Aplysia limacina myoglobin cDNA cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis.
نویسندگان
چکیده
The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back transplanting the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)-containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved.
منابع مشابه
Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins.
In the globin family, similarities in the folding mechanism have been found among different mammalian apomyoglobins (apoMb). The best-characterized intermediate of sperm whale apoMb, called I(AGH), is mainly stabilized by nativelike contacts among the A, G, and H helices involving a cluster of hydrophobic residues that includes two conserved tryptophans. To verify the hypothesis of a common int...
متن کاملMolecular Cloning and Mutagenesis of Rat Glucocerebrosidase Gene
Purpose: The aim of this study was cloning the Gba enzyme in pUCBM21 plasmid, and making frame mutation on it and sequencing it. Materials and methods: mRNA was extracted from mouse spleen and glucocerebrosidase cDNA was synthesized and amplified by PCR with specific primers. cDNA was cloned in pUCBM21 and analyzed by restriction enzymes. A fragment of its sequence was deleted using MscI restr...
متن کاملHaem disorder in two myoglobins: comparison of reorientation rate.
The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237, 613-616] for sperm wha...
متن کاملSite-Directed Mutagenesis in Human Granulocyte-colony Stimulating Factor, Cloning and Expression in Escherichia coli
Human granulocyte colony stimulating factor (hG-CSF) induces proliferation and differentiation of granulocyte progenitor cells. This glycoprotein is currently being used for treatment of neutropenia, in patients who have undergone bone marrow transplantation. So far, different researchers have tried to enhance hG-CSF biological activity and stability. In this study, Polymerase Chain Reaction (P...
متن کاملSerratia marcescens B4A chitinase thermostability enhancement by S390I QuikChange site directed mutagenesis
Thermostable chitinases are useful for industrial and biotechnological applications. This paper reports the stabilization of chitinase from Serratia marcescens B4A through rational mutagenesis. Changing of Ser 390 to Ile in S. marcescens. The stabilization was enhanced through entropic stabilization by reduction of the loop length and also by increasing of the beta chain length. With this repla...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The Biochemical journal
دوره 314 ( Pt 1) شماره
صفحات -
تاریخ انتشار 1996