The Role of -Sheet Interactions in Domain Stability, Folding, and Target Recognition Reactions of Calmodulin
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- 作者：J. Peter Browne ; Molly Strom ; Stephen R. Martin ; and Peter M. Bayley
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：August 5, 1997
- 年：1997
- 卷：36
- 期：31
- 页码：9550 - 9561
- 全文大小：480K
- 年卷期：v.36,no.31(August 5, 1997)
- ISSN：1520-4995
文摘
Single-residue mutations have been made of thehydrophobic Ile or Val residue in position 8of each of the four calcium-binding loop sequences (sites I-IV) ofDrosophila calmodulin. These highlyconserved residues are part of the hydrophobic core of eithercalmodulin domain and are involved in thestructural link of two calcium-binding sites via a short antiparallel
-sheet. In the apo-form, the replacementof Ile (or Val) by Gly causes a significant destabilization, shown bythe unfolding of the secondary structureof the domain carrying the mutation. In the presence of calcium,the deficiency in 
-helical structure at20 
C is restored for the mutants at site I, II, or III but not atsite IV, which requires the further bindingof a high-affinity target peptide to re-establish the nativeconformation. The extent of the destabilizationis seen in the depression of the melting temperature of individualdomains, which can be as large as 80C in the case of Ca4-CaM(V136G). However,because of low values of the unfolding enthalpy forcalmodulin domains, only relatively low values of <2 kcal/mol areimplied for 
G, the free energy ofdestabilization due to mutation. Consistent with this, thesecondary structure of any unfolded mutantdomain is highly sensitive to solvent composition and is largelyrefolded in the presence of 12.5% (v/v)aqueous trifluoroethanol. Compared to wild-type calmodulin, theaffinities of the mutants for calciumand target peptides from sk-MLCK at 20 C are significantly reducedbut the effects are relatively small.These results indicate that the conformation of calmodulin can bedramatically altered by mutation of asingle highly conserved residue but that changes in solvent or thebinding of a target sequence can readilycompensate for this, restoring the wild-type properties. Theresults also suggest that the integrity of boththe apo- and holo-forms of calmodulin is important for the maintenanceof its biological function andconfirm the importance of conserving the structural function of theresidues involved in the -sheetinteractions.
-sheet. In the apo-form, the replacementof Ile (or Val) by Gly causes a significant destabilization, shown bythe unfolding of the secondary structureof the domain carrying the mutation. In the presence of calcium,the deficiency in -helical structure at20 C is restored for the mutants at site I, II, or III but not atsite IV, which requires the further bindingof a high-affinity target peptide to re-establish the nativeconformation. The extent of the destabilizationis seen in the depression of the melting temperature of individualdomains, which can be as large as 80C in the case of Ca4-CaM(V136G). However,because of low values of the unfolding enthalpy forcalmodulin domains, only relatively low values of <2 kcal/mol areimplied for G, the free energy ofdestabilization due to mutation. Consistent with this, thesecondary structure of any unfolded mutantdomain is highly sensitive to solvent composition and is largelyrefolded in the presence of 12.5% (v/v)aqueous trifluoroethanol. Compared to wild-type calmodulin, theaffinities of the mutants for calciumand target peptides from sk-MLCK at 20 C are significantly reducedbut the effects are relatively small.These results indicate that the conformation of calmodulin can bedramatically altered by mutation of asingle highly conserved residue but that changes in solvent or thebinding of a target sequence can readilycompensate for this, restoring the wild-type properties. Theresults also suggest that the integrity of boththe apo- and holo-forms of calmodulin is important for the maintenanceof its biological function andconfirm the importance of conserving the structural function of theresidues involved in the -sheetinteractions.