Gay Grant L, Lindhout Darrin A, Sykes Brian D
Canadian Institutes of Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Canada.
Protein Sci. 2004 Mar;13(3):640-51. doi: 10.1110/ps.03412704.
The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.
研究了利用顺磁性镧系离子使溶液中的肌钙蛋白C部分排列，作为确定结合的肌钙蛋白I肽结构的工具的可能性。这些研究的一个先决条件是了解镧系离子在蛋白质金属结合位点中的占据顺序。二维[(1)H, (15)N] HSQC核磁共振光谱已被用于研究Ce(3+)、Tb(3+)和Yb(3+)与人心脏肌钙蛋白C（cTnC）的脱辅基和全蛋白形式以及Ce(3+)与全蛋白鸡骨骼肌肌钙蛋白C（sTnC）的结合顺序。HSQC谱中交叉峰共振的消失被用于确定每种镧系元素在cTnC和sTnC中结合位点的占据顺序。对于所测试的镧系元素，结合顺序遵循结合位点残基净电荷从最负到次负的顺序；N结构域钙结合位点首先被占据，随后是C结构域位点。鉴于镧系离子的这种结合顺序，已证明有可能创建一种cTnC物种，其中N结构域位点中有一个镧系元素，C结构域结合位点中有两个Ca(2+)离子。通过使用cTnC.Yb(3+).2 Ca(2+)物种，有可能使结合的人心脏肌钙蛋白I（cTnI）肽产生部分排列。通过使用二维[(1)H, (15)N]同相-反相（IPAP）核磁共振光谱测量了结合的(15)N标记的cTnI(129 - 148)中共振的剩余偶极耦合（RDC）。
