Tài liệu Báo cáo khoa học: The conformational stability of the Streptomyces coelicolor

The conformational stability of the Streptomyces coelicolor

histidine-phosphocarrier protein

Characterization of cold denaturation and urea–protein interactions

Jose´ L. Neira1,2 and Javier Go´ mez1

1

Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Elche (Alicante); 2

Instituto de Biocomputacio´n y Fı´sica

de los Sistemas complejos, Zaragoza, Spain

Thermodynamic parameters describing the conformational

stability of the histidine-containing phosphocarrier protein

from Streptomyces coelicolor, scHPr, have been determined

by steady-state fluorescence measurements of isothermal

urea-denaturations, differential scanning calorimetry at

different guanidinium hydrochloride concentrations and,

independently, by far-UV circular dichroism measurements

of isothermal urea-denaturations, and thermal denatura￾tions at fixed urea concentrations. The equilibrium unfold￾ing transitions are described adequately by the two-state

model and they validate the linear free-energy extrapolation

model, over the large temperature range explored, and the

urea concentrations used. At moderate urea concentrations

(from 2 to 3 M), scHPr undergoes both high- and low￾temperature unfolding. The free-energy stability curves have

been obtained for the whole temperature range and values of

the thermodynamic parameters governing the heat- and

cold-denaturation processes have been obtained. Cold￾denaturation of the protein is the result of the combination

of an unusually high heat capacity change (1.4 ± 0.3

kcalÆmol)1

ÆK)1

, at 0 M urea, being the average of the fluor￾escence, circular dichroism and differential scanning calori￾metry measurements) and a fairly low enthalpy change upon

unfolding at the midpoint temperature of heat-denaturation

(59 ± 4 kcalÆmol)1

, the average of the fluorescence, circular

dichroism and differential scanning calorimetry measure￾ments). The changes in enthalpy (mDHi

), entropy (mDSi

) and

heat capacity (mDCpi), which occur upon preferential urea

binding to the unfolded state vs. the folded state of the

protein, have also been determined. The mDHi and the mDSi

are negative at low temperatures, but as the temperature is

increased, mDHi makes a less favourable contribution than

mDSi to the change in free energy upon urea binding. The

mDCpi is larger than those observed for other proteins; how￾ever, its contribution to the global heat capacity change upon

unfolding is small.

Keywords: calorimetry; denaturant–binding interactions;

histidine-phosphocarrier; protein stability.

A full understanding of the physical interactions underlying

the structure, folding and the function of a protein requires

a detailed description of its conformational stability in

terms of the free energy of unfolding. Such a thermo￾dynamic description relies on the quantitative analysis of

denaturant-induced or thermally induced folding-unfolding

transitions, measured either spectroscopically or calorimet￾rically. In both cases, data analyses involves the extra￾polation of the thermodynamic parameters to standard

conditions, usually 298 K in the absence of denaturant. To

extrapolate thermal denaturation data, the change in DCp,

and its temperature dependence must be known [1,2]. The

extrapolation of data from chemical-denaturation [with

either urea or guanidinium hydrochloride (Gdm Cl) as

denaturants] is carried out using either the linear free

energy model, LEM [3–5], or the binding model [6]. The

LEM is by far th e most commonly used model, and it has

been found to be valid for several proteins [7–9]. Combined

analysis of the LEM with thermal denaturation data,

assuming a temperature-independent DCp and the thermo￾dynamic equivalence between the thermally and chemically

denatured states, have been reported for several proteins

[10, 7 and references therein]. These analyses yield the

thermodynamic parameters governing the conformational

Correspondence to J. L. Neira and J. Go´mez, 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 966658459, + 34 966658459, Tel.: + 34 966658467,

E-mail: [email protected] and [email protected]

Abbreviations: CD, circular dichroism; DSC, differential scanning

calorimetry; Gdm Cl, guanidinium hydrochloride; DCp, the heat

capacity change; mDCpi , the heat capacity change upon preferential

urea-binding to the unfolded protein vs. the protein folded state; DHm,

the calorimetric enthalpy change at Tm; mDHi , the enthalpy change

upon preferential urea-binding to the unfolded protein vs. the protein

folded state; HPr, histidine phosphocarrier protein of the PTS;

scHPr, HPr from S. coelicolor; bsHPr, HPr from B. subtilis;

ecHPr, HPr from E. coli; LEM, linear extrapolation method;

PTS, the phosphoenolpyruvate-dependent sugar

phosphotransferase system; DSm, the calorimetric entropy

change at Tm; mDSi , the entropy change upon preferential

urea-binding to the unfolded protein vs. the protein folded

state; Tm, thermal denaturation midpoint.

Dedication: This paper is dedicated to the memory of Jose´ Laynez.

(Received 27 January 2004, revised 24 March 2004,

accepted 2 April 2004)

Eur. J. Biochem. 271, 2165–2181 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.4142.x