RESUMO
Five mutant alpha-lactalbumins, with one or two amino acid substitution(s) in the B helix, were engineered to examine the relation between the stability of the molten globule state and the hydrophobicity of these amino acids. The mutation sites (Thr29, Ala30 and Thr33) have been chosen on the basis of comparison of the amino acid sequences of goat, bovine and gunea pig alpha-lactalbumin, in which the guinea pig protein shows a remarkably more stable molten globule than the other proteins. The recombinant proteins were expressed Escherichia coli and then purified and refolded efficiently to produce the active proteins. The stability of the molten globule state of these engineered proteins has been investigated by urea-induced unfolding transition under an acidic condition (pH 2.0), where the molten globule state is stable in the absence of urea. The results show that the molten globule state is stabilized by the amino acid substitutions which raise the hydrophobicity of the residues, suggesting that the hydrophobic core in a globular protein plays an important role in the stability of the molten globule state. The change in stabilization free energy of the molten globule state caused by each amino acid substitution has been evaluated, and molecular mechanisms of stabilization of the molten globule state are discussed.
Assuntos
Lactalbumina/química , Mutação , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Dicroísmo Circular , Escherichia coli/genética , Cabras , Cobaias , Lactalbumina/genética , Modelos Moleculares , Dados de Sequência Molecular , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Ureia/farmacologiaRESUMO
A cDNA clone was isolated that encodes the partial sequence of a putative endoplasmic reticulum Ca(2+)-ATPase of tobacco. The 1.497-kb insert had an open reading frame of 1.149 kb. The deduced peptide had the greatest homology to the endoplasmic reticulum Ca(2+)-ATPases of Drosophila and Artemia, followed by the mammalian and avian enzymes (SERCA2 and 3). The cDNA insert hybridized to a single mRNA of 4.4 kb from tobacco cultured cells or plant tissues. The level of this transcript was induced about 2-fold by NaCl shock in 428 mm NaCl-deadapted tobacco cells that were maintained in medium without salt, but not in unadapted cells. The level of this transcript was 3- to 4-fold higher in 428 mm NaCl-adapted cells growing in salt than in unadapted cells growing without salt.
RESUMO
A cDNA clone encoding the 70-kilodalton subunit of the tobacco (Nicotiana tabacum var Wisconsin 38) tonoplast ATPase has been isolated. The 1.656 kilobase insert contains only open reading frame that represents more than 80% of the carrot cDNA coding region. The deduced amino acid sequence has greater than 95% sequence identity with the homologous carrot sequence. A transcript of approximately 2.7 kilobase was detected on Northern blots of tobacco poly(A)(+) selected or total RNA using labeled probe produced from this clone. The gene was expressed throughout the growth cycle in unadapted and 428 millimolar NaCl adapted cells. Transcription of the 70-kilodalton subunit gene or mRNA stability was induced by short-term NaCl treatment in NaCl adapted cells or by abscisic acid treatment in both adapted and unadapted cells. Southern analysis indicated the presence of up to four genes encoding the 70-kilodalton subunit.
