denaturant (GdmCl) (A) WT* in GdmCl and urea (298 K in 25 m M acetate buffer, pH 5.0) Dotted lines extend the unfolding arms to the y-axis to determine the unfolding rate constants in buffer alone, [denaturant] ¼ 0 The estimated sta-bility constants from DG ¼ –RT ln(k fold /k unfold ) are )25.5 kJÆmol )1 (GdmCl) and )25.8 kJÆmol )1 (urea); m-values are estimated as follows in urea, m fold ¼ 1604 ± 88 JÆmol)1Æ M )1 and m unfold ¼
2919 ± 43 J mol)1Æ M )1 (B) Refolding and unfolding data for WT* as
in (A) and in the presence of 0.4 M Na 2 SO 4 The data for the latter were fitted to a three-state on-pathway model (U«I«N) in which the intermediate state is significantly populated with an equilib-rium constant K UI ¼ 204 Rate constants and m-values are as follows: m UI ¼6992 ± 250 JÆmol)1Æ M )1 , k IN ¼ 468 ± 70 s)1, m IN ¼
1001 ± 378 JÆmol)1Æ M )1 , k NI ¼ 0.0034 ± 0.0011 s)1 and m NI ¼
3103 ± 168 JÆmol)1Æ M )1
Trang 6of the protein may allow some flexibility in accommodating
these side chains NMR analysis of Ha chemical shifts for
the SQ and QL mutants, in line with structural studies
described above, confirms that only relatively small local
perturbations to the structure have taken place Detailed
kinetic analysis shows that the reduction in stability of these
mutants is largely manifested in perturbations to the
unfolding rates with the degree of compactness of the
TSE (aD) and linearity of the chevron plots very similar to
WT* (Fig 6)
The analysis of multiple mutations at a common site (P37/P38) is conveniently expressed in terms of a Brønsted plot, allowing the relationship to be examined between the logarithm of the refolding and unfolding rates and the effect on protein stability [50] Such a relationship should enable us to assess the extent to which P37 and P38 are involved in native-like contacts in the TSE Linear Brønsted plots have been interpreted as indicating that the residues at the mutation site give rise to the same degree of partial structure in the transition state as in WT*, and that the substitutions are not significantly perturbing the position of the TSE along the folding pathway [51,52]
We have considered the P37/P38 mutations simultaneously and constructed the Brønsted plot shown in Fig 7 on the basis of the following:
lnkfold¼ lnkfold bfDDG=RT ð6Þ lnkunfold¼ lnkunfold þ ð1 bfÞDDG=RT ð7Þ where kfold and kunfold are the rate constants for folding and unfolding of WT*, kfoldand kunfoldare the folding and unfolding rates of the mutants derived from the chevron plot analysis, and bfis a constant describing the degree of native-like structure formation in the TSE at the P37/P38 site The plots of kfold and kunfold vs DDG/RT (both DDGeq/RT and DDGkin/RT; Fig 7) are linear demonstra-ting that all mutants show the same degree of structure formation in the TSE, which appears to be tolerant to the variety of changes introduced Values of bf¼ 1 have been interpreted as evidence that residues at the mutation site occupy a highly native-like environment in the TSE, whereas much smaller values (close to zero) suggest that these residues are largely unstructured in the rate-limiting step for folding The linear plots in Fig 7 indicate a bf-value
of 0.09 supporting the latter model We see that the proline mutations produce very small effects on the folding rate of ubiqutin with only a two-fold difference between the fastest and slowest folding mutants In contrast, we see a 26-fold range in the rate of unfolding
This trend is also reflected in the effects of the S19P mutation on the kinetics The significant stabilizing effect
of this mutation ()5.3 kJÆmol)1) is also manifested largely
in a deceleration of the unfolding rate By analogy with the above analysis,F-values provide an estimate, on the scale
of 0–1, of the extent to which a side chain interaction formed in the native state, and which is deleted through mutation, is present (F ¼ 1) or absent (F ¼ 0) in the TSE for folding [53,54] Formerly, the F-value was calculated as:
U¼ RT lnðkfold WT=kfoldmutÞ=DDGeq ð8Þ where kfoldWT* and kfoldmut are the folding rates for the WT* and mutant protein, and DDGeqis the difference in equilibrium stability between mutant and WT* The single point S19P mutation leads to aF ¼ 0.37, which points to the stabilizing effect of this mutation not being realized in the folding TSE, indicative of the loop between the N-terminal b-hairpin and the main a-helix remaining flexible in the TSE, with native-like contacts and back-boneF,w angles becoming consolidated at a late stage in the folding process
Fig 5 Amplitude of the raw fluorescence signal for the refolding of
WT* ubiquitin In the absence (A) and presence of 0.4 M Na 2 SO 4 (B) at
298 K in 25 m M acetate buffer, pH 5.0 The black dots and solid line
are the fit to the refolding data enabling a two-state equilibrium
unfolding curve to be constructed The dashed line (circles) is a linear
fit in (A) to the denaturant dependence of the fluorescence signal of the
unfolded state In (B), in the presence of stabilizing salt, the
fluores-cence signal of the unfolded state (dashed line, circles) shows deviations
from a linear extrapolation, providing evidence for a burst phase
around 1 M GdmCl where the fluorescence intensity increases
signifi-cantly as the collapsed state is destabilized by the denaturant This is
consistent with the curvature observed in the corresponding chevron
plot in Fig 4B and formation of an intermediate collapsed state at low
denaturant concentrations.
Trang 7Activation parameters for folding
The temperature-dependence of the refolding kinetics were
examined in detail for WT* and the A37A38 double mutant
under fixed refolding conditions (0.4MGdmCl) to
deter-mine thermodynamic parameters for formation of the
folding TSE Because formation of the TSE buries a
significant hydrophobic surface area (aDvalues 0.66–0.71),
the temperature dependence of the refolding rate should be
associated with a nonzero change in heat capacity [8,30]
The experimental data show a pronounced curvature,
consistent with the large aDvalues observed (Fig 8)
data were fitted to Eqn (3) over the temperature range 283–
310 K to give DCp values of)2.1 (± 0.3) and )2.4 (± 0.5)
kJÆK)1Æmol)1, respectively The activation enthalpy and
entropy terms for folding are also very similar for the two
proteins The positive entropy change (25 ± 4 and
28 ± 6 JÆK)1Æmol)1, respectively) reflects a small
favour-able stabilization of the TS, however, the enthalpy term
(66 ± 2 and 67 ± 2 kJÆmol)1, respectively) is highly
unfa-vourable to folding and dominates the size of the activation barrier, DG [9]
Discussion
Context-dependent effects of proline residues
on protein stability Ubiquitin is highly conserved across species with the yeast and human forms differing in only three residues (S19P, E24D and A28S) The first of these is located in a loop region which connects the N-terminal b-hairpin sequence (residues 1–17) to the main a-helix (residues 24–33) (Fig 1) The E24D and A28S substitutions lie within the main a-helix Both structures have conserved prolines (P37 and P38) in adjacent positions at the N terminus of a short a-helix (residues 38–40) in an otherwise extended loop region connecting the C terminus of the main a-helix to subsequent strands of b-sheet (Fig 1) We have investigated the context-dependent effects of mutations at these sites on
Fig 6 Chevron plot analysis of the logarithm
of the refolding and unfolding rates vs concen-tration of denaturant (GdmCl) Data shown for WT* and all ubiquitin mutants studied (298 K
in 25 m M acetate buffer, pH 5.0) Refolding and unfolding were monitored by changes in tryptophan fluorescence at 358 nm Kinetic data were determined by fitting to Eqn (2); results are shown in Table 2.
Table 2 Kinetic data for the refolding (U fi N)/unfolding (N fi U) of ubiquitin mutants (298K, pH 5.0 in 25 m M acetate buffer) monitored by changes in tryptophan fluorescence using GdmCl denaturant a D -values determined from m UN /(m UN + m NU ).
Mutant kN fi U(s)1)
m N fi U (JÆmol)1Æ M )1 ) k U fi N (s)1)
m U fi N (JÆmol)1Æ M )1 ) a D
Trang 8protein stability, and their involvement in the folding
pathway from studies of refolding/unfolding kinetics While
the single point mutation P37A is destabilizing by
4.5 kJÆmol)1, in contrast the P38A mutation produces
an equal and opposite enhancement of stability of
)4.6 kJÆmol)1 The reduction in stability of the A37A38
double mutant approximates to the additive effects of the
single point mutations (1.1 kJÆmol)1) Thus, the observed
changes in stability cannot be interpreted purely in terms of
entropic effects on the flexibility of the polypeptide back-bone since in one case removal of proline leads to an enhancement of stability The side chain solvent accessibility
of P37 and P38 is quite similar in the native protein (53% and 46%, respectively) Truncation of the P37 side chain removes van der Waals contacts with the side chain of Q40, and these may account for some loss of stability In contrast, structural analysis suggests that removal of the P38 side chain, which substantially enhances stability by )4.6 kJÆmol)1, favours greater solvent accessibility of the partially buried Q41 side chain and this may be a contributing factor to the stability changes Further, proline
is a good helix capping residue and P38 is found to N-cap the short three-residue helix spanning residues 38–40 The S19P mutation produces a substantial increase in stability ()5.3 kJÆmol)1) which we can also attempt to rationalize on the basis of the X-ray structure of human ubiquitin which already has Pro at this position The structure shows that the Pro19 side chain forms significant hydrophobic contacts with the side chain of Met1, which becomes more solvent accessible when Pro is replaced with Ser There may also be solvation implications for the Ser hydroxyl group, which may also contribute a small destabilizing effect The contrasting effects of the S19P, P37A and P38A mutations
on stability appear to reflect a complex balance between entropic factors relating to changes in backbone flexibility, changes in hydrophobic surface burial, effects on solvent accessibility to other polar groups and changes in intrinsic backbone conformational preferences These observations are consistent with those of others that proline residues play
a variety of context-dependent roles in modulating protein stability [10–12,16,19]
Apparent two-state model for folding of ubiquitin There have been conflicting reports as to whether ubiquitin folds via an apparent two-state model or via a more complex process involving a significantly populated inter-mediate, which forms rapidly in the dead-time of the stopped-flow experiment [13,14,33] In the case of the yeast protein described here, the linear dependence of the folding and unfolding rates on denaturant concentration (both GdmCl and urea), and the lack of a burst phase change in fluorescence intensity at low denaturant concentrations, is indicative of an apparent two-state model in which any intermediate state is too high in energy to be significantly populated [34,35] However, kinetic experiments at low temperature, using multiple probes including CD and SAXS, suggest rapid formation of a compact ensemble which is invisible by fluorescence [55] All of the mutants studied here by fluorescence conform to the two-state model Only in the presence of stabilizing inorganic salts (0.4MNa2SO4) do we see any evidence for nonlinear effects consistent with rapid collapse to a compact intermediate [15,33,35] Recent results describing folding studies of human ubiquitin have established that transient aggregation effects are an important factor in accounting for nonlinear effects on refolding rates [35] Possible errors in determining rate constants near the limit of detection, further compli-cated by slow isomerization-limited phases, have also been proposed to result in roll-over effects in chevron-plot analysis [13,14]
Fig 8 Temperature dependence of the refolding rate for WT* yeast
ubiquitin and the proline-free A37A38 mutant Data collected in 0.4 M
GdmCl at pH 5.0 in 25 m M acetate buffer The logarithm of the
observed rate constant vs 1/T shows distinct curvature reflecting a
significant change in heat capacity associated with TS formation Solid
lines represent the best fit to Eqn (3) from which activation parameters
(DH, DS and DC p ) have been determined.
Fig 7 Brønsted plot showing logarithm of the observed rate (refolding
and unfolding) vs change in stability (DDG/RT) for the family of P37/
P38 mutants DDG values were estimated from both equilibrium
(cir-cles) and kinetic data (squares) Data were fitted to the linear
corre-lations represented by equations 6 and 7 A b f value of 0.09 indicates
that the loop region containing the two adjacent proline residues is
largely unstructured in the rate-limiting step for folding.
Trang 9A description of the TSE for folding of ubiquitin, at the
level of a detailedF-value analysis to map out interactions
present in the TSE, has not yet been reported However,
human ubiquitin has been studied by Krantz et al [56] using
a combination of w-value analysis and protein engineering
methods to introduce bis-His metal coordination sites to
identify native noncovalent interactions involved in the
folding TSE [57] This approach, through metal
complex-ation, enables the degree of partial structure formation at
specific sites to be continuously varied over a wide range of
relative populations such that the effects on the rate-limiting
step can be determined The conclusions of this novel
approach are that ubiquitin folds through a native-like TSE
with a common nucleus but with heterogeneous structural
features populated according to their relative stability A
broad TSE, and pathway diversity, reflects the variable
degrees of structure formation which appears to be formed
around a common folding nucleus consisting of part of the
major helix docked against native-like b-strand structure
Previously, HX exchange studies have suggested that the
formation of hydrogen bonded structure (and hence
pro-tection against NH/ND exchange) occurs in a single
co-operative event from which all of the major secondary
structure emerges [36], suggesting a loose TSE driven by
hydrophobic collapse in which secondary structure is yet to
be consolidated
Analysis of the kinetic data for the single and double
P37P38 mutants using the Brønsted analysis [48,50]
dem-onstrates that all mutants show the same degree of structure
formation in the transition state, with a b-value close to zero
(0.09) The data indicate that these residues are largely
unstructured in the rate-limiting step for folding, forming
native like contacts at a late stage along the folding
co-ordinate We draw a similar conclusion from the S19P
single point mutation where we obtain an estimatedF-value
of 0.37 [53,54] Although the w-value analysis described by
Krantz et al has implicated the N-terminal b-hairpin
sequence (residues 1–17) and part of the main a-helix
(Fig 1) in the folding nucleus, the loop connecting the two
elements of secondary structure does not appear to be
significantly ordered Similarly, P37 and P38 in adjacent
positions at the N terminus of a short a-helix (residues
38–40) in an otherwise extended loop region connecting the
C terminus of the main a-helix to subsequent strands of
b-sheet (Fig 1), also appears to play a passive role in the
rate-limiting step for folding
Activation parameters for folding and formation
of a compact transition state
The temperature-dependence of the refolding rate provides
thermodynamic insights into the nature of the TSE for
folding Curvature in the plot of 1/T vs ln kfold is
characteristic of a change in heat capacity associated with
burial of hydrophobic surface area The aDvalues derived
from the denaturant dependence of kfold and kunfold,
namely from the mfold and munfold values, are consistent
with a compact TSE (aD in the range 0.66–0.71) The
temperature dependence of the refolding rate enables us to
estimate a DCp of)2.1 (± 0.3) to )2.4 (± 0.5) kJÆK)1Æ
mol)1 for WT and the A37A38 double mutant Despite
the small fitting errors, the estimated DC values are
subject to the uncertainties of having measured the refolding rates over a relatively narrow range (283–
310 K) where the total curvature of the plot is small
Literature estimates of DCpUN for the full U–N folding transition from DSC and van’t Hoff analysis are close to
5000 JÆK)1Æmol)1[58,59] It is not entirely clear whether burial of 66–71% of the hydrophobic surface area of the native state should account for all of the observed DCp for folding, and how other factors relating to desolvation
of polar groups, conformational dynamics and hydrogen bonding also contribute [60] The observation of a positive entropy of activation (DS) suggests that the favourable entropic contribution from release of ordered water associated with the hydrophobic effect is able to overcome the conformational entropy term associated with ordering the flexible polypeptide chain in TSE formation The large positive enthalpy of activation also attributed to the thermodynamic consequences of the hydrophobic effect [9], dominates DG for TSE formation Thus, a positive DS, a positive DH, a significant negative DCp and large
aD are all consistent with hydrophobic surface burial driving the folding polypeptide chain over the transition state energy barrier We have shown that the proline residues play a passive role in the apparent two-state folding of ubiquitin, forming native-like contacts at a late stage in the folding process, despite the observation that mutations produce significant and highly context-depend-ent effects on protein stability
Acknowledgements
MDC thanks the University of Nottingham, Astex Technology Ltd and Roche Products Ltd for funding, GWP thanks the EPSRC and GlaxoSmithKline for financial support, and RB acknowledges the EU for a Marie-Curie individual research fellowship.
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