LamA is inactive at room temperature and shows maximum enzymatic activity at 104C, where ‘normal’ proteins from mesophilic organisms are already denatured [2,16].. In the simplified pictu
Trang 1hyperthermostable protein
Sotirios Koutsopoulos1, John van der Oost2and Willem Norde1,3
1 Laboratory of Physical Chemistry and Colloid Science, Wageningen University, the Netherlands
2 Laboratory of Microbiology, Wageningen University, the Netherlands
3 Department of Biomedical Engineering, University of Groningen, the Netherlands
Hyperthermophilic microorganisms, which often
belong to the Archaea, are able to grow optimally at
100C or higher [1] After their discovery, it was
nec-essary to revise our ideas about the mechanisms
involved in the maintenance of protein structural
integ-rity and function at elevated temperatures [2,3] For
the stabilization of proteins at high temperatures,
a concerted optimization of structural features is
employed These include reduced solvent-exposed
sur-face area [4], increased packing density [5–7], increased
core hydrophobicity [8,9], decreased length of surface
loops [6] and extended ion-pair networks [10–13]
Nat-ure uses different combinations of the same structural
features to stabilize proteins that are adjusted to other
environmental conditions [14]
In this work, we have investigated the unfolding⁄
refolding process of the extracellular
endo-b-1,3-glu-canase (LamA) from the hyperthermophilic micro-organism Pyrococcus furiosus that flourishes in the surroundings of low-depth undersea volcanic areas at temperatures ranging from 70 to 103C [15] Proteins evolve through a balanced compromise between struc-tural rigidity, allowing for the maintenance of the native conformation at the physiological temperature
of the organism, and flexibility, which is required for functionality LamA is inactive at room temperature and shows maximum enzymatic activity at 104C, where ‘normal’ proteins from mesophilic organisms are already denatured [2,16] In the simplified picture introduced 70 years ago by Anson and Mirsky [17], protein heat denaturation was described as a two-state transition between the native and the denatured state Nowadays, the idea of one or more intermediate states
is well established in many cases of heat-induced
Keywords
calorimetry; endo-b-1,3-glucanase;
hyperthermostable enzyme; protein
refolding
Correspondence
S Koutsopoulos, Center for Biomedical
Engineering, Massachusetts Institute of
Technology, NE47-Room 307,
500 Technology Square, Cambridge,
MA 02139-4307, USA
Fax: +31 617 258 5239
Tel: +31 617 324 7612
E-mail: sotiris@mit.edu
(Received 30 July 2007, accepted
21 September 2007)
doi:10.1111/j.1742-4658.2007.06114.x
The thermal denaturation of endo-b-1,3-glucanase from the hyperthermo-philic microorganism Pyrococcus furiosus was studied by calorimetry The calorimetric profile revealed two transitions at 109 and 144C, correspond-ing to protein denaturation and complete unfoldcorrespond-ing, respectively, as shown
by circular dichroism and fluorescence spectroscopy data Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow Furthermore, pre-viously denatured protein samples gave well-resolved denaturation transi-tion peaks and showed enzymatic activity after 3 and 9 months of storage, indicating slow refolding to the native conformation over time
Abbreviations
DSC, differential scanning calorimetry; DNS, 3,5-dinitrosalicylic acid; DH cal , calorimetrically determined enthalpy change; DH vH , van’t Hoff enthalpy change; LamA, endo-b-1,3-glucanase; Td, denaturation temperature.
Trang 2protein denaturation: the native state is tightly folded,
the intermediate state(s) is functionally inactive and
partially folded in a non-native conformation(s), and
the unfolded state is characterized by significant
amounts of loosely structured domains In terms of
biological functioning, the last two states are
dena-tured, but only the latter resembles the random coiled
conformation A major question is whether the
transi-tions between these states belong to a sequence of
reversible processes that can be described
thermody-namically
Apart from the profound applications of
thermo-zymes in biocatalysis and biotechnology, by studying
the thermal resistance properties of proteins we aim to
tackle one of the most challenging problems in modern
biophysics: what is the mechanism used by these
pro-teins to stabilize their three-dimensional structure and
sustain biological function at anthropocentrically
extreme temperatures?
Results
Calorimetry
Calorimetric studies of LamA in 0.01 m phosphate
buffer showed a single denaturation transition peak
The denaturation temperature Td was dependent on
pH and shifted from c 112C at pH 4.0 to 109 C at
pH 7.0 to 104C at pH 8.5 [18] A typical thermogram
of LamA is shown in Fig 1A (line a) Variation of the
scanning rate between 6 and 90CÆh)1 did not affect
the Td, the shape of the endothermic peak or the
enthalpy associated with the transition This suggests
that the thermal denaturation of LamA is not
kineti-cally controlled [19,20]
The calorimetric criterion introduced by Privalov &
Khechinashvili [21] to judge a two-state transition
requires that the calorimetrically determined enthalpy
change DHcal is equal to the van’t Hoff enthalpy
change DHvH, which may be calculated from the
dif-ferential scanning calorimetry (DSC) thermogram
using the equation
DHvH¼ 4RT2Cp;max
DHcal
ð1Þ
where R is the ideal gas constant, Td is the
denatur-ation temperature and cp,max is the maximum heat
capacity, with regard to the peak baseline, which is
observed at the denaturation temperature A two-state
model implies that transient intermediate states, which
should be distinguished from thermodynamically stable
intermediates such as the molten globule, are not
pop-ulated at the transition temperature [22] The validity
of this criterion has often been argued and therefore care should be taken when it is applied [20] In the case of LamA, the DHcal⁄ DHvH ratio deviates from unity, yielding a value of c 0.5, suggesting a non-two-state transition Furthermore, the standard functions integrated in the microcal origin dsc software (MicroCal Inc., Northampton, MA, USA) could not
fit the endotherms as a two-state transition
The reversibility of the thermal transitions was tested by cooling the protein sample to room tempera-ture Using different cooling rates between 15 and
90CÆh)1, no exothermic transition suggesting protein refolding was observed (Fig 1A, line a¢) Reheating the protein solution in the calorimeter cell, after cooling to room temperature, did not show an endo-thermic peak (Fig 1A, line b) Heating LamA to
C B A
Fig 1 Heat capacity as a function of temperature for 0.5 mgÆmL)1 LamA in 0.01 M phosphate buffer at pH 7.0 (A, B) Cooling and reheating of LamA shows no reversible denaturation peaks (C) Transition observed when the first run is stopped just below the denaturation temperature The scan rates tested were between 6 and 90 CÆh)1 and, after each heating step, the sample was allowed to cool to room temperature.
Trang 3temperatures just above Td did not result in a
revers-ible transition peak (Fig 1B) In another experiment,
the sample was heated up to exactly Tdand was then
allowed to cool to 25C Subsequent reheating
revealed a transition at the same Td value (Fig 1C),
which, however, was characterized by a heat exchange
which was c 50% decreased relative to that associated
with the first peak This suggests that approximately
half of the LamA molecules are irreversibly denatured
during the first partial transition (curve a) [20] These
are standard experiments from which we may conclude
irreversible unfolding
The first experimental evidence of the reversible
thermal denaturation of LamA was observed at very
slow cooling rates As mentioned previously, fast
cool-ing did not result in an exothermic refoldcool-ing peak
However, when very slow cooling of 0.1CÆh)1 was
applied, a refolding transition peak was observed at
the same temperature at which denaturation occurred
during the first heating step (Fig 2) Furthermore, the
enthalpy released during refolding was similar to that
absorbed on denaturation Notably, this protein
sam-ple, which was obtained during slow cooling, was
found to be enzymatically active and, on reheating, a
denaturation peak of slightly lower intensity was
observed at the same temperature as before
Calorimetric tests of long-stored samples of
dena-tured LamA (i.e after 3 and 9 months of storage at
) 20 C) showed well-resolved transition peaks at the
same temperatures as those observed during the first
heating step (Fig 3) However, the enthalpies
associ-ated with these transitions were considerably lower
This indicates that the number of refolded LamA
mol-ecules is smaller than that which initially gave the first
strong endothermic peak and, furthermore, that refold-ing is time dependent It is intrigurefold-ing to suggest that, if the system were given more time, more denatured enzyme molecules would be natively refolded How-ever, it was not possible to test this because, in the absence of antimicrobial agents, the denatured protein sample was not stable for longer periods
Transition phenomena similar to those shown in Figs 1–3 were also observed for LamA in solutions at
pH 6.5 and pH 8.5
Enzymatic activity The specific enzymatic activity of LamA is 1547.6 unitsÆmg)1 at 90C LamA samples derived from very slow cooling experiments were tested and showed recovered activity up to 83% (Fig 4) compared with the activity of the untreated enzyme In the fast-cooled denatured samples of LamA from a standard DSC experiment, the enzymatic activity was completely sup-pressed However, storage of these samples for 3 and
9 months resulted in a notable increase in the enzymatic activity by 8% and 19%, respectively The relatively long time required for the LamA molecules to show measurable activity reflects the slow kinetics of the refolding process to the native conformation
After heat incubation at 150C, samples of LamA did not show detectable activity, even after 6 months
of storage at 4C
Circular dichroism The secondary structural characteristics of LamA in solution were determined using far-UV CD (Fig 5,
Fig 2 Effect of cooling rate on the refolding of 0.5 mgÆmL)1LamA
in 0.01 M phosphate buffer at pH 7.0 Denaturation on heating (a)
at a heating rate of 0.1 CÆh)1 and the exothermic peak (b)
observed on cooling with the same scanning rate.
Fig 3 LamA refolding as a function of time: (a) heat denaturation
of 0.5 mgÆmL)1native LamA in 0.01 M phosphate buffer at pH 7.0; (b) reheating the same sample 3 months later; (c) reheating the same sample 9 months later Scan rate was 30 CÆh)1.
Trang 4top panel) Spectral analysis suggested that the
second-ary structure of native LamA mainly consisted of
b-sheets and turns, up to c 96% Heat-denatured
LamA at 110C still contained 86% of b-structures,
but the amount of a-helices and random coils
increased to 4% and 10%, respectively The far-UV
CD spectrum of LamA after heat treatment in the CD
cell unit at 110C resembled that obtained from the
same protein sample after cooling to 25C Prolonged
heat incubation for 30 min at 150C resulted in the
collapse of the secondary structure, and the
polypep-tide chain of LamA appeared to be unordered (Fig 5,
top panel, curve c)
Following the ellipticity of LamA in a closed cell at
220 nm as a function of temperature, we observed a
transition between 105 and 110C (Fig 5, top panel,
inset) This transition could not be monitored further
because of the instrument’s temperature limitations At
pH 7.0, the denaturation of LamA occurs at c 109C,
which is just below the maximum scanning
tempera-ture of the instrument
Fluorescence spectroscopy
The tryptophan fluorescence emission spectrum of
native LamA shows a maximum at 335 nm (Fig 5,
bottom panel, curve a) Increasing the temperature of
the solution resulted in a gradual decrease in the
fluo-rescence intensity without a shift in the emission
maxi-mum Such a decrease in the intensity is attributed to
increased tryptophan quenching as a result of thermal
motion [23] In comparison with the spectrum of
native LamA at 25C, the spectral profile of
dena-tured LamA at 110C (Fig 5, bottom panel, curve b,
corrected for the temperature effect on the intensity)
showed slightly decreased intensity with a red shift in the emission maximum to 344 nm This indicates a structural distortion, accompanied by partial exposure
of previously confined tryptophan(s) to the solvent After incubation for 30 min at 150C and cooling
to 25C, the emission maximum shifted to 357 nm (curve c) and the intensity decreased three-fold, sug-gesting a collapsed tertiary structure [24]
Discussion
The unfolding⁄ refolding process of LamA was moni-tored using calorimetry Analysis of the thermograms
Fig 4 Enzymatic activity of native and heat-treated LamA at
differ-ent storage times The activity was measured at 90 C in 0.01 M
phosphate buffer at pH 7.0.
Fig 5 Far-UV CD (top panel) and fluorescence emission (bottom panel) spectra of 0.25 mgÆmL)1 LamA in 0.01 M phosphate buffer
at pH 7.0 Curve a, native state (recorded at 25 C); curve b, heat-denatured at 110 C (recorded at 110 C; fluorescence spectrum was corrected for the tryptophan emission yield which decreases
as a function of temperature); curve c, after heat incubation for
30 min at 150 C (recorded at 25 C) Top panel inset: thermal tran-sition of LamA monitored by the molar ellipticity at 220 nm.
Trang 5suggested that the denaturation of LamA could not be
described as a two-state transition, because the
calori-metric criterion, i.e the DHcal⁄ DHvH ratio, deviated
from unity However, the difference between DHcal
and DHvHmay be caused by inherent difficulties in the
precise determination of the peak height and the
inte-grated area of the peak, which has an unusual shape
This point needs further discussion The heat capacity
of denatured LamA, represented by the post-transition
baseline, was unexpectedly low when compared with
the heat capacity of the native state (pre-transition
baseline) It should be noted that, in Fig 1C, line b,
the second up-scan of pre-heated LamA and, in Fig 3
line c, the slowly refolded protein showed the same
negative-like Dcp profile Unless we assume that this is
caused by an instrument artefact operating at the
extreme end of its detection limit (i.e 127C), such a
profile is commonly attributed to aggregation of
dena-tured protein molecules
Previously reported time-resolved anisotropy data
did not show a significant increase in the
hydrody-namic radius of the heat-denatured LamA molecule
compared with the size of the native protein [25] In
addition, the fact that slow cooling resulted in an
exothermic refolding transition suggests that the
aggregation of LamA is not very likely (unless we
assume that protein aggregation is a reversible
pro-cess) In the absence of aggregation, similar
denatur-ation profiles have been reported, but not discussed,
for other hyperthermostable proteins [26–30] The
observed negative-like Dcp upon thermal denaturation
of LamA at 109C may stem, in part, from the
physicochemical properties of liquid water at
temper-atures approaching 110C (H Klump, University of
Cape Town, South Africa, personal communication)
[31] Several lines of evidence support this hypothesis:
(a) extrapolation of calorimetric data by Privalov
[32], in his review on calorimetry in 1979, showed
that the specific entropy of unfolding of several
pro-teins intersects at c 110C; (b) Baldwin’s
hydrocar-bon model predicted that the entropy of mixing DS
of a nonpolar compound with water is negative at
ambient temperatures, but approaches zero at
c 113C [33] (when DS is zero, the solution shows
ideal entropy of mixing and hydrophobic moieties
may be readily dissolved in hydrophilic medium); (c)
Shinoda [34] suggested that the increased solubility of
hydrocarbons at high temperatures also depends on
the enthalpy, which reflects the changes in the
hydro-gen bonding interactions in water surrounding the
nonpolar compound; (d) according to Ne´methy and
Scheraga [35–37], the interaction of hydrocarbons
with water at high temperatures results in changes in
the local structure of the clustered water molecules adjacent to the hydrophobic surfaces (e.g these water molecules show less hydrogen bonding and, therefore, are less hydrophilic) If the exposure of hydrophobic groups to water on denaturation does not contribute much to Dcp, perhaps other factors, such as solvation
of the protein’s polar groups, become more impor-tant
Moreover, we cannot exclude the possibility that, on denaturation, the conformational changes in the pro-tein molecule are such that, from the propro-tein core, more polar (compared to hydrophobic) amino acids are exposed to the polar solvent The resulting struc-turally distorted, partially unfolded equilibrium inter-mediates are probably related to the kinetic folding intermediate reported by Park et al [38] Indeed, the surface of the native LamA molecule contains a large nonpolar fraction A similar post-transition decrease in heat capacity, lower than that expected for a com-pletely unfolded polypeptide, was also observed in the denaturation of the recombinant human growth hor-mone [39] Therein, it was suggested that the protein retained residual structure and, hence, was not fully hydrated after thermal denaturation
Whatever the case may be in the LamA system, these conjectures suggest that a negative Dcp on pro-tein denaturation and unfolding may be possible It is also possible that the observed transition profile rests
on an eluding component or mechanism that has not been considered so far
A more detailed characterization of the state of the protein at temperatures beyond 110C was not possi-ble because of limitations in the existing instrumenta-tion, which is not designed to operate at such biologically extreme temperatures We were able to show that the state of denatured LamA was signifi-cantly different from that of the native protein with regard to secondary and tertiary structural elements (Fig 5) However, as the transition could not be completely monitored by CD or fluorescence spec-troscopy, we could not unambiguously determine whether the post-transitional state of the protein rep-resents unfolding, or if it is just an intermediate which unfolds completely only upon heating to even higher temperatures
To answer the question about the state of LamA
at temperatures above the denaturation point (i.e at
109C), we used a calorimeter with a scanning tem-perature efficiency up to 200C (MC-DSC 4100, Cal-orimetry Sciences Corporation, Lindon, UT, USA) This experiment revealed that, following the main transition peak at 109C, another small exothermic peak appeared at 144C (Fig 6), which suggests that
Trang 6the first peak does not represent complete unfolding,
and that specific protein domains remain folded up to
the temperature of the second transition This implies
that refolding proceeds through an intermediate state
Notably, this partially folded non-native state of
LamA, after thermal denaturation at 109C, may
refold to the native conformation either by ultra-slow
cooling immediately after denaturation or by storing
it for prolonged times Slow cooling resulted in an
exothermic refolding peak, indicating protein
refold-ing This was confirmed not only by enzymatic
activ-ity tests but also by calorimetric studies: (a) on
reheating the refolded protein, a DSC denaturation
profile similar to that observed in the first heating
step was found; and (b) heating a previously
dena-tured LamA sample gave, after long storage,
well-resolved denaturation peaks with partial recovery of
the enthalpy exchange
In this work, we have shown that the
temperature-induced transition of LamA from the native to the
denatured state can be reversed if sufficient time is
given for the system to equilibrate Irreversible
dena-turation is commonly observed on heating of
pro-teins However, the effect of time is rarely considered,
even though theoretical studies have predicted that, in
the absence of aggregation, reversible transitions are
possible when slow relaxation is involved [40] When
the system relaxes more slowly than the time window
of the measurement, i.e the duration of the
experi-ment, we ‘see’ the process as irreversible, but, given
sufficient time, it may well be restored to the original
state This is the case for the refolding of LamA In the article by Kaushik et al [41], the unfold-ing⁄ refolding kinetics were investigated, and it was shown that a predenatured hyperthermophilic pepti-dase from P furiosus could refold completely after
36 h of incubation at 32C Refolding required a few days on incubation at lower temperatures These experiments resemble those presented here, where we showed reversible transition in long-stored frozen LamA samples It is interesting to speculate that this behaviour may also be found in other heat-denatured proteins: refolding to the native conformation may be possible if sufficient time is given to the system
By contrast with the above-mentioned study, we were able to observe by calorimetry an exothermic refolding peak on very slow cooling of denatured LamA It is not clear yet whether the denatured, par-tially folded state of LamA is kinetically trapped as a result of slow relaxation refolding kinetics, or whether this state is a thermodynamically stable form trapped
in a local energy minimum of the energy distribution funnel We can speculate that one of the reasons for the slow refolding process may be the relatively slow cis⁄ trans isomerization of one or more of the 18 prolines of the protein [42] This process requires hundreds of seconds to be completed [43] LamA con-tains only one cysteine, and therefore post-transitional improper intramolecular disulfide bond formation is not possible, which would result in irreversible pro-tein denaturation Chemical modification of amino acids on protein denaturation is possible in denatur-ation processes occurring at such high temperatures However, temperature-induced deamidation of gluta-mines and asparagines could not be detected by mass spectroscopy, because this chemical reaction did not lead to significant changes in the protein mass It is conceivable that this possibility is not very likely because LamA was able to refold to the active pro-tein conformation: extensive chemical changes on deamidation would irreversibly prevent correct protein folding
In conclusion, the calorimetric analysis showed that the transition of LamA from the native state to a par-tially unfolded intermediate was reversible if conditions were selected to give the system sufficient time There
is a strong biological argument supporting the conclu-sions presented here: extracellular LamA is exposed to temperature changes occurring in the microorganism’s environment (e.g volcanic underwater milieu) During
an environmental temperature change, such a system relaxes to the initial state very slowly During this per-iod, denatured LamA molecules may recover their native conformation and biological activity
Fig 6 Heat capacity as a function of temperature up to 200 C of
0.7 mgÆmL)1LamA in 0.01 M phosphate buffer at pH 7.0 (scan rate
was 30 CÆh)1).
Trang 7Experimental procedures
Purification of LamA
The gene encoding LamA (GenBank accession
no.AF013169) was expressed in Escherichia coli (strain BL 21
DE3) and cloned into pGEF+ under control of the T7
pro-moter [16] Further purification was achieved by gel filtration
(Superdex 200, Amersham Pharmacia, Uppsala, Sweden)
The purity of the enzyme was tested using high-performance
liquid chromatography and matrix-assisted laser desorption
ionization-time of flight mass spectroscopy (PerSeptive
Bio-systems Voyager DE-RP mass spectrometer, Framingham,
MA, using sinapinic acid crystallized on a gold-coated well
plate; spectra were calibrated with protein standards) LamA
is a single domain globular-ellipsoid protein with a molecular
mass of 30 085 Da Its isoelectric point is at pH 4.4, as
deter-mined by isoelectric focusing Pure LamA was stored at
) 20 C in 0.01 m phosphate buffer at pH 7.0, without
anti-microbial agents, which might affect the protein’s
physico-chemical characteristics
Differential scanning calorimetry
Calorimetric studies were carried out in a VP-DSC
calori-meter (MicroCal Inc.) Very small heat exchanges of LamA
were recorded between 20 and 130C using, as reference,
the buffer solution All samples were degassed under
vac-uum for 15 min prior to loading the cells, which were
main-tained under a pressure of 2.5 bar to avoid boiling of the
sample The concentration of LamA was 0.5 mgÆmL)1;
experiments were also performed at different concentrations
between 0.1 and 2 mgÆmL)1 with the same results
normal-ized per mass of the enzyme Unless stated otherwise, the
temperature was increased at a rate of 30CÆh)1and, after
reaching the maximum desired value, the sample was
allowed to cool to room temperature Heating rates
between 6 and 90CÆh)1were also used A very slow
cool-ing rate of 0.1CÆh)1(i.e 0.002CÆmin)1) was also tested
The normalized excess heat capacity functions were
obtained after baseline subtraction and data processing
using the formulation of Privalov [32]
Enzymatic activity tests
The enzymatic activity of LamA before and after heat
treat-ment was measured using the colorimetric reagent
3,5-dini-trosalicylic acid (DNS) [44] This method is based on the
spectrophotometric determination of the hydrolysed ends of
oligosaccharides resulting from degradation of the substrate
(i.e laminarin) For the assay, the hyperthermostable
enzyme and the substrate in 0.01 m phosphate buffer at
pH 6.5 were incubated for 10 min at 90C The enzymatic
reaction was stopped by rapidly cooling the sample at room
temperature After the addition of DNS, the sample was
incubated at 100C for 5 min and diluted (1 : 5, v ⁄ v) in water The sample was then allowed to cool to room tem-perature and the absorbance was measured at 595 nm
Circular dichroism measurements
CD spectroscopy was used to investigate the secondary structure of LamA before and after heat treatment
Far-UV (190–260 nm) CD spectra of 0.25 mgÆmL)1 LamA in quartz cuvettes (path length, 0.1 cm) were recorded in a J-715 spectrophotometer (JASCO, Tokyo, Japan) The scan rate was 100 nmÆmin)1, with a resolution of 0.2 nm and response time of 0.25 s Spectra were recorded in a closed metal-caged quartz cuvette under pressure to prevent the evaporation of water The CD spectra of LamA after heat incubation at 150C were collected on samples which had been previously heated and then cooled to room tempera-ture (heat incubation for 30 min at 150C was performed
in a temperature-controlled oil bath using thick-walled glass tubes with a lid capable of withstanding the vapour pres-sure of water) After subtraction of blank spectra, data analysis was performed by fitting the spectra to reference spectra using contin software [45,46]
Fluorescence spectroscopy Fluorescence emission spectra of 0.025 mgÆmL)1 LamA in quartz cuvettes (path length, 1 cm) were recorded in the range 300–400 nm in a Varian Cary Eclipse spectrophotometer (Palo Alto, CA) Spectra of denatured LamA at 110C were recorded in a closed cuvette under pressure to prevent solvent evaporation Fluorescence spectra of LamA after heat incu-bation at 150C were collected on samples that had been cooled to room temperature Excitation was set at 300 nm to excite only the tryptophans The excitation and emission slit widths were 5.0 and 2.5 nm, respectively All spectra were corrected for the background emission peak of water
Acknowledgements
The authors gratefully acknowledge discussions with Horst Klump (University of Cape Town, South Africa)
on the shape of the denaturation profile of the protein from calorimetric data This research was supported
by an Individual Marie Curie Fellowship of the Euro-pean Community programme ‘Improving Human Research Potential and the Socio-Economic Knowl-edge Base’ to S.K
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