title: Consequences of a six residual deletion from the N-terminal of rabbit muscle creatine kinase.
abstract: A mutant of dimeric rabbit muscle creatine kinase (CK), in which six residues (residues 2-7) at the N-terminal were removed by the PCR method, was studied to assess the role of these residues in dimer cohesion and to determine the structural stability of the protein. The specific activity of the mutant was 70.39% of that of the wild-type CK, and the affinity for Mg-ATP and CK substrates was slightly reduced compared with the wild-type protein. The structural stability of the mutant was investigated by a comparative equilibrium urea denaturation study and a thermal denaturation study. The data acquired by intrinsic fluorescence and far-UV circular dichroism (CD) during urea unfolding indicated that, the secondary and tertiary structures of the mutant were more stable than those of wild-type CK. Furthermore, results of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence demonstrated that the hydrophobic surface of the mutant CKND(6) was more stable during urea titration. Data from size exclusion chromatography (SEC) experiments indicated that deletion of the six N-terminal residues resulted in a relatively loose molecular structure, but the dissociation of the mutant CKND(6) occurred later during the unfolding process than for wild-type CK. Consistent with this result, the differential scanning calorimetry (DSC) profiles demonstrated that the thermal stability of the enzyme was increased by removal of the six N-terminal residues. We conclude that a more stable quaternary structure was obtained by deletion of the six residues from the N-terminal of wild-type CK.
authors.affiliation: Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China.
authors.forename: Shu-Yuan
authors.initials: SY
authors.lastname: Guo
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Shao-Wei
authors.initials: SW
authors.lastname: Ni
authors.forename: Xi-Cheng
authors.initials: XC
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D014508:Urea / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D014508:Urea; D003402:Creatine Kinase