RESUMO
The effects of negatively charged phosphatidylserine-prepared membranes (PS) and neutral phosphatidylcholine-prepared membranes (PC) on the structure of wild-type and mutant bovine pancreatic trypsin inhibitor (BPTI) at neutral pH were investigated. The presence of PC did not have any effect on the protein structure while PS induced a non-native structure in three mutant BPTI proteins. However, the negatively charged membrane did not have any effect on wild-type BPTI. The findings revealed that (i) elimination of some disulphide bonds results in dramatic change in protein structure, and, (ii) that this biochemical interaction is surface-driven and electrostatic interactions may play a very strong role in influencing the fore-stated changes in protein structure. Of further interest were the results obtained from investigating the possible role of PS fluidity and concentration in altering mutant. When the value of Gibbs free-energy change of unfolding (DeltaG(U)) was positive, various non-native structures were formed in a concentration-dependent manner. However, when the value of DeltaG(U) was negative, only two types of non-native structures were formed: one with high beta structure content at low PS fluidity state, and the other with a high alpha-helical content at high PS fluidity state. Our study reveals how particular combinations of phospholipid:protein interactions can induce a protein conformation transition from a native to a non-native one at neutral pH, especially when the native structure is predestabilized by amino acid substitutions. This revelation may open up opportunities to explore alternative ways in which phospholipids may play a role in protein mis-folding and the related pathologies.
Assuntos
Aprotinina/química , Proteínas Mutantes/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Temperatura , Animais , Aprotinina/genética , Aprotinina/metabolismo , Bovinos , Dicroísmo Circular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Conformação Proteica , TermodinâmicaRESUMO
Ion pairs have been considered to be general stabilizing factors in hyperthermophilic proteins, but the present experimental data cannot fully explain how ion pairs and ion-pair networks contribute to the stability. In this paper, we show experimental evidence that not all of the internal ion pairs contribute to the thermal and thermodynamic stability, using O(6)-methylguanine-DNA methyltransferase from Thermococcus kodakaraensis KOD1 (Tk-MGMT) as a model protein. Of three mutants in which an inter-helical ion pair was disrupted, only one mutant (E93A) was shown to be destabilized. Delta G of E93A was lower by approximately 4 kJ mol(-1) than that of the wild type, and E93A unfolded one order of magnitude faster than did the wild type and other variants. Glu 93 has unique properties in forming an ion-pair network that bridges the N- and C-terminal domains and connects three helices in the protein interior.
Assuntos
O(6)-Metilguanina-DNA Metiltransferase/química , Mutação Puntual , Thermococcus/enzimologia , Aminoácidos/química , Aminoácidos/genética , Dicroísmo Circular , Simulação por Computador , Estabilidade Enzimática/genética , Vetores Genéticos/genética , Guanidina/química , Temperatura Alta , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , O(6)-Metilguanina-DNA Metiltransferase/genética , Reação em Cadeia da Polimerase , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Eletricidade Estática , Temperatura , Termodinâmica , Transfecção , Temperatura de TransiçãoRESUMO
Tertiary structure of the recombinant glutamate dehydrogenase from Thermococcus kodakaraensis KOD1 (Tk-rGDH) converts into an intact form induced by the heat treatment. This phenomenon, heat-induced structural maturation, means that high temperature plays an important role in the proper folding and oligomerization of Tk-rGDH. In this work, we analyzed the heat-induced structural maturation of Tk-rGDH by differential scanning microcalorimetry (DSC), circular dichroism (CD), and activity measurements. In DSC measurements, the peak of adsorption of non-heated Tk-rGDH (nh-Tk-rGDH) was two times smaller than that of Tk-rGDH heated at 70 degrees C for 30 min (h-Tk-rGDH). The transition temperature (T(m)) of h-Tk-rGDH was 115 degrees C, which was about 3 degrees C higher than that of nh-Tk-rGDH. In the presence of 0.5 M NaCl, the nh-Tk-rGDH showed two peaks at 107 degrees C and 114 degrees C, while the h-Tk-rGDH showed a single peak at 115.7 degrees C. The heat-induced conformational change process was monitored by changes in CD intensity at 222 nm, and the result showed that heat-induced structural maturation is irreversible. The heat treatment at 70 degrees C showed the highest enhancement in activity, which was 15% larger than that of heat-treated Tk-rGDH at 40 degrees C. The results indicate that heat-induced structural maturation involves an irreversible process, transforming the non-heated form to the stable and active form.
