Ca2+ Coordination to Backbone Carbonyl Oxygen Atoms in Calmodulin and Other EF-Hand Proteins: 15N Chemical Shifts as Probes for Monitoring Individual-Site Ca2+ Coordin
详细信息 查看全文
- 作者：Rodolfo R. Biekofsky ; Stephen R. Martin ; J. Peter Browne ; Peter M. Bayley ; and James Feeney
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：May 19, 1998
- 年：1998
- 卷：37
- 期：20
- 页码：7617 - 7629
- 全文大小：270K
- 年卷期：v.37,no.20(May 19, 1998)
- ISSN：1520-4995
文摘
Examination of the NMR 15N chemical shifts of a numberof EF-hand proteins shows that theshift value for the amido nitrogen of the residue in position 8 of acanonical EF-hand loop (or position10 of a pseudo EF-hand loop) provides a good indication of metaloccupation of that site. The NH of theresidue in position 8 is covalently bonded to the carbonyl of residue7, the only backbone carbonyl thatcoordinates to the metal ion in a canonical EF-hand loop. Uponmetal coordination to this carbonyl,there is an appreciable deshielding of the 15N nucleus atposition 8 (+4 to +8 ppm) due to the polarizationof the O(7)=C(7)-N(8) amido group and the correspondingreduction in the electron density of the nitrogenatom. This deshielding effect is effectively independent of thebinding of metal to the other site of anEF-hand pair, allowing the 15N shifts to be used as probesfor site-specific occupancy of metal bindingsites. In addition, a Ca2+-induced change inside-chainH
hars/alpha.gif" BORDER=0>-Chars/alpha.gif" BORDER=0>-Chars/beta2.gif" BORDER=0 ALIGN="middle">-Hhars/beta2.gif" BORDER=0 ALIGN="middle">torsion angle for isoleucine or valineresidues in position 8 can also contribute to the deshielding of theamide 15N nucleus. This conformationaleffect occurs only in sites I or III and takes place upon binding aCa2+ ion to the other site of an EF-handpair (site II or IV) regardless of whether the first site is occupied.The magnitude of this effect is in therange +5 to +7 ppm. A Ca2+ titration of15N-labeled apo-calmodulin was performed using 2D1H-15NHSQC NMR spectra. The changes in the 15N chemicalshifts and intensities for the peaks correspondingto the NH groups of residues in position 8 of the EF-hand loops allowedthe amount of metal bound atsites II, III and IV to be monitored directly at partial degrees ofsaturation. The peak corresponding tosite I could only be monitored at the beginning and end of thetitration because of line broadening effectsin the intermediate region of the titration. Sites III and IV bothtitrate preferentially and the resultsdemonstrate clearly that sites in either domain fill effectively inparallel, consistent with a significantpositive intradomain cooperativity of calcium binding.
hars/alpha.gif" BORDER=0>-Chars/alpha.gif" BORDER=0>-Chars/beta2.gif" BORDER=0 ALIGN="middle">-Hhars/beta2.gif" BORDER=0 ALIGN="middle">torsion angle for isoleucine or valineresidues in position 8 can also contribute to the deshielding of theamide 15N nucleus. This conformationaleffect occurs only in sites I or III and takes place upon binding aCa2+ ion to the other site of an EF-handpair (site II or IV) regardless of whether the first site is occupied.The magnitude of this effect is in therange +5 to +7 ppm. A Ca2+ titration of15N-labeled apo-calmodulin was performed using 2D1H-15NHSQC NMR spectra. The changes in the 15N chemicalshifts and intensities for the peaks correspondingto the NH groups of residues in position 8 of the EF-hand loops allowedthe amount of metal bound atsites II, III and IV to be monitored directly at partial degrees ofsaturation. The peak corresponding tosite I could only be monitored at the beginning and end of thetitration because of line broadening effectsin the intermediate region of the titration. Sites III and IV bothtitrate preferentially and the resultsdemonstrate clearly that sites in either domain fill effectively inparallel, consistent with a significantpositive intradomain cooperativity of calcium binding.