Lakowicz J R, Gryczynski I, Cheung H C, Wang C K, Johnson M L, Joshi N
Department of Biological Chemistry, University of Maryland, Baltimore School of Medicine 21201.
Biochemistry. 1988 Dec 27;27(26):9149-60. doi: 10.1021/bi00426a012.
We used resonance energy transfer to examine the distribution of distances between two sites on troponin I (TnI). The donor (D) was the single tryptophan residue at site 158 (Trp 158), and the acceptor (A) was cysteine 133 (Cys 133) which was labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IE). A distribution of D-A distances results in a distribution of donor decay times, which were resolved by using frequency-domain fluorometry. In the native state we recovered a relatively narrow distribution of D-A distances. The widths of the distance distributions were found to increase progressively and dramatically with increasing concentrations of guanidine hydrochloride. Binding of calcium-free troponin C (TnC) to troponin I did not alter the distance distribution. Addition of Ca2+ to the TnI.TnC complex resulted in a sharper distance distribution and protected against the guanidine hydrochloride induced increase in the width of the distance distribution. Additionally, the same distance distributions were recovered for native and denatured TnI when the Forster distance for energy transfer was decreased by acrylamide quenching. These results demonstrate that distance distributions can be recovered with good accuracy, to the extent of revealing modest changes due to binding of other components. This technique should have widespread applications in studies of protein folding.
我们利用共振能量转移来研究肌钙蛋白I（TnI）上两个位点之间的距离分布。供体（D）是位点158处的单个色氨酸残基（Trp 158），受体（A）是用N-（碘乙酰基）-N'-（1-磺基-5-萘基）乙二胺（IE）标记的半胱氨酸133（Cys 133）。供体-受体距离的分布导致供体衰减时间的分布，通过频域荧光法得以解析。在天然状态下，我们得到了相对较窄的供体-受体距离分布。发现随着盐酸胍浓度的增加，距离分布的宽度逐渐显著增加。无钙肌钙蛋白C（TnC）与肌钙蛋白I的结合并未改变距离分布。向TnI.TnC复合物中添加Ca2+导致距离分布更窄，并防止盐酸胍引起的距离分布宽度增加。此外，当通过丙烯酰胺猝灭降低能量转移的福斯特距离时，天然和变性的TnI得到了相同的距离分布。这些结果表明，距离分布能够以较高的准确度得到，能够揭示由于其他成分结合而产生的适度变化。这项技术在蛋白质折叠研究中应该有广泛的应用。
