当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin.

Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin.

Abstract：

:We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Sun H,Yin D,Coffeen LA,Shea MA,Squier TC

doi

10.1021/bi0104266

subject

Has Abstract

pub_date

2001-08-14 00:00:00

pages

9605-17

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

杂志文章

在线工具

Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin.