Role of Hydration in the Closed-to-Open Transition Involved in Ca2+ Binding by Troponin C
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- 作者：Marisa C. Suarez ; Cosme José ; V. Machado ; Luí ; s Maurí ; cio T. R. Lima ; Lawrence B. Smillie ; Joyce R. Pearlstone ; Jerson L. Silva ; Martha M. Sorenson ; and Dé ; bora Foguel
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 13, 2003
- 年：2003
- 卷：42
- 期：18
- 页码：5522 - 5530
- 全文大小：170K
- 年卷期：v.42,no.18(May 13, 2003)
- ISSN：1520-4995
文摘
Troponin C (TnC) is the Ca2+-binding subunit of the troponin complex of vertebrate skeletalmuscle. It consists of two structurally homologous domains, N and C, connected by an exposed 
-helix.The C-domain has two high-affinity sites for Ca2+ that also bind Mg2+, whereas the N-domain has twolow-affinity sites for Ca2+. Previous studies using isolated N- and C-domains showed that the C-domainapo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol usingfluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability ofthe protein to urea (0-8 M) in the absence of Ca2+. Furthermore, the ability to expose hydrophobicsurfaces normally promoted by Ca2+ binding or low temperature under pressure was partially lost in thepresence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca2+ affinity of the N-domain insolution. From the ln Kobs versus ln aH2O, we obtained the number of water molecules (63.5 ± 8.7) involvedin the transition N 
N·Ca2 that corresponds to an increase in the exposed surface area of 571.5 ± 78.3Å2. In skinned fibers, the affinity for Ca2+ was also reduced by glycerol, although the effect was muchless pronounced than in solution. Our results demonstrate quantitatively that the stability of this proteinand its affinity for Ca2+ are critically dependent on protein hydration.
-helix.The C-domain has two high-affinity sites for Ca2+ that also bind Mg2+, whereas the N-domain has twolow-affinity sites for Ca2+. Previous studies using isolated N- and C-domains showed that the C-domainapo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol usingfluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability ofthe protein to urea (0-8 M) in the absence of Ca2+. Furthermore, the ability to expose hydrophobicsurfaces normally promoted by Ca2+ binding or low temperature under pressure was partially lost in thepresence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca2+ affinity of the N-domain insolution. From the ln Kobs versus ln aH2O, we obtained the number of water molecules (63.5 ± 8.7) involvedin the transition N N·Ca2 that corresponds to an increase in the exposed surface area of 571.5 ± 78.3Å2. In skinned fibers, the affinity for Ca2+ was also reduced by glycerol, although the effect was muchless pronounced than in solution. Our results demonstrate quantitatively that the stability of this proteinand its affinity for Ca2+ are critically dependent on protein hydration.