pH-dependent relationship between thermodynamic and kinetic stability in the denaturation of human...

lable at ScienceDirect

Biochimie xxx (2014) 1e9

Contents lists avai

Biochimie

journal homepage: www.elsevier .com/locate/biochi

Research paper

pH-dependent relationship between thermodynamic and kineticstability in the denaturation of human phosphoglycerate kinase 1

Angel L. Pey*

Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av./Fuentenueva s/n, E-18071 Granada, Spain

a r t i c l e i n f o

Article history:Received 18 January 2014Accepted 27 March 2014Available online xxx

Keywords:Phosphoglycerate kinaseProtein thermodynamic stabilityProtein kinetic stabilityProtein denaturation mechanism

Abbreviations: PGK, phosphoglycerate kinase; hPGkinase 1; Ea, activation energy; Tm, temperature of tdenaturation transition; DSC, differential scanning ca* Tel.: þ34 686469926; fax: þ34 958272879.

E-mail address: [email protected].

http://dx.doi.org/10.1016/j.biochi.2014.03.0150300-9084/� 2014 Elsevier Masson SAS. All rights re

Please cite this article in press as: A.L. Pey,human phosphoglycerate kinase 1, Biochim

a b s t r a c t

Human phosphoglycerate kinase 1 (hPGK1) is a glycolytic enzyme essential for ATP synthesis, and it isimplicated in different pathological conditions such as inherited diseases, oncogenesis and activation ofdrugs for cancer and viral treatments. Particularly, mutations in hPGK1 cause human PGK1 deficiency, arate metabolic conformational disease. We have recently found that most of these mutations causeprotein kinetic destabilization by significant changes in the structure/energetics of the transition state forirreversible denaturation. In this work, we explore the relationships between protein conformation,thermodynamic and kinetic stability in hPGK1 by performing comprehensive analyses in a wide pHrange (2.5e8). hPGK1 remains in a native conformation at pH 5e8, but undergoes a conformationaltransition to a molten globule-like state at acidic pH. Interestingly, hPGK1 kinetic stability remainsessentially constant at pH 6e8, but is significantly reduced when pH is decreased from 6 to 5. We foundthat this decrease in kinetic stability is caused by significant changes in the energetic/structural balanceof the denaturation transition state, which diverge from those found for disease-causing mutations. Wealso show that protein kinetic destabilization by acidic pH is strongly linked to lower thermodynamicstability, while in disease-causing mutations seems to be linked to lower unfolding cooperativity. Theseresults highlight the plasticity of the hPGK1 denaturation mechanism that responds differently tochanges in pH and in disease-causing mutations. New insight is presented into the different factorscontributing to hPGK1 thermodynamic and kinetic stability and the role of denaturation mechanisms inhPGK1 deficiency.

� 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Phosphoglycerate kinase (PGK; ATP: 3-phosphoglycerate 1-phosphotransferase; EC 2.7.2.3) is glycolytic enzyme present in allliving organisms. PGK catalyzes the reversible phosphotransfer fromATP to 3-phosphoglycerate yieldingADPand1,3-biphosphoglycerate,an essential step for ATP synthesis in the glycolytic pathway [1]. PGKthree-dimensional structure is highly conserved among prokaryotes,yeast andmammals, showing two similarly sized domains (N- and C-domains) connected by a hinge-bending linker [2]. The N-domainbinds 1,3-BPG/3-PG, while the C-domain binds the nucleotides.Ligand binding triggers conformational transitions between different

K1, human phosphoglyceratehe maximum in the thermallorimetry.

served.

pH-dependent relationship bie (2014), http://dx.doi.org/10

open and closed conformations [3,4]. These structural transitionsseem to correspond to functional transitions from low to high activitystates, which in turn can be mimicked by macromolecular crowdingagents in vitro and inside cells [5].

PGK enzymes have been model systems to study protein sta-bility and folding mechanisms in two-domain proteins (recentlyreviewed by Ref. [6]). Equilibrium denaturation by chemical de-naturants of PGK enzymes from different species show significantdiversity in folding mechanisms, from simple two-state to morecomplexmodels involving equilibrium intermediates [7e9]. Kineticfolding/unfolding studies reveal the presence of kinetic in-termediates, especially in the refolding pathways, with evidence fordownhill folding in some cases [10e13]. Several studies have alsoaddressed the relationships between thermodynamic and kineticstabilities using comprehensive analyses of chemical, thermaldenaturation and proteolysis, showing remarkable differences innative state dynamics between PGK from different species that maycontrol protein kinetic stability [7,8,11].

etween thermodynamic and kinetic stability in the denaturation of.1016/j.biochi.2014.03.015

Delta:1_given name

mailto:[email protected]

www.sciencedirect.com/science/journal/03009084

http://www.elsevier.com/locate/biochi

http://dx.doi.org/10.1016/j.biochi.2014.03.015



A.L. Pey / Biochimie xxx (2014) 1e92

PGK exists in humans in two isoforms (hPGK1 and 2) which arefunctionally and structurally alike [2,14,15]. hPGK1 is found in allsomatic and premeiotic cells, while hPGK2 is expressed in meiotic/postmeiotic spermatogenic cells. Beyond its fundamental role inglycolysis, hPGK1 is also involved in oncogenesis and tumordevelopment, DNA replication and repair, and activation of L-nucleoside analogs for anticancer and antiviral treatment (see Ref.[6] and references therein).

Mutations in the PGK-1 gene are associated with hPGK1 defi-ciency, a rare X-linked recessively inherited metabolic disease(OMIM ID 311800). Nearly 40 cases have been reported so far, and21 different mutations found in patients with this disease [6].Recent studies have addresses the role of protein stability in thepathogenesis of this disease, showing that a large fraction of themutations cause protein kinetic destabilization (i.e. higher rates ofthermal denaturation; [7,16,17]. Mutation-induced kinetic desta-bilization seems to be linked to changes in the denaturation tran-sition state, which becomes more native-like as the protein isdestabilized (a Hammond effect; [7]). Moreover, studies usingchemical denaturant and proteolysis kinetics have revealed thatdisease-associated mutations may also decrease unfolding coop-erativity ([11]; Pey, submitted). Interestingly, strong correlationsbetween kinetics of denaturation and proteolysis and the aggre-gation propensity of hPGK1 enzymes in cells have been found,suggesting that folding and stability defects in disease-causinghPGK1 enzymes may translate into their ability to properly foldin vivo (Pey, submitted).

In this work, we explore the complex relationships betweenthermodynamic and kinetic stability in hPGK1 using unfoldingthermodynamic and kinetic analysis by spectroscopic and calori-metric techniques in awide range of pH values. Our studies supportthat pH-dependent relationships between thermodynamic andkinetic stabilities are arised from effects on the energetics of thenative state and the relevant denaturation transition state. We thusprovide a more complete picture on stability of hPGK1 which mayalso be useful to understand hPGK1 deficiency as a loss-of-functionconformational disease.

2. Materials and methods

2.1. Protein expression, purification and preparation

WT hPGK1 was expressed and purified as recently described[7,16]. Experiments performed at different pHs used the followingbuffers (at 50 mM): glycine (pH 2.5e3.5), sodium acetate (pH 4e5);MES (4-morpholineethanesulfonic acid; pH 5.5e6.5), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; pH 7e8). The pHwas adjusted using concentrated HCl or NaOH solutions. Proteinconcentration was measured spectrophotometrically at 280 nmusing a ε ¼ 27,960 M�1 cm�1.

2.2. Spectroscopic analyses

All spectroscopic analyses were recorded at 25 �C usingthermostatized devices. Circular dichroism spectra were ac-quired in a Jasco-710 spectropolarimeter, in a 200e260 nm rangeat a 50 nm/min scan rate, using 1 mm-path length cuvettes and5 mM in protein subunit. Fluorescence spectra were acquired in aCary Eclipse spectrofluorometer using 3 mm path length cu-vettes and 5 mM protein subunit. Trp-emission fluorescencespectra were acquired in a 300e450 nm at a 100 nm/min scanrate using a lem ¼ 295 nm and 5 nm slits. ANS-emission fluo-rescence spectra were acquired in a 425e550 nm range at a100 nm/min scan rate using a lem ¼ 380 nm and 5 nm slits.Dynamic light scattering (DLS) was carried out in a DynaPro MSX

Please cite this article in press as: A.L. Pey, pH-dependent relationship bhuman phosphoglycerate kinase 1, Biochimie (2014), http://dx.doi.org/10

instrument (Wyatt) using 1.5 mm path length cuvettes and 5e10 mM protein in subunit at 25 �C. 50 spectra were acquired foreach DLS analysis, averaged and used to determine the hydro-dynamic radius and polydispersity using the average autocor-relation function.

2.3. Differential scanning calorimetry (DSC)

Calorimetric measurements were performed on a capillary VP-DSC microcalorimeter (GE Healthcare) with a cell volume of0.135 ml. Protein samples were prepared in the different buffers ata 10 mM concentration in protein monomer. Experiments wereperformed in a 4e80 �C temperature range at scan rates of 1e4 �C/min. DSC profiles were analyzed using a two-state irreversibledenaturation model (N / F) as described previously [7,18]. Briefly,this model considers the irreversible denaturation of the nativeprotein (N) as a kinetic conversion to a final state (F) that cannotfold back. This conversion is characterized by a stronglytemperature-dependent first-order rate constant, and its temper-ature dependence follows the Arrhenius equation (with an acti-vation energy, Ea).

For experiments carried out in the presence of urea, a stocksolution of 8 M urea was prepared in the corresponding bufferand diluted to a final concentration up to 1 M, except for exper-iments at pH 5 in which the final urea concentration was 0.25 M.The kinetic m values were determined from the urea concentra-tion dependence of the Tm and Ea as described [7,19]. Thecontribution to the activation energy/enthalpy (Ea/DHz) from“unfolding barriers” is determined as DH$ms=m, where mz is thekinetic m value, m is the theoretical equilibrium m value deter-mined as described [7] and DH is the denaturation enthalpy orheat. The contribution from “solvation barriers” is determined asthe difference between the activation enthalpies and the contri-bution from unfolding barriers [7,19]. The energetic and structuraldifferences between the native and the transition state for therate-limiting step of thermal denaturation were determined asdescribed elsewhere [7]. The changes in activation free energiesand entropies are determined based on the transition state theoryas described [7].

2.4. Urea induced unfolding

Equilibrium unfolding profiles were evaluated by double jumpunfolding assays essentially as described [11]. Briefly, hPGK1(40 mM) was incubated at pH 5e7 and 25 �C in the presence of 0e3 M urea and 1 mM TCEP (Tris(2-carboxyethyl)phosphine) for 2 hand then diluted 20-fold into HEPES 50 mM pH 7 and a final ureaconcentration of 6.5 M, resulting in the quick unfolding of thenative hPGK1 present at equilibrium (with an unfolding rate con-stant kU of 0.55 � 0.02 min�1 at 25 �C; 50 experiments). Theamplitude of these jumps monitored by Trp-emission fluorescencespectroscopy (exc. 295 nm; em. 350 nm) was obtained from afitting to a single exponential function, and it is proportional to thefraction of native protein present at equilibrium [11,20,21].Unfolding free energies (DGU) were calculated from the Cm andequilibriumm values determined as described previously assuminga two-state unfolding model [11,21].

Global unfolding kinetics was determined by manually mixing ahPGK1 solution (20 mM) into urea solutions (3e6.5 M) prepared inbuffers with a pH of 5e7 and previously thermostatized at 25 �C. Asingle exponential fitting of the corresponding traces provides theunfolding rate constants kU at different urea concentrations whichare used to determine the unfolding rate constant at zero dena-turant (kU(0M)) by linear extrapolation [7,11]. The kinetic m valuesare RT times the slopes of these linear plots.


A.L. Pey / Biochimie xxx (2014) 1e9 3

3. Results

3.1. hPGK1 populates a partially folded state at pH < 5

We have investigated the effect of pH on the conformation ofhPGK1 by spectroscopic methods (Fig. 1). The Far-UV circular di-chroism (CD) spectra show a native like secondary structure con-tent at pH 5e8, while at more acidic pH the spectra is significantlyaltered (Fig. 1A). Plots of the Far-UV CD signals at 205 and 220 nmvs. pH show an apparent pK z4.0 for this structural transition. Thetertiary structure of hPGK1 as a function of pH has been alsoinvestigated by Trp-emission fluorescence (Fig. 1C). The fluores-cence intensity only slightly changes with pH, in agreement withthe low sensitivity of this property for detecting unfolding of hPGK1(see for instance, the small changes upon global unfolding by urea;

Fig. 1. Conformation of hPGK1 as a function of pH. A) Far-UV CD spectra; B) pH dependencelines are shown as in panel A; D) pH dependence of fluorescence intensity at 350 nm (edependence fluorescence intensity of ANS (the dotted line indicates ANS without protein) (edynamic light scattering.


[7,11]). However, the maximum of the fluorescence spectra do varywith pH (Fig. 1C). This shift can be accurately monitored as the ratioof fluorescence intensities between different wavelengths (330 and370 nm; Fig. 1D). Similarly to Far-UV CD spectroscopy results, theseratios are kept constant at pH 5e8, while change at pH lower that 5,indicating a transition to a non-native tertiary structure. Accord-ingly, ANS fluorescence is also strongly enhanced at mild acidic pHs(Fig. 1E), consistent with a partially folded state of hPGK1 at pHvalues lower than 5. We have also assessed the hydrodynamicbehavior of hPGK1 as a function of pH by dynamic light scattering(Fig. 1F). The hydrodynamic radius remains constant (r z 3.1 nm,consistent with a monomer) at pH 5e8, while significantly in-creases at lower pH values, which suggests the formation of anexpanded and partially unfolded state at pH < 5 (Fig. 1F). Overall,these results indicate hPGK1 remains in a native conformation

of the Far-UV CD signals at 222 nm and 205 nm; C) Trp-fluorescence emission spectra;xc. at 295 nm); inset: fluorescence intensity ratios between 370 and 330 nm; E) pHxc. 380 nm); F) pH dependence of the apparent hydrodynamic radii as determined by



between pH 5 and 8, while it undergoes a pH dependent unfoldingtransition with a pK z4, possibly due to the titration of the sidechains of acidic residues (aspartic and glutamic acids).

3.2. hPGK1 is kinetically destabilized towards thermal denaturationat pH < 6

The thermal stability of hPGK1 has been evaluated by DSC in apH range of 2.5e8 (see Fig. 2). Analysis of thermal denaturationprofiles (at 4 �C/min scan rate) using a two-state irreversibledenaturation model yield the parameters shown in Fig. 3. Controlexperiments show that these parameters are strongly dependenton pH but weakly dependent on the buffer used at a given pH (seeTable S1), with the exception of phosphate buffer which enhancesthe stability of hPGK1 possibly due to specific binding to the nativeprotein (Fig. S1). The Tm values, denaturation enthalpies or heats(DH) and activation energies (Ea) remain essentially constant in the5.5e8.0 pH range, but all three parameters decreases at lower pHs,and at pH < 3.5, no detectable transition is found (Fig. 3AeC). Thesimultaneous decrease in Tm and Ea at acidic pH translates into alarge kinetic destabilization (i.e. higher denaturation rate constantat 37 �C) of the enzyme towards thermal denaturation (Fig. 3D).Interestingly, hPGK1 displays a native-like conformation at pH 5but shows a large decrease in kinetic stability, with a change inactivation free energy of z4 kcal mol�1 at 37 �C, which implies anincrease in denaturation rate constant of about three orders ofmagnitude. At lower pH values, the decrease in kinetic stabilitymust be caused at least by partial unfolding of the hPGK1 at lowtemperature (Fig. 1).

Thermal denaturation of hPGK1 at pH ranging from 5 to 7.5 hasbeen further investigated (Fig. 4). In this pH range, the two-stateirreversible denaturation model provides an excellent descriptionof the thermal profiles (see the consistencies tests based on [18],and compiled in Table 1). The large kinetic destabilization found atpH 5 (Fig. 4A) stems from large and opposite changes in the acti-vation enthalpies and entropies that largely cancel out (Fig. 4BeD).

Fig. 2. Differential scanning calorimetry profiles at the indicated pH values. The scanrate was 4 �C/min.


A similar enthalpy/entropy compensation in the denaturation freeenergy barrier of hPGK1 has been recently described for disease-causing mutants [7].

3.3. Mild acidic pH affects the solvation barriers associated to thedenaturation of hPGK1 leading to kinetic destabilization

It is interesting that disease-causing mutations and mild acidicpH inWT hPGK1 causes protein kinetic destabilization due to fairlysimilar large changes in the enthalpic and entropic contribution todenaturation free energies (Fig. 4 and [7]). To get further insightinto pH mediated kinetic destabilization, we have performedadditional DSC analyses in the presence of urea, that allow deter-mine the structural/energetic changes occurring in the denatur-ation free energy barriers due to unfolding coupled to solvation(“unfolding barrier”) and solvation barriers (regions with brokeninternal contact but not yet solvated; “solvation barrier”)[7,19,22,23]. First, we have determined the kineticm values (i.e. theurea concentration dependence of the denaturation free energy,which are proportional to the difference in solvent exposure be-tween the native and transition state) at different pH values,finding that they are almost pH-independent in the range 5e7.5(Fig. 5A). These results imply that the amount of surface areaexposed in the denaturation transition state are not significantlyaffected by the pH, and thus, its contribution to the changes inactivation enthalpies must be small (Fig. 5B). Thus, the difference inthe activation enthalpy due to pH changes must primarily stemfrom changes in the solvation barrier contributions (Fig. 5B).Accordingly, the dependence of the solvation barrier contributionon the changes in activation free energy is 5.4-fold higher that thedependence of the unfolding barrier contribution (slopes inFig. 5C). As previously discussed [7,19,24], these changes in theunfolding and solvation barriers contributions are linked to struc-tural changes occurring between the native and the transition statefor irreversible denaturation (Fig. 5D). Interestingly, pH inducedkinetic destabilization ofWT hPGK1 seems to occur mostly throughchanges in the solvation barrier contribution, in contrast to theeffect of disease-causingmutants whichmostly affect the unfoldingbarrier contribution [7].

3.4. A pH-dependent linkage between decreased thermal kineticstability and thermodynamic stability

A simple mechanism compatible with the two-state irreversiblemodel that describes thermal unfolding of hPGK1 is the three-statemechanism:

N5U0F

where N and U are the native and unfolded states (which are atequilibrium, and this equilibrium described by an equilibriumconstant KU), and the unfolded state U undergoes the irreversibledenaturation step to the final state F (characterized by a rate con-stant kirr) [7,25,26]. As long as the unfolded state is not significantlypopulated along the thermal denaturation process, this mechanismcannot be distinguished from the phenomenological two-stateirreversible model. As we have recently discussed for hPGK1, ifthe irreversible step is rate-limiting (i.e. slow compared to therefolding step U / N), the overall denaturation rate is determinedby the values of the equilibrium constant KU and the rate of theirreversible step kirr [7,11]. We have thus determined the thermo-dynamic stability of hPGK1 at pH values ranging 5e7, to seewhether thermodynamic stability is linked to the changes in kineticstability found by DSC at pH 5. We have used double jumpunfolding assays [20,21] which allow determine the fraction of


Fig. 3. Energetic parameters for thermal denaturation of hPGK1 as a function of pH determined from single DSC scans shown in Fig. 2. Data are from fittings to a two-stateirreversible kinetic model. In panel D, DDGz values are determined using the denaturation rate constant at pH 3.5 as a reference.


native protein as a function of urea concentration (Fig. 6A,B). Byassuming a two-state unfoldingmechanism (i.e. only the native andthe unfolded states are significantly populated), it is straightfor-ward to quantify the thermodynamic stability. As shown in Fig. 6C,thermodynamic stability is largely decrease at pH below 6, reachinga thermodynamic destabilization of about 6 kcal mol�1 at pH 5compared to pH 7, which arises from a large decrease in the mid-point urea concentration (Fig. 6D). The fact that the equilibriumm value is not largely affected support that the two-state reversiblemodel is a good description of reversible unfolding of hPGK1 atdifferent pHs, and that the structural changes (difference in solventexposed surface area between native and unfolded states) occur-ring upon denaturation are not very different at the pHs tested(based on the correlations reported by Ref. [27]).

The results described above support that global unfolding ki-netics is not rate-limiting in the irreversible denaturation rate ofhPGK1 at different pHs. Thus, it would be expected that the largekinetic destabilization observed by DSC would not correlate withsimilar changes in global unfolding kinetics. Indeed, we observeonly modest changes in the global unfolding rate constantsextrapolated to zero denaturant at pH lower than 6 (about 5-foldfaster unfolding rate constants at pH 5 than at neutral pH; Fig. 7).These changes amount to a z1 kcal mol�1 kinetic destabilization,


which is much smaller than the z4 kcal mol�1 found for theirreversible denaturation (Figs. 3D and 4A). Thus, these resultsfurther support that thermodynamic stability plays a pivotal role inthe kinetic destabilization towards irreversible denaturation foundat pH 5. Interestingly, the kinetic m values found for globalunfolding (Fig. 7B) are 10-fold smaller than those found for theirreversible denaturation (Fig. 5A), supporting the different struc-tural properties of the relevant transition state for global unfoldingand for irreversible denaturation of hPGK1.

4. Discussion

In this work, we characterize the pH-dependent relationshipsbetween thermodynamic and kinetic stability in hPGK1, a proteinof biomedical interest due to its relationship with inherited disease(hPGK1 deficiency), oncogenesis, and activation of agents foranticancer and antiviral treatment.We show that hPGK1 remains ina native-like conformation between pH 5 and 8, but undergoes adramatic decrease in kinetic stability (up to 3-orders of magnitude)towards irreversible denaturation at pH < 6, due to significantchanges in the energetic/structural balance of the relevant dena-turation transition state. Using a simple three-state mechanism,this decrease in kinetic stability may be explained by either a large


Table 1Activation energy (Ea) values for hPGK1 thermal denaturation determined using theconsistency test proposed by Ref. [18]. Data are from four independent measure-ments at different scan rates.

pH Ea (kcal mol�1)

Fittings Arrhenius Ln n/Tm2 vs. 1/Tm Average

7.5 155 � 7 155 � 3 180 � 20 163 � 147 166 � 7 166 � 3 182 � 4 171 � 96.5 164 � 7 164 � 3 190 � 18 172 � 156 152 � 7 152 � 2 171 � 18 158 � 115.5 148 � 5 148 � 3 158 � 11 151 � 65 110 � 3 111 � 2 110 � 11 111 � 1

Fig. 4. Effect of pH on the activation energetic parameters for thermal denaturation of hPGK1. pH dependence of the activation free energy (DDGz; panel A), enthalpy (DDHz, panel B)and entropy (�TDDSz, panel C). In panel D the enthalpy/entropy contributions to denaturation free energy barriers are shown using the values at pH 7.5 as reference.


decrease in thermodynamic stability or in the kinetic stability to-wards global unfolding. However, equilibrium/kinetic unfoldingexperiments support that thermodynamic stability plays a majorrole in the stability of hPGK1 towards irreversible denaturation.These results allow to obtain a detailed picture of the relationshipsbetween kinetic and thermodynamic stability in hPGK1, but also tocompare these pH effects with those recently described for muta-tions associated with hPGK1 deficiency [6,7,11].

Disease-associated mutants reduce the kinetic stability towardsirreversible denaturation by up to 5-orders of magnitude causingchanges in the structure/energetics of the denaturation transitionstate [7], but these changes are different to those found by mildacidic pH (pH 5). Disease-causing mutations strongly decrease theamount of solvent exposed surface in the denaturation transition


state (i.e. “unfolding barrier”) with little effect on the amount ofbroken internal interactions not yet satisfied by solvation (i.e.“solvation barrier”) [7]. Conversely, at mild acidic pH hPGK1 showsa significant decrease in the contribution from solvation barriers,with only small changes in the contribution from unfolding bar-riers. Moreover, in the case of disease-causing mutations, the lowerkinetic stability could be explained by a decrease unfolding coop-erativity (which could explain the lower kinetic m values found formutants by thermal denaturation; [6,7,11]), while the pH effectsseems to arise from a simple thermodynamic destabilization of theprotein which still follows two-state reversible denaturation(which could explain the high equilibrium m values from ureadenaturation and kinetic m values from DSC, which are large andunaffected by pH). Thus, the mechanism of kinetic destabilizationby pH and disease-causing mutations may also differ: in thedisease-causing mutations, partially folded states may contributesignificantly to the rate of irreversible denaturation [6,11], while inthe pH range 5e8, the population of the unfolded state at equilib-rium determines the rate of irreversible denaturation.

Evolution seems to have selected proteins with high kineticstability (i.e. a high activation free energy towards irreversibledenaturation) to preserve their function in an appropriate physio-logical time scale [26]. In the case of hPGK1, as well as in yeast andpig muscle PGK [28], it seems that a high kinetic stability at 37 �C isprovided by high Ea values (120e200 kcal mol�1) and moderate Tmvalues (w53e55 �C). Noteworthy, the kinetic stability of hPGK1reaches a plateau at pH 6e8, possibly by optimization of surfaceelectrostatic interactions in this pH range (note that the theoretical


Fig. 5. pH dependence of the contributions from unfolding and solvation barriers to the kinetic stability of hPGK1. A) Kineticm values determined from DSC scans in the presence ofurea; B) contributions to the activation energies (Ea) from unfolding and solvation barriers as a function of pH. C and D) contributions from unfolding and solvation barriers to theactivation energies/enthalpies (C) and in structural terms (D) as a function of the changes in the denaturation free energy barriers (taking pH 7.5 as a reference).

Fig. 6. Equilibrium unfolding of hPGK1 by urea at different pH values. A) Profiles of the amplitudes obtained in the double jump unfolding assays (DI) as a function of ureaconcentration at pH 7 (circles), 6 (triangles) and 5 (squares). B) The fraction of native protein as a function of urea concentration determined from the data in panel A; the symbolshave the same meaning than in panel A; CeE) pH dependence of the unfolding free energies (DGU, panel C), midpoint denaturant concentration (Cm, panel D) and equilibrium mvalues (panel E). Lines in panels A and B are fits to a two-state unfolding model. Lines in panels CeE are only meant to guide the eye.


Please cite this article in press as: A.L. Pey, pH-dependent relationship between thermodynamic and kinetic stability in the denaturation ofhuman phosphoglycerate kinase 1, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.03.015

Fig. 7. Kinetics of global unfolding of hPGK1 by urea at different pH values. A) Urea-induced unfolding branches at pH 7 (squares), 6 (triangles) and 5 (circles). The linear fits areused to obtain the kinetic m values (panel B) and the unfolding rate constant at zero denaturant (kU(0M); panel C) as a function of pH.


pI is 8.3). Conversely, disease-causing mutants in hPGK1 deficiency[7] as well as in other metabolic inherited diseases [29] may causeenzyme loss-function by large changes on the activation energieswhich exponentially translate into the half-lives for irreversibledenaturation at physiological temperature. All this suggests thatmutations leading to enzyme loss-of-function in hPGK1 (andpossible others inherited metabolic diseases; see Refs. [25,29]) maytarget residues which have a strong impact in protein kineticthrough significant changes in the structure of the denaturationtransition state upon mutation. Accordingly, sequence-alignmentstatistics analyses provide evolutionary information that allowsmodulate protein kinetic stability. Mutations frequent in thealignment enhance protein kinetic stability, while rare mutationsdecrease it (such as disease-causing mutations; [30e32]).

The changes in thermodynamic and kinetic stability with pH canbe linked to differences in the protons released or taken up uponunfolding (DQ(NeU)pH) using the following linkage relationship[33,34]:

DQðN-UÞpH ¼ vDGðN-UÞpHvpH

$1

2:3$R$T

If we consider the changes in thermodynamic stability fromurea induced denaturation at pH 7 and 5, then about 2.3 moreprotons are taken up by the unfolded at pH 5 than at pH 7. If thechanges in denaturation rates are considered, then we obtain 1.5(from DSC measurements) and 0.4 (from urea unfolding kinetics)more protons taken up by the denaturation transition state at pH 5than at pH 7. These analyses have several interesting implications:1) the denaturation transition state for irreversible denaturation(DSC) is largely denatured, since the changes in the number ofprotons at low pH is close to those for the unfolded state. Thiscorrelates well with the high kinetic m value close to the equilib-rium m value (i.e. corresponding to the unfolding state); 2) thedenaturation transition state for global unfolding kinetics is quitenative-like, as seen by the small increase in the number of protonsuptaken by the denaturation transition state at low pH as well as bythe low kinetic m values.

In conclusion, we dissect here the pH-dependent relationshipsbetween thermodynamic and kinetic stabilities for hPGK1, showingthat thermodynamic stability determines to a large extent the ki-netic stability in a pH range 5e8, while at lower pH the enzymeundergoes pH-mediated partial denaturation. Our results allowcomparison with the effects of disease-causing mutants on theserelationships allowing obtain a more clear picture of the stability ofhPGK1 in health and disease.


Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank Dr. Jose Manuel Sanchez-Ruiz for support. This workwas supported by grants from Ministry of Economy and Competi-tiveness (CSD2009-00088 and BIO2012-34937), Junta de Andalucia(CTS11-07187) and FEDER Funds. A.L.P. is recipient of a Ramon yCajal contract from MINECO (RYC2009-04147).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biochi.2014.03.015.

References

[1] E. Beutler, PGK deficiency, Br. J. Haematol. 136 (2007) 3e11.[2] M.J. Cliff, M.W. Bowler, A. Varga, J.P. Marston, J. Szabo, A.M. Hounslow,

N.J. Baxter, G.M. Blackburn, M. Vas, J.P. Waltho, Transition state analoguestructures of human phosphoglycerate kinase establish the importance ofcharge balance in catalysis, J. Am. Chem. Soc. 132 (2010) 6507e6516.

[3] Z. Palmai, L. Chaloin, C. Lionne, J. Fidy, D. Perahia, E. Balog, Substrate bindingmodifies the hinge bending characteristics of human 3-phosphoglycerate ki-nase: a molecular dynamics study, Proteins 77 (2009) 319e329.

[4] M. Vas, A. Varga, E. Graczer, Insight into the mechanism of domain move-ments and their role in enzyme function: example of 3-phosphoglyceratekinase, Curr. Protein Pept. Sci. 11 (2010) 118e147.

[5] A. Dhar, A. Samiotakis, S. Ebbinghaus, L. Nienhaus, D. Homouz, M. Gruebele,M.S. Cheung, Structure, function, and folding of phosphoglycerate kinase arestrongly perturbed by macromolecular crowding, Proc. Natl. Acad. Sci. U. S. A.107 (2010) 17586e17591.

[6] G. Valentini, M. Maggi, A.L. Pey, Protein stability, folding and misfolding inhuman PGK1 deficiency, Biomolecules 3 (2013) 1030e1052.

[7] A.L. Pey, N. Mesa-Torres, L.R. Chiarelli, G. Valentini, Structural and energeticbasis of protein kinetic destabilization in human phosphoglycerate kinase 1deficiency, Biochemistry 52 (2013) 1160e1170.

[8] T.A. Young, E. Skordalakes, S. Marqusee, Comparison of proteolytic suscepti-bility in phosphoglycerate kinases from yeast and E. coli: modulation ofconformational ensembles without altering structure or stability, J. Mol. Biol.368 (2007) 1438e1447.

[9] J.M. Yon, M. Desmadril, J.M. Betton, P. Minard, N. Ballery, D. Missiakas,S. Gaillard-Miran, D. Perahia, L. Mouawad, Flexibility and folding of phos-phoglycerate kinase, Biochimie 72 (1990) 417e429.

[10] M.L. Galisteo, P.L. Mateo, J.M. Sanchez-Ruiz, Kinetic study on the irreversiblethermal denaturation of yeast phosphoglycerate kinase, Biochemistry 30(1991) 2061e2066.

[11] A.L. Pey, The interplay between protein stability and dynamics in conforma-tional diseases: the case of hPGK1 deficiency, Biochim. Biophys. Acta 1834(2013) 2502e2511.

[12] D. Missiakas, J.M. Betton, A. Chaffotte, P. Minard, J.M. Yon, Kinetic studies ofthe refolding of yeast phosphoglycerate kinase: comparison with the isolatedengineered domains, Protein Sci. 1 (1992) 1485e1493.




http://refhub.elsevier.com/S0300-9084(14)00108-4/sref1

















































[13] J. Sabelko, J. Ervin, M. Gruebele, Observation of strange kinetics in proteinfolding, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6031e6036.

[14] H.F. Willard, S.J. Goss, M.T. Holmes, D.L. Munroe, Regional localization of thephosphoglycerate kinase gene and pseudogene on the human X chromosomeand assignment of a related DNA sequence to chromosome 19, Hum. Genet.71 (1985) 138e143.

[15] J.R. McCarrey, K. Thomas, Human testis-specific PGK gene lacks introns andpossesses characteristics of a processed gene, Nature 326 (1987) 501e505.

[16] L.R. Chiarelli, S.M. Morera, P. Bianchi, E. Fermo, A. Zanella, A. Galizzi,G. Valentini, Molecular insights on pathogenic effects of mutations causingphosphoglycerate kinase deficiency, PLoS One 7 (2012) e32065.

[17] E. Fermo, P. Bianchi, L.R. Chiarelli, M. Maggi, G.M. Mandara, C. Vercellati,A.P. Marcello, W. Barcellini, A. Cortelezzi, G. Valentini, A. Zanella, A newvariant of phosphoglycerate kinase deficiency (p.I371K) with multiple tissueinvolvement: molecular and functional characterization, Mol. Genet. Metab.106 (2012) 455e461.

[18] J.M. Sanchez-Ruiz, J.L. Lopez-Lacomba, M. Cortijo, P.L. Mateo, Differentialscanning calorimetry of the irreversible thermal denaturation of thermolysin,Biochemistry 27 (1988) 1648e1652.

[19] D. Rodriguez-Larrea, S. Minning, T.V. Borchert, J.M. Sanchez-Ruiz, Role ofsolvation barriers in protein kinetic stability, J. Mol. Biol. 360 (2006) 715e724.

[20] M. Mucke, F.X. Schmid, A kinetic method to evaluate the two-state character ofsolvent-induced protein denaturation, Biochemistry 33 (1994) 12930e12935.

[21] B. Ibarra-Molero, J.M. Sanchez-Ruiz, Are there equilibrium intermediate statesin the urea-induced unfolding of hen egg-white lysozyme? Biochemistry 36(1997) 9616e9624.

[22] M. Costas, D. Rodriguez-Larrea, L. De Maria, T.V. Borchert, A. Gomez-Puyou,J.M. Sanchez-Ruiz, Between-species variation in the kinetic stability of TIM pro-teins linked to solvation-barrier free energies, J. Mol. Biol. 385 (2009) 924e937.

[23] A.L. Pey, A. Martinez, Iron binding effects on the kinetic stability and unfoldingenergetics of a thermophilic phenylalanine hydroxylase from Chloroflexusaurantiacus, J. Biol. Inorg. Chem. 14 (2009) 521e531.


[24] D. Rodriguez-Larrea, B. Ibarra-Molero, J.M. Sanchez-Ruiz, Energetic andstructural consequences of desolvation/solvation barriers to protein folding/unfolding assessed from experimental unfolding rates, Biophys. J. 91 (2006)L48eL50.

[25] A.L. Pey, Protein homeostasis disorders of key enzymes of amino acidsmetabolism: mutation-induced protein kinetic destabilization and newtherapeutic strategies, Amino Acids 45 (2013) 1331e1341.

[26] J.M. Sanchez-Ruiz, Protein kinetic stability, Biophys. Chem. 148 (2010) 1e15.[27] J.K. Myers, C.N. Pace, J.M. Scholtz, Denaturant m values and heat capacity

changes: relation to changes in accessible surface areas of protein unfolding,Protein Sci. 4 (1995) 2138e2148.

[28] A. Varga, B. Flachner, E. Graczer, S. Osvath, A.N. Szilagyi, M. Vas, Correlationbetween conformational stability of the ternary enzyme-substrate complexand domain closure of 3-phosphoglycerate kinase, FEBS J. 272 (2005) 1867e1885.

[29] A. Fortian, D. Castano, G. Ortega, A. Lain, M. Pons, O. Millet, UroporphyrinogenIII synthase mutations related to congenital erythropoietic porphyria identifya key helix for protein stability, Biochemistry 48 (2009) 454e461.

[30] R. Godoy-Ruiz, F. Ariza, D. Rodriguez-Larrea, R. Perez-Jimenez, B. Ibarra-Molero, J.M. Sanchez-Ruiz, Natural selection for kinetic stability is a likelyorigin of correlations between mutational effects on protein energetics andfrequencies of amino acid occurrences in sequence alignments, J. Mol. Biol.362 (2006) 966e978.

[31] A.L. Pey, D. Rodriguez-Larrea, S. Bomke, S. Dammers, R. Godoy-Ruiz,M.M. Garcia-Mira, J.M. Sanchez-Ruiz, Engineering proteins with tunablethermodynamic and kinetic stabilities, Proteins 71 (2008) 165e174.

[32] D. Rodriguez-Larrea, R. Perez-Jimenez, I. Sanchez-Romero, A. Delgado-Del-gado, J.M. Fernandez, J.M. Sanchez-Ruiz, Role of conservative mutations inprotein multi-property adaptation, Biochem. J. 429 (2010) 243e249.

[33] C. Tanford, Protein denaturation, Adv. Protein Chem. 23 (1968) 121e282.[34] C. Tanford, Protein denaturation. C. Theoretical models for the mechanism of

denaturation, Adv. Protein Chem. 24 (1970) 1e95.






















































































Date post:	30-Dec-2016
Category:	Documents
Upload:	angel-l
View:	212 times
Download:	0 times

pH-dependent relationship between thermodynamic and kinetic stability in the denaturation of human...

Documents