RESUMO
Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.
Assuntos
Archaeoglobus fulgidus , Proteínas de Membrana , Desnaturação Proteica , Dodecilsulfato de Sódio , Termodinâmica , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/farmacologia , Archaeoglobus fulgidus/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Espectrometria de Fluorescência , Estabilidade Proteica , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Naftalenossulfonato de Anilina/química , Naftalenossulfonato de Anilina/metabolismo , Triptofano/química , Triptofano/metabolismo , Cobre/química , Cobre/metabolismo , Dobramento de Proteína , Conformação ProteicaRESUMO
Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.
Assuntos
Adenosina Trifosfatases , Biocatálise , Cobre , Detergentes , Micelas , Dodecilsulfato de Sódio , Adenosina Trifosfatases/metabolismo , Archaeoglobus fulgidus/enzimologia , Biocatálise/efeitos dos fármacos , Calorimetria , Cobre/metabolismo , Detergentes/farmacologia , Hidrólise/efeitos dos fármacos , Legionella pneumophila/enzimologia , Dodecilsulfato de Sódio/farmacologia , Temperatura , TermodinâmicaRESUMO
Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG()=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH()=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH()=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP.
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas Arqueais/química , Archaeoglobus fulgidus/química , Cobre/química , Monoéster Fosfórico Hidrolases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/enzimologia , Biocatálise , Domínio Catalítico , Cátions Bivalentes , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Temperatura Alta , Cinética , Magnésio/química , Modelos Moleculares , Nitrofenóis/química , Compostos Organofosforados/química , Fosfolipídeos/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue alpha-helical membrane protein that is involved in transporting Cu(+) throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =0.93 kJ mol(-1) K(-1). These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.
Assuntos
Archaeoglobus fulgidus/química , Detergentes/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Fosfolipídeos/química , Dobramento de Proteína , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Archaeoglobus fulgidus/metabolismo , Dicroísmo Circular , Cinética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise EspectralRESUMO
Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.