Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Subunit conformation and dynamics in a heterodimeric protein: studies of the hybrid isozyme of creatine kinase
Introduction
The cytoplasmic isozymes of creatine kinase (EC 2.7.3.2) occur in three tissue specific, dimeric forms designated CK-MM (muscle), CK-BB (brain) and CK-MB (heart) [1]. Creatine kinase MM, which is found in heart as well, is both soluble and a particulate constituent of the M-line structure, whereas CK-MB is found only in the soluble part of the myocardial cell [2]. While the M and B subunits of CK are approximately 80% sequence homologous [3], CK-MM and CK-BB have several different physical properties [1, 4], including complete antigenic non-crossreactivity [5]. An interesting question to pose is how the same subunits which constitute the homodimers are able to comply with the structural requirements for formation of the heterodimer. Subunit conformation in the hybrid may be similar to that in the homodimers or adjustments may be required for activity and subunit-subunit stability. Comparisons of the three isozymes then, affords an opportunity to determine the types of conformational adaptations required for subunit hybridization and addresses the more basic issue of the nature of protein-protein interaction.
Several physical characteristics suggest that conformational changes in subunits occur as the result of hybridization, including relative isoelectric points and electrophoretic mobilities [6], fluorescence resonance energy between the two active sites [7, 8, 9] and antigenicities [5]. An important start in understanding subunit interdigitation of CK at the structural level has been made with the determination of the crystallographic three-dimension conformation of the mitochondrial octameric CK [10]. As more structures are detailed, it would be useful to complement these with an analysis of conformational flexibility. In this study, we have compared elements of secondary, tertiary and quaternary structure of CK-MB with the homodimers. Results indicate significant conformational and dynamic changes upon subunit-subunit interaction in the assembly of the heterodimer.
Section snippets
Materials and methods
Rabbit CK-MB from heart [11] and CK-BB from brain [6] were purified as described previously. Rabbit CK-MM was obtained from Boehringer/Mannheim (Indianapolis, IN). Creatine kinase MB was also prepared by in vitro hybridization [7]. Equal activities of CK-MM and CK-BB in 0.05 M Tris-HCl buffer, pH 8.0 and 2 mM DTT were mixed, then treated with sufficient crystalline, enzyme grade urea, to bring the final concentration to 6 M urea. After 1 h at room temperature, buffer was added to the denatured
Purification of CK-MB from rabbit heart and comparison with CK-MB prepared by in vitro hybridization
Creatine kinase MB isolated from rabbit heart and CK-MB prepared by in vitro hybridization exhibited identical electrophoretic mobilities in agarose thin film electrophoresis and on polyacrylamide gels containing SDS. In the latter method, each protein appeared as two closely migrating bands, with molecular weights approximately 40 kDa. Isozymes MM, BB, MB (purified) and MB (prepared by hybridization) exhibited elution times (±0.01 min) of 6.57, 6.54, 6.59, and 6.59 min, respectively, on an
Discussion
In this investigation, we identify specific conformational changes at the secondary and tertiary levels of protein organization in response to subunit hybridization. The spectral characteristics of CK-MB, whether absorbance, fluorescence or circular dichroism, are not an average of those of CK-MM and CK-BB. Had the conformation of the subunits in the hybrid of CK-MB been the same as those in the homodimers, the difference in absorbance between CK-MB and a mixture of CK-MM+CK-BB should be
Acknowledgements
Supported in part by a grant from the American Heart Association, Florida Affiliate.
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