Donor–Donor Energy Migration (DDEM) for Determining Intramolecular Distances in Proteins. II. the Effect of Partial Labeling

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• 作者：Karolin ; Jan ; Hä ; gglö ; f ; Peter ; Ny ; Tor ; Johansson ; Lennart B. &Aring ; .
• 关键词：Donor– ; donor energy migration ; intramolecular distance ; proteins ; partial labeling
• 刊名：Journal of Fluorescence
• 出版年：1997
• 出版时间：1997
• 年：1997
• 卷：07
• 期：4
• 页码：331-339
• 全文大小：1.09 MB

文摘

By using site-specific mutagenesis it is possible to prepare a protein molecule that can be labeled with two identical fluorescent probes at different positions.^(1,2) To calculate intramolecular distances between the two fluorescent donors in a protein, a recently developed DDEM model can be applied.^(3,4) Here we have studied the influence of incomplete donor labeling on the calculated distances. For this purpose, the previous model has been extended and compared with experiments performed on three mutants (V106C, M266C, and V106C-M266C) of plasminogen activator inhibitor type 1 (PAI-1) labeled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl) iodo-acetamide (SBDY). The Cb>αb> of the residues to which the SBDYs are covalently linked are separated by 55.1 Å, as determined by X-ray diffraction on the wild-type PAI-1. To examine the reliability of the extracted parameters, synthetic data were generated and reanalyzed with the same model as used to analyze real experiments. It is concluded that, even for a low degree of labeled double mutant (≈60%), a distance of 54 ± 3 Å is found for both models.