Báo cáo khoa học: The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pH ppt

Experiments carried out at different protein concentrations 6–20 lM did not show any difference in the thermodynamic parameters obtained data not shown.. Experiments carried out at diffe

The histidine-phosphocarrier protein of Streptomyces coelicolor

folds by a partially folded species at low pH

Gregorio Ferna´ndez-Ballester1, Javier Maya1, Alejandro Martı´n1, Stephan Parche2,*, Javier Go´mez1, Fritz Titgemeyer2and Jose´ L Neira1,3

1

Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Elche (Alicante), Spain;2Lehrstuhl fu¨r Mikrobiologie, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Germany;3Instituto de Biocomputacion y Fisica de los sistemas complejos, Zaragoza, Spain

The folding of a 93-residue protein, the

histidine-phospho-carrier protein of Streptomyces coelicolor, HPr, has been

studied using several biophysical techniques, namely

fluo-rescence, 8-anilinonaphthalene-1-sulfate binding, circular

dichroism, Fourier transform infrared spectroscopy, gel

filtration chromatography and differential scanning

calori-metry The chemical-denaturation behaviour of HPr,

fol-lowed by fluorescence, CD and gel filtration, at pH 7.5 and

25°C, is described as a two-state process, which does not

involve the accumulation of thermodynamically stable

intermediates Its conformational stability under those

con-ditions is DG¼ 4.0 ± 0.2 kcalÆmol)1 (1 kcal¼ 4.18kJ),

which makes the HPr from S coelicolor the most unstable

member of the HPr family described so far The stability of

the protein does not change significantly from pH 7–9, as

concluded from the differential scanning calorimetry and

thermal CD experiments Conformational studies at low pH

(pH 2.5–4) suggest that, in the absence of cosmotropic

agents, HPr does not unfold completely; rather, it accumu-lates partially folded species The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa¼ 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluores-cence However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilino-naphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-fil-tration studies) It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration These series of intermediates have not been reported previously in any member of the HPr family

Keywords: folding; molten-globule; protein stability; PTS; structure

The phosphoenolpyruvate phosphotransferase system

(PTS) catalyzes the uptake and phosphorylation of

carbo-hydrates in most bacterial species, via a cascade of several

proteins [1] Enzyme I (EI), the first protein in the cascade,

is autophosphorylated by phosphoenolpyruvate, yielding

phosphorylated EI (P-EI) P-EI acts as a phosphoryl donor

to the histidine-phosphocarrier protein (HPr)

Phosphoryl-ated HPr, in turn, donates the phosphoryl moiety to a group

of specific sugar-transporter proteins, known as enzymes II (EII) The transfer of the phosphoryl moiety from EII to the sugar occurs concomitantly to its transport into the cell HPr, the smallest protein in the cascade, is thought to be the key component in that cascade, because it phosphorylates all sugar-specific EII proteins EI phosphorylates HPr at the imidazole ring of the highly conserved histidine [1] This phosphorylation is common to both Gram-positive and Gram-negative bacteria However, in Gram-positive bac-teria there is also an additional regulatory phosphorylation site in HPr at the conserved Ser46 This regulatory site is thought to be involved in carbon catabolite repression of several genes, and as a transcription regulator of several operons in Gram-positive bacteria [2]

Streptomycesare soil-dwelling, high GC Gram-positive actinomycetes which grow on a variety of carbon sources, such as cellulose and many monosaccharides and disaccha-rides They are the source of approximately two-thirds of all natural antibiotics currently produced by the pharmaceu-tical industry Despite their importance, our knowledge on nutrient sensing, carbohydrate transport and regulation is very poor The complete sequence of Streptomyces coeli-colorhas been sequenced, showing the largest number of genes found in any bacteria [3] The presence of the different components of the PTS in S coelicolor has been reported, and the corresponding proteins cloned and expressed [4–9]

EI and HPr proteins from S coelicolor are similar, in

Correspondence to J L Neira, Instituto de Biologı´a Molecular y

Celular, Edificio Torregaita´n, Universidad Miguel Herna´ndez,

Avda del Ferrocarril s/n, 03202, Elche (Alicante), Spain.

Fax: +34 966658758, Tel.: +34 966658459,

E-mail: jlneira@umh.es

Abbreviations: ANS, 8-anilinonaphthalene-1-sulfonate ammonium

salt; DSC, differential scanning calorimetry; DC p , heat capacity

change; DH cal , the calorimetric enthalpy change; DH VH , the van’t Hoff

enthalpy change; EI, enzyme I; EII, enzyme II; FTIR, Fourier

trans-form infrared spectroscopy; GdmCl, guanidine hydrochloride; HPr,

histidine-phosphocarrier protein; PTS, the

phosphoenolpyruvate-dependent sugar phosphotransferase system; T m , thermal

denaturation midpoint.

*Present address: Nestle´ Research Center, Vers-chez-les-Blanc,

CH-100, Lausanne 26, Switzerland.

(Received 7 January 2003, revised 5 March 2003,

accepted 26 March 2003)

molecular size and activity, to those corresponding proteins

of other microorganisms previously characterized [4]

Because of its key role in the PTS and its small size, we

are studying HPr from S coelicolor as a model for:

(a) advancing our knowledge of the PTS enzymatic activity,

and then, its involvement in antibiotic production; and

(b) research in protein folding

The structures of HPrs from several species have been

studied by NMR spectroscopy [10–12 and references therein]

and X-ray diffraction [13,14,12 and references therein] HPrs

from those species show a classical open-face b-sandwich

fold consisting of three a helices packed against a

four-stranded antiparallel b sheet; this fold has been related to

other proteins with no apparent relationship in function,

such as ferredoxin and diphosphate kinase [15,16] HPr

from S coelicolor contains 93 amino acid residues; it lacks

cysteine and tyrosine, but it has a large number of alanine,

valine and leucine residues Furthermore, the protein only

contains one tryptophan residue and one phenylalanine

residue, which makes it a good model to follow its folding

mechanism and other biochemical features by using

fluor-escence spectroscopy Assignment and preliminary NMR

studies of the HPr from S coelicolor indicate that its

structure is similar to that observed in other members of the

HPr family (J L Neira, unpublished results) As the HPr

from S coelicolor has a similar structure, but a completely

different amino acid sequence to those HPrs from

Escheri-chia coli or Bacillus subtilis, whose structure and folding

properties have been described, it is important to understand

if the structure, the sequence or both determine the

conformational stability and biochemical properties in the

HPr family There is much current interest in determining

the extent to which related proteins share stability and

folding features [17] The exploration of the folding and

stability among the different HPr members will allow us to

decide whether there is a common thermodynamic

equili-brium behaviour in this important family

In this study, we use several biophysical techniques (CD,

fluorescence, 8-anilinonaphthalene-1-sulfate (ANS) binding,

differential scanning calorimetry (DSC), thermal CD, FTIR

and gel filtration chromatography) to follow the folding of

HPr from S coelicolor Our findings indicate that the folding

of HPr can be adequately described as a two-state process

without the accumulation of intermediates at neutral and

moderately basic conditions (pH 7–9) at 25°C The stability

of the protein, at pH 7.5 and 25°C, as obtained by

chemical and thermal denaturation experiments, is low:

DG¼ 4.0 ± 0.3 kcalÆmol)1 At moderately acid pH values

(pH 2.5–4), in the absence of cosmotropic agents, the protein

undergoes noncooperative thermal denaturations and it

accumulates partially folded species Although extensive

folding studies have been carried out with the E coli HPr

[16], no such species have been previously observed nor

characterized Thus, this is the first member of the HPr where

partially folded species have been found

Experimental procedures

Materials

Urea and guanidine hydrochloride (GdmCl) ultra-pure

were from ICN Biochemicals Urea and GdmCl molecular

biology grade, imidazole, trizma base, NaCl and ANS were from Sigma 2-Mercaptoethanol was from Bio-Rad, and the Ni2+-resin was from Invitrogen Glutaraldehyde was from Fluka Dialysis tubing was from Spectrapore with a molecular mass cut-off of 3500 Da Standard suppliers were used for all other chemicals Water was deionized and purified on a Millipore system Urea and GdmCl stock solutions were prepared gravimetrically and filtered using 0.22-lm syringe driven filters from Millipore Exact con-centrations of urea stock solutions were calculated from the refractive index of the solution using an Abbe 325 refractometer [18]

Protein expression and purification The HPr clone comprises residues 1–93 (with the extra methionine at the N terminus) and the His6-tag at the

N terminus We have carried out all the studies with this construction as its structure, as observed by NMR (unpub-lished results), is similar to that found in other members of the HPr family and the His6-tag is disordered in solution, making no contacts with the rest of the protein Recom-binant protein was expressed in E coli C43 strain [19], and purified using Ni2+-chromatography To eliminate any protein or DNA bound to the resin, coeluting with the protein, an additional gel filtration chromatography step was carried out by using a Superdex 75 16/60 gel filtration column, running on an AKTA-FPLC (Amersham Bio-sciences) system The yield was 25–30 mg of protein per litre

of culture Protein was more than 99% pure as judged

by SDS protein-denaturing gels Also, mass spectrometry analysis was carried out in a MALDI-TOF instrument; only one peak was observed The samples were dialyzed exten-sively against water and stored at )80 °C Samples were prepared by dissolving the lyophilized protein in deionized water (unfolding) or in 8M urea (in the refolding experi-ments) and adding the proper buffer solution Protein concentration was calculated from the absorbance of stock solutions measured at 280 nm, using the extinction coeffi-cients of model compounds [20]

Protein without the His6-tag was obtained using the thrombin-cleavage capture kit from Novagen (Germany) This protein was used as a control experiment to address the importance of the His6-tag in the biophysical properties of the protein under different pH conditions

Cross-linking experiments Cross-linking reactions were performed at 25°C in the corresponding buffers at different protein concentrations by addition of glutaraldehyde to a final concentration of 4 mM Reactions were stopped after 15 min by addition of SDS-buffer

Fluorescence measurements All fluorescence spectra were collected on a SLM 8000 spectrofluorometer (Spectronics Instruments, Urbana, IL), interfaced with a Haake water bath, at 25°C All measure-ments were corrected for wavelength dependence on the exciting-light intensity through the use of the quantum counter rhodamine B in the reference channel [21] Sample

concentration was in the range 6–20 lM, and the final

concentration of the buffer was, in all cases, 10 mM A

0.5-cm path-length quartz cell (Hellma) was used

Steady state fluorescence measurements All protein

sam-ples were excited at 280 nm, as excitation at 295 nm yielded

the same spectrum with smaller intensity (data not shown)

The slit width was typically equal to 4 nm for the excitation

light, and 8nm for the emission light The fluorescence

experiments were recorded between 300 and 400 nm The

signal was acquired for 1 s and the increment of wavelength

was set to 1 nm Blank corrections were made in all spectra

The urea titrations, followed either by fluorescence or

CD, were carried out by two different procedures: (a)

dilution of the proper amount of the 8Mdenaturant stock

solution and leaving the samples at 25°C, for at least 8h

prior to performing the experiments; or (b) by directly

titrating the protein with urea No differences were observed

between the procedures As the concentration of urea was

increased, the fluorescence spectra were red-shifted and their

intensities decreased (data not shown) Experiments carried

out at different protein concentrations (6–20 lM) did not

show any difference in the thermodynamic parameters

obtained (data not shown)

In the pH-induced unfolding experiments, followed either

by fluorescence or CD, the pH was measured after

comple-tion of the experiments with an ultra-thin Aldrich electrode in

a Radiometer (Copenhagen) pH meter The pH range

explored using both techniques was 1.5–12 The buffers were:

pH 1.5–3.0, phosphoric acid; pH 3.0–4.0, formic acid;

pH 4.0–5.5, acetic acid; pH 6.0–7.0, NaH2PO4; pH 7.5–

9.0, Tris acid; pH 9.5–11.0, Na2CO3; pH 11.5–12, Na3PO4

Fluorescence quenching experiments Quenching of

intrin-sic tryptophan fluorescence by iodide or acrylamide [21] was

examined at different pH values Excitation was at 280 nm,

and emission was measured from 300 to 400 nm In

experiments employing KI as a quencher, ionic strength

was kept constant by addition of KCl; also, Na2S2O3was

added to a final concentration of 0.1Mto avoid formation

of I3 The slit width was set at 8nm for both excitation and

emission The dynamic and static quenching constants for

acrylamide were obtained by fitting the data from different

wavelengths (in the range 330–340 nm) to the Stern–Volmer

equation, which includes an exponential term to account for

static quenching [21]:

F0

F ¼ 1 þ Ksv½Xeðv½XÞ ð1Þ where Ksv is the Stern–Volmer constant for collisional

quenching and v is the static quenching constant Iodide

quenching did not show a significant static component, and

then the exponential term was not included in the fitting of

Eqn (1) The range of concentrations used in both

quenc-hers was 0–0.7M Experiments carried out at different

protein concentrations did not show any difference in the

parameters obtained (data not shown)

ANS binding ANS binding was measured by collecting

fluorescence spectra at different pH values in the presence of

50 lMdye Excitation wavelength was 380 nm, and

emis-sion was measured from 400 to 600 nm Slit widths were

4 nm for excitation and 8nm for emission Stock solutions

of ANS were prepared in water and diluted into the samples

to the above final concentration In all cases, the blank solutions were subtracted from the corresponding spectra Experiments carried out a different protein concentrations did not show any difference (data not shown)

Circular dichroism Circular dichroism spectra were collected on a Jasco J810 spectropolarimeter fitted with a thermostated cell holder and interfaced with a Neslab RTE-111 water bath The instrument was periodically calibrated with (+)10-cam-phorsulfonic acid Isothermal wavelength spectra at differ-ent pH values were acquired with a scan speed of

50 nmÆmin)1, and a response time of 2 s and averaged over four scans at 25°C Far-UV measurements were performed using 14–295 lMof protein in 10 mMbuffer, using 0.1- or 0.2-cm pathlength cells (Hellma) During the pH titration experiments no significant changes either in the shape or in the molar ellipticity were observed as the concentration of protein was increased; thus, we can rule out the presence

of concentration-dependence at those pH values Near-UV spectra were acquired using 30–40 lMof protein in a 0.5-cm pathlength cell (Hellma) All spectra were corrected by subtracting the proper baseline To allow for comparison at different pH values and different urea concentrations, raw ellipticity was converted to molar ellipticity [22]

Thermal-denaturation experiments were performed at constant heating rates of 60°CÆh)1 and 30°CÆh)1; the response time was 8s Thermal scans were collected in the far-UV region at 222 nm from 25°C (or 5 °C) to 90 °C (or

95°C) in 0.1-cm pathlength cells (Hellma) with a total protein concentration of 40–100 lM Conditions were the same as those reported in the steady-state far-UV experi-ments The reversibility of thermal transitions was tested by recording a new scan after cooling down to 5°C the thermally denatured samples To check also for reversibility,

we carried out the reheating experiments at different speeds

to the heating measurements; no differences among the scans acquired at different speeds were observed at those pH values where HPr unfolds reversibly Every thermal dena-turation experiment was repeated at least three times with fresh new samples at different concentrations The measured thermodynamic parameters did not change when experi-ments were acquired at different protein concentrations In all cases, after the reheating experiment, the samples were transparent and no precipitation was observed The possi-bility of drifting of the CD spectropolarimeter was tested by running two samples containing buffer, before and after the thermal experiments No difference was observed between the scans

In the urea-denaturation experiments, far-UV CD spec-tra were acquired at a scan speed of 50 nmÆmin)1and four scans were recorded and averaged at 25°C The response time was 2 s The pathlength cell was 0.1 cm, with protein ranging in 10–30 lM Spectra were corrected by subtracting the proper baseline in all cases The chemical denaturation reaction was fully reversible, as demonstrated by the agreement between the folding and unfolding curves (data

not shown) Each chemical denaturation experiment was

repeated at least three times with new samples Experiments

carried out at different protein concentrations did not show

any difference

Analysis of the pH- and chemical-denaturation curves,

and free energy determination

The average energy of emission (or the intensity weighted

average of the inverse wavelengths) in the fluorescence

spectra, <k>, was calculated as defined in [23]

The pH-denaturation experiments were analyzed

assu-ming that both species, protonated and deprotonated,

contributed to the fluorescence (or CD) spectrum:

X¼ðXaþ Xb10

ðpHpK a ÞÞ ð1 þ 10ðpHpK a ÞÞ ð2Þ where X is the physical property being observed (ellipticity

or fluorescence), Xais the physical property being observed

at low pH values (that is, the fluorescence or ellipticity of the

acid form), Xbis the physical property observed at high pH

values, and pKais the apparent pK of the titrating group

The apparent pKa reported was obtained from three

different measurements, prepared with new samples In

the fluorescence experiments, the determinations were

carried out using either the <k> or the maximum

wavelength in fitting the Eqn (2) In the CD experiments,

the ellipticity at 222 nm was the chosen parameter, either in

the pH-denaturation or chemical-denaturation experiments

To facilitate comparison among the different biophysical

techniques, data were converted to the fraction of folded

and unfolded molecules [24]

The denaturation data obtained by fluorescence or CD

were fit to the two-state equation:

X¼ðXNþ XDe

ðDG=RTÞÞ

where XN and XD are the corresponding fractions of the

folded (N) and unfolded states (U), respectively, which were

allowed to change linearly with either the denaturant

(XN¼ aN+ bN[D], and XD¼ aD+ bD[D]), or the

tem-perature (that is, XN¼ aN+ bNTand XD¼ aD+ bDT),

Ris the gas constant and T is the temperature in K

Chemical-denaturation curves were analyzed using a

two-state unfolding mechanism, according to the linear

extrapolation model: DG¼ m([D]50%) [D]) [20], where DG

is the free energy of denaturation, and [D]50%is the

denat-urant concentration at the midpoint of the transition The

chemical-denaturation-binding model [25,26] was also used

for the fitting of the chemical denaturation data, but no

reliable parameters were obtained either for DG or m (data

not shown) Thus, the linear extrapolation method was used

in all the conformational stability calculations

The change in free energy upon temperature in Eqn (3) is

given by the Gibbs–Helmholtz equation:

DGðT Þ ¼ DHVH 1 T

Tm

 DCp ðTm TÞ þ T ln T

Tm

ð4Þ

where DHVH is the van’t Hoff enthalpy change By substitution of this expression in Eqn (3), we obtain DHVH,

Tmand DCpof the thermal experiments

Fittings by nonlinear least-squares analysis to Eqns (1, 2 and 3) were carried out by using the general curve fit option

ofKALEIDAGRAPH(Abelbeck software)

Gel filtration chromatography Analytical gel filtration experiments were carried out by using an analytical gel filtration Superdex 75 HR 16/60 (Amersham Biosciences) running on an AKTA FPLC system at 25°C Flow rates of 0.8mLÆmin)1(at high urea concentrations) or 1 mLÆmin)1 were used The elution buffers for the pH experiments were those described above with 150 mM NaCl added to avoid non-specific inter-actions with the column To check for the presence of aggregated species at low pH values, protein concentra-tions ranged from 20 to 60 lM No differences in the elution volumes were observed among the different concentrations used

The chemical denaturation experiments were acquired at

pH 7.5, 10 mM phosphate buffer and 150 mM NaCl Protein concentration was 20–60 lM and absorbance was monitored at 280 nm No differences in the elution volumes were observed when the protein concentration was increased The column was calibrated using the gel filtration low relative molecular mass calibration kit (Amersham Biosciences) The standards used and their corresponding Stokes radii were: ribonuclease A (16.4 A˚); chymotrypsi-nogen (20.9 A˚); ovoalbumin (30.5 A˚), and bovine serum albumin (35.5 A˚) [27]

The elution of a macromolecule in gel filtration experi-ments is usually given by the partition coefficient, r, which

is defined as the fraction of solvent volume within the gel matrix accessible to the macromolecule [28] The r of protein standards and HPr were calculated by:

r¼ðVe VoÞ

Vi

ð5Þ

where Veis the elution volume of the protein, and, Voand Vi

are the void and internal volumes of the column, respect-ively The values of those volumes are, respectively, 8.13 ± 0.06 mL and 28.43 ± 0.03 mL The Vo and Vi volumes were, respectively, determined using Blue dextran (5 mgÆmL)1, in 10 mM phosphate buffer plus 150 mM

NaCl) andL-tryptophan (0.5 mgÆmL)1, in the above buffer)

by averaging four measurements for each agent

There is a linear relationship between the molecular Stokes radius, Rs, and the inverse error function comple-ment of r (erfc)1(r)), given by [28,29]:

Rs¼ a þ bðerfc1ðrÞÞ ð6Þ where a and b are the calibration constants for the column Fitting of the calculated erfc)1(r) to the above equation by linear least-squares analysis was carried out with KALEIDA-GRAPH (Abelbeck software) working on a PC computer Once the calibration parameters are obtained, the Stokes radius of any macromolecule can be obtained by using Eqn (6)

Fourier transform infrared spectroscopy

The protein was lyophilized and dissolved in deuterated

buffer at different pH values The buffer was composed of

0.1M NaCl, 0.1 mM ethylenediaminetetracetate, 0.02%

NaN3, 10 mM sodium acetate, 10 mM

N-(1-morpholino)-propane-sulfonic acid, and 10 mM

3-(cyclohexylamino)-1-propane-sulfonic acid No corrections were done for the

isotope effects in the measured pH Samples of HPr at a

final concentration of 5–6 mgÆmL)1were placed between a

pair of CaF2windows separated by a 50-lm thick spacer

in a Harrick Ossining demountable cell Spectra were

acquired on a Nicolet 520 instrument equipped with a

deuterated triglycine sulfate detector and thermostated

with a Braun water bath at 25°C The cell container was

continuously filled with dry air Usually 600 scans per

sample were taken, averaged, apodized with a Happ–

Genzel function, and Fourier transformed to give a final

resolution of 2 cm)1 The contributions of buffer spectra

were subtracted, and the resulting spectra used for analysis

after smoothing The spectra smoothing was carried out

by using the maximum entropy method [30] Derivation of

FTIR spectra was performed using a power of 3 and a

breakpoint of 0.3 Fourier self-deconvolution was

per-formed using a Lorentzian bandwidth of 18cm)1 and a

resolution enhancement factor of 2 [30] The prediction of

protein secondary structure was quantified by

deconvolu-tion of the amide I band, as described elsewhere [31],

yielded essentially the same percentages of a helix, b turns

and b sheet, which have been observed in the NMR

structure (unpublished results)

Thermal denaturation experiments followed by FTIR

were performed at a protein concentration of 6 mgÆmL)1,

with a scanning rate of 50°CÆh)1, and acquired every 5°C

Differential scanning calorimetry

DSC experiments [32] were performed with a MicroCal

MC-2 differential scanning calorimeter interfaced to a

computer equipped with a Data Translation

DT-2801 A/D converter board for instrument control and

automatic data collection Lyophilized protein was

dis-solved in 10 mM phosphate buffer, pH 7.5 and dialyzed

extensively against 2 L of the same buffer (twice) at 4°C

Protein concentration was calculated from the absorbance

of the solution at 280 nm [20] Samples were degassed

under vacuum for 10–15 min with gentle stirring prior to

being loaded into the calorimetric cell DSC experiments

were performed under a constant external pressure of

1 bar in order to avoid bubble formation, and samples

were heated at a constant scan rate of 60°CÆh)1 Once the

first scan was completed, the samples were cooled in situ

down to 10°C for 40 min and rescanned under the same

experimental conditions in order to check the reversibility

of the heat-induced denaturation reaction Experimental

data were corrected from small mismatches between the

two cells by subtracting a buffer vs buffer baseline prior to

data analysis After normalizing to concentration, a

chemical baseline calculated from the progress of the

unfolding transition was subtracted The excess heat

capacity functions were then analyzed using the software

package (Microcal Software, Inc.)

Results

pH-induced unfolding of HPr

To examine how the secondary and tertiary structure of HPr changes with the pH, we used multiple spectroscopic techniques, which give complementary information about the melting of the secondary and/or tertiary structure of the protein

Fluorescence experiments We used fluorescence to map any change in the tertiary structure of the protein upon pH changes [33] HPr has one tryptophan residue, which is at the C-terminal region of the first b strand The emission fluorescence spectrum of native HPr showed a maximum at

337 nm at neutral pH (Fig 1A), indicating Trp burial As the pH decreased, the maxima wavelength were red-shifted towards 350 nm, and the fluorescence intensities increased

Fig 1 pH-induced unfolding of HPr followed by fluorescence and ANS binding (A) Steady-state fluorescence: the average energy of emission (filled circles) and the maxima wavelength (open circles) are repre-sented vs the pH (B) ANS-binding experiments: the average energy (filled circles) and the maxima wavelength (open circles) are repre-sented vs the pH (C) The low-pH region of the steady-state fluores-cence experiments (the symbols are the same as in A) The conditions were: 6 l M of protein, and 40 l M ANS, when required, at

25 (± 0.1) °C; buffer concentration was 10 m M in all cases; spectra were acquired in 0.5-cm pathlength cells The lines are fittings to Eqn (2).

Conversely, as the pH increased, the maxima wavelength

and the spectral intensities remained constant up to high pH

(Fig 1A)

The profile of <k> vs pH (Fig 1A) showed a sigmoidal

behaviour at low pH, and a plateau region above pH 4 The

maxima wavelength followed the same pattern than the

<k> The apparent pKawas 3.2 ± 0.3 (Fig 1A)

The protein without the His6-tag showed the same

behaviour at the different pH values than that observed

for the His6-tagged protein (data not shown)

Examination of tryptophan exposure by fluorescence

quenching To further check whether the tertiary structure

around the sole Trp residue changes upon pH, we examined

iodide and acrylamide quenching by excitation at 280 nm,

which provided us with information about burial of the

indole moiety Acrylamide-quenching experiments, carried

out at pH 3.3 and 7.5, yielded exponential Stern–Volmer

plots At pH 7.5, where as judged by fluorescence, CD and

FTIR measurements the protein was folded, the Ksvand v

were small, indicating burial of the tryptophan moiety

(Table 1) These results are in agreement with the

observa-tion that the maxima wavelength of spectra appeared at

337 nm at that pH Conversely, at low pH, both quenching

parameters were larger, indicating solvent-exposure of the

aromatic ring This is also in agreement with previous

observations on the shift of the maxima wavelength at low

pH values Larger values of the quenching parameters were

also observed at pH 7.5 in the presence of 6Murea, where

the protein was completely unfolded The fact that the

values of Ksvin the presence and in the absence of urea, as

measured by acrylamide, are very similar within the error

(Table 1) is not fully understood, but it could be due to the

larger size of acrylamide when compared to that of KI

Therefore, we can conclude that as the pH decreases, the

Trp is more solvent-exposed

The use of KI as a quenching agent yielded similar results,

but experiments at very low pH values could not be carried

out because of precipitation We do not know why HPr

precipitates, but it might be due to the presence of the

negative charge of the I–and the large amount of positive

charges at low pH, as happens in other molten-globule-like

species [34] Here, conversely to that observed in acrylamide

experiments, only linear plots were found A small Ksvvalue

was found when the protein is folded, and larger values were

observed when the protein was completely unfolded (6M

urea)

It is interesting to note here, that the Ksv(in acrylamide and KI) and v (in acrylamide) values for folded (and unfolded) HPr are similar to those found in other folded (an unfolded) proteins [35,36]

ANS binding fluorescence ANS binding is used to monitor the extent of exposure of protein hydrophobic regions, and to detect the existence of non-native partially folded conformations When ANS is bound to solvent-exposed hydrophobic patches of proteins, its quantum yield

is enhanced and the maxima of the emission spectra is shifted from 520 nm to 480 nm [37] At low pH values, the intensity of the ANS in the presence of HPr was largely enhanced and the maxima wavelength appeared at 482 nm (Fig 1B) As the pH was increased, the spectra intensity was reduced and the maxima wavelength shifted towards 528nm These results suggest that: (a) ANS was bound to HPr at low pH values, probably because of the presence of solvent-exposed hydrophobic regions and (b) those hydro-phobic patches were probably buried in the pH range 4–7,

as concluded from the titration curve measured (Fig 1B) The apparent pKa was 5.3 ± 0.5 The ANS-binding experiments carried out with the protein without the His6-tag showed the same behaviour (data not shown) Far-UV and near-UV CD We used far-UV CD in the analysis of the unfolding of HPr as a spectroscopic probe that is sensitive to the presence of protein secondary structure [22] Its CD spectrum was intense and showed the typical features of an a-helical protein, with intense minima at 222 and 208nm (Fig 2A), although interference from the absorbance of tryptophan and histidine residues could not be ruled out at 222 nm [22] The shape of the CD spectrum of S coelicolor HPr was similar to that observed for E coli HPr [16] This shape did not change substantially over the pH range explored (Fig 2A), but the intensity at low pH values was smaller than at neutral pH values However, the ellipticity at low pH values (pH 3.0) was not

as small as that observed at high urea concentrations, where the protein was completely unfolded suggesting that the protein has residual structure at low pH values The apparent pKa was 3.5 ± 0.3 (Fig 2B), which is, within the error, the same as that determined by fluorescence

We used near-UV CD to detect possible changes in the asymmetric environment of aromatic residues [22] The near-UV of HPr was very weak and no intense bands were observed (Fig 2C), probably due to the low content of aromatic residues The spectrum showed a small band at

292 nm and a shoulder at 285 nm corresponding to the vibronic components of the 1Lbtransition in tryptophan residues [38] Because of lack of intense distinctive features,

we did not use further the near-UV spectrum to map any temperature, pH or chemical denaturant changes

FTIR spectroscopy FTIR is a powerful method for investigation of protein secondary structure The main advantage in comparison with CD is that FTIR is more sensitive to the presence of b structure or random-coil Measurements were only carried out at selected pH values from pH 2.5–7.5, because of the large amounts of protein

Table 1 Quenching constants for HPr in acrylamide and KI Errors are

data fit errors to Eqn (1) with (acrylamide) or without (KI) the

exponential factor The K sv and v were obtained by fitting of

fluores-cence intensity at 335 nm vs concentration of quenching agent (similar

values were obtained by fitting the intensities at 336, 337 and 338nm,

data not shown) Experiments were carried out at 25 °C The value

with a –’ could not be measured due to HPr precipitation.

Conditions

K sv ( M )1 ) v ( M )1 ) K sv ( M )1 )

used in the FTIR experiments and the impossibility of

sample recovery As the pH increased the maxima of the

amide I bands moved from 1651.0 to 1645.6 cm)1(Fig 3B)

The titration, as concluded from the position of the amide I

band, followed the same sigmoidal behaviour than that

observed by CD and fluorescence, but a reliable value for

the pKamidpoint could not be determined

Deconvolution of the bands at the extreme explored pH

values (pH 2.5 and 7.5) indicates that the helical structure

remained basically unaltered (50%); conversely, antiparallel

b structures (the 1628and the 1666 cm)1 bands) were

increased at lower pH values (it changed from 20% at

pH 7.5–27% at low pH) Although the exact reasons of the increase in the b sheet bands are unknown, it could be due to formation of soluble oligomers at low pH values The presence of those oligomer species would explain also the apparent thermal titration observed at low pH values in the FTIR experiments

Gel-filtration chromatography Gel filtration provides a measurement of the compactness (hydrodynamic volumes)

of the polypeptide chain [39] HPr eluted at neutral pH at

Fig 2 CD of HPr under different conditions (A) Far-UV CD spectra at different conditions (filled circles, pH 3; open circles, neutral pH; squares,

6 M urea) The conditions were: 20 l M of protein at 25 (± 0.1) °C; buffer concentration was 10 m M in all cases; spectra were acquired in 0.1-cm pathlength cells (B) pH-induced unfolding of HPr following the ellipticity at 222 nm in the far-UV CD The line is the fitting to Eqn (2) (C)

Near-UV of HPr Continous line (circles) is the near-Near-UV spectra at pH 7.5, 10 m M phosphate buffer Dotted line (squares) is the near-UV at 6 M urea,

pH 7.5, 10 m M phosphate buffer Conditions were: 60 l M protein, 25 °C, spectra were acquired in 0.5-cm pathlength cells (D) CD spectra at different concentrations at pH 3.0, after normalization by the smaller concentration used (10 l M ) Open circles (black lines) are spectra acquired at

10 l M ; open squares (red lines) are at 20 l M and filled circles (blue lines) at 40 l M protein concentration The ellipticity units on the y-axis are the normalized raw ellipticity.

Fig 3 FTIR of HPr at different pH values (A) Green (pH 2.5), blue (pH 3), red (pH 5) and black lines (pH 7) Vertical bars indicate absorbance units (B) Position (cm)1) of the amide I band at different pH values Protein concentration was 6 mgÆmL)1; all other con-ditions as described under the Experimental procedures section.

the volume expected for a globular folded protein of its size,

13.92 mL The Rs determined from Eqn (6) is 16.35 A˚,

within the range expected for a globular protein of its size

(Fig 4A) At low pH values, the protein eluted close to

the void volume of the column as a single peak (Fig 4B),

8.55 mL at pH 3.5 From pH 4–4.5, HPr eluted in two

different peaks: (a) the first peak eluted at the same volume

as the pH-unfolded protein and (b) the second peak was

very broad These data indicate that the interconversion

between the unfolded and native forms of the protein is slow

as compared to the column retention time [39] Similar

behaviour has been observed in other partially folded

species [40]

The fact that the elution volume of HPr at low pH values

is close to the void volume suggests that either HPr is an

oligomer or it has lost most of this globular shape We can

rule out the first explanation as: (a) glutaraldehyde

cross-linking at pH 3.0 did not show the presence of oligomers at

the concentrations explored, 20–100 lM(data not shown);

(b) far-UV experiments at different concentrations, ranging

from 14 to 295 lM, at pH 3.0 did not show any change

either in the shape or the raw ellipticity after being

normalized by the smallest concentration used (Fig 2D);

(c) if there was a large population of aggregated forms, two

intense bands at 1620 cm)1and 1685 cm)1[30,31] should

appear in the FTIR spectrum; at pH 2.5, even at the large

amounts of protein used in the FTIR experiments, these

bands were not found at 25°C (Fig 6B); however, the

presence of an small amount of aggregated forms could

explain the increase (7%) in the b sheet structure observed

at this pH; (d) the gel filtration experiments carried out

at different protein concentrations (20–60 lM) at these low pH values did not show any difference in the elution volume and (e) the <k> is very sensitive to changes in oligomerization processes [23], and the value observed at these pH values is close (2.8 4 lm)1) (Fig 1A) to that observed for urea-unfolded HPr (where aggregated forms are not present); furthermore, only below pH 2 a large increase in the <k> was observed (from pH 0.22–0.87,

<k> shifted from 2.90 to 2.86 lm)1, respectively); also, the maxima wavelength decreased as the pH was reduced (from

pH 0.22–0.87, it shifted from 340 to 348 nm, respectively) (Fig 1C); these findings indicate the presence of aggregated forms at pH values below 2 Then, all these probes suggest that at pH 3.0, HPr is monomeric, and the small elution volume indicates that HPr has lost most of its globular form Below this pH small populations of aggregated species cannot be ruled out, but their contribution to the spectral properties is insignificant as suggested by the FTIR spectra

Thermal-denaturation experiments at different

pH values

In order to obtain the thermodynamic parameters charac-terizing the unfolding transition of HPr, we carried out thermal-denaturation experiments followed by CD, FTIR and DSC Measurements trying to obtain a complete set of thermodynamic parameters by using fluorescence failed due

to the large temperature dependence of the intrinsic

Fig 4 pH-induced unfolding of HPr followed by gel-filtration chromatography (A) Determination of the Stokes radius by gel filtration chroma-tography on a HR Superdex G75 (Amersham Biosciences) The elution volume of HPr is indicated by an arrow and a square The numeration corresponds, respectively, to the elution volumes of ribonuclease A (1), chymotrypsinogen A (2), ovalbumin (3), and albumin (4) (B) Elution volume vs pH, the filled circles observed from pH 4–4.5 indicate one of the two peaks observed during protein elution The open circles and squares indicate the elution volumes of two different measurements (C) Chromatograms at selected pH values: continuous black lines and open circles,

pH 3; dotted blue lines and open squares pH 4.3; dotted-and-dashed red lines and filled circles pH 7 Conditions were: 20–60 l M of protein at

25 °C, in 10 m M of the corresponding buffer and 150 m M NaCl.

fluorescence of both the native and unfolded states of the

protein (data not shown)

Far-UV CD We explored the thermal-denaturation

beha-viour of HPr from pH 2.0–11.0 (Fig 5), by following the

ellipticity at 222 nm We found four different pH regions,

according to the reversibility and sigmoidal behaviour (a)

Between pH 2.0 and 3.0 the heating transition did not have

a sigmoidal behaviour, but it was reversible (b) Between

pH 3.5 and pH 4.5 the heating transition was

noncooper-ative and irreversible (c) Between pH 5.0 and pH 5.5 the

heating transition showed a sigmoidal behaviour, but was

not reversible (d) Between pH 6 and pH 9 the heating

transition had a sigmoidal behaviour and it was reversible

The reversibility at those pH values was approximately

90–100%, as measured from the relative ellipticity recovery

in the reheating experiments At higher pH values, the

thermal transitions were not reversible, probably due to deamidation processes [16,41] A reliable determination of the DCpand DHcalbetween pH 6.0–9.0 could not be carried out, because of the absence of baseline in the unfolded state

at some pH values The thermal unfolding of HPr was pH-independent from pH 7.0–9.0, as concluded from the identical Tmvalues (64.6 ± 0.6°C), suggesting that protein stability was similar in that pH range

It is interesting to note here that the transition at pH 3.5 (Fig 5A) showed a low degree of cooperativity at low temperatures, although not a proper and complete sig-moidal behaviour was observed This could suggest the presence of a molten-globule species, as has been seen in a-lactalbumin and other proteins [42]

FTIR spectroscopy Upon heating, the shape of the amide I band of HPr changed dramatically at pH 2.5 and 7.5, as shown by: (a) a substantial loss in the integrated intensity of bands arising from a helix and (b) the appearance of a strong and weak bands at 1620 and

1680 cm)1, respectively, which correspond to interactions between extended chains, and have been related to aggre-gation of thermally unfolded proteins [30] (Fig 6) The measurement of the whole band width at half-height upon temperature allowed the characterization of the melting curve at pH 7.5, which resembled that found by CD experiments (Figs 5 and 6) Similar sigmoidal transitions were also obtained by following the change in intensity in the bands at 1652 cm)1(where the a helix appears) and at

1630 cm)1 (where the b sheet is absorbing) (data not shown) At pH 7.5, the transitions were irreversible, precluding the determination of Tm The lack of reversibi-lity, when compared to CD and DSC results, was probably due to the high protein concentrations used

At pH 2.5 a thermal transition was also observed (Fig 6A), in contrast to the experimental findings obtained

by CD The presence of this transition at pH 2.5 is not understood, but it could be due to aggregation processes occurring at the high protein concentrations and tempera-tures used in the FTIR experiments

DSC experiments We studied the heat-induced denatur-ation of the protein by DSC at pH 7.5 The protein (1 mgÆmL)1, 8 7.5 lM) was heated at a constant scanning rate (60°CÆh)1) up to 95°C (scan), cooled down, and reheated under identical conditions (re-scan) The scan and re-scan experiments are equally well fitted by the two-state model [43] with van’t Hoff to calorimetric enthalpy ratios,

DHVH/DHcal, of 1.02 and 1.01, respectively The DSC results indicate that the heat-induced unfolding of HPr was characterized, under these experimental conditions, by a melting temperature, Tm, of 65.4 ± 0.5°C, a calorimetric enthalpy change upon unfolding, DHcal¼ 60.3 ± 1.5 kcalÆmol)1 and an entropy change upon unfolding: DS(Tm)¼ DH(Tm)/Tm¼ 177.8calÆK)1Æmol)1(Fig 7) Chemical denaturation experiments

As the stability of HPr does not change significantly around neutral pH, as concluded from thermal denatur-ation experiments, we decided to follow the chemical denaturation at pH 7.5 to compare with the stability

Fig 5 Thermal denaturation profiles of HPr followed by far-UV CD at

222 nm Continuous line (circles) is the heating experiment and the

dotted line (squares) is the reheating scan at (A) pH 3.5; (B) pH 5; and

(C) pH 7.5 The ellipticity units on the y-axis are arbitrary The

con-ditions were: 20 l M of protein; buffer concentration was 10 m M ;

spectra were acquired in 0.1-cm pathlength cells The scan rate was

60 °CÆh)1in all cases.

results obtained in other HPr family members Figure 8

shows the chemical denaturation curves of HPr at pH 7.5,

10 mM phosphate, followed by fluorescence, far-UV and

gel filtration chromatography at 25°C The agreement

between the three probes suggests that the

chemical-denaturation can be described as a two-state model

Thermodynamic parameters from gel-filtration and CD

measurements had a large error We do not know the

reasons for those large errors, but they could be due to the

spread observed in the baselines of the transitions Similar

large slopes in chemical denaturation experiments followed

by NMR have also been observed by following the

GdmCl chemical-denaturation in the E coli HPr [16], but

they do not yield large errors in the thermodynamic

parameters Chemical denaturation followed by ANS

binding did not show any sigmoidal titration (data not shown), suggesting that no intermediate with close solvent-exposed hydrophobic patches accumulated during the denaturation The free energy, determined from the fluorescence measurements using the linear extrapolation method approach, yielded a value of 4.0 ± 0.2 kcalÆmol)1, indicating that HPr is not a highly stable protein The denaturation experiments were reversible in all cases

Discussion

Equilibrium-unfolding of HPr at neutral and high

pH values follows a two-state mechanism The chemical-denaturation folding of S coelicolor follows a two-state mechanism at neutral and high pH values, as happens in other small proteins [44] The denaturation of HPr can be described as a two-state reaction at neutral pH from the following evidence (a) All the unfolding data can

be fitted to a single transition curve using Eqn (3) (b) The denaturant transitions appear to be independent of the biophysical probe (fluorescence, far-UV CD and gel-filtra-tion chromatography) used (Fig 8), and reversible for either folding and unfolding (data not shown) (c) The ratio

of the van’t Hoff enthalpy of denaturation and the calorimetric enthalpy obtained from DSC is close to unity for the heating and the reheating scans (Fig 7)

The conformational stability of S coelicolor is the smallest among that of the other family members reported

so far (Table 2) This low stability is also confirmed by hydrogen exchange measurements at pH 7.5 using FTIR and NMR experiments, where most of the protons exchanged within 20 min (data not shown) Comparison

of the sequence among 31 HPr homologues in the protein data bank indicates that the HPr of S coelicolor forms a completely different cluster of sequence equally distant to the rest of the members of the family [9] (Fig 9) The differences in stability, as the structure of HPr of S coeli-color (unpublished results) is nearly identical to those of

E coli and B subtilis, must rely on differences in the packing of their different side-chains

Fig 7 Excess heat capacity function of HPr at pH 7.5 in 10 m M

phosphate buffer The continuous lines represent the fitting of the experimental data to a two-state reversible model.

Fig 6 Thermal denaturation profiles of HPr followed by FTIR (A)

Thermal denaturation profiles of HPr at pH 2.5 (filled squares) and

pH 7.5 (filled circles) The lines at both pH values are drawn to guide

the eye (B) Thermal denaturation profiles of HPr at pH 7.5 (left side)

and pH 2.5 (right side) at selected temperatures Protein concentration

was 6 mgÆmL)1; all other conditions as described under the

Experi-mental procedures section.