A 13C Labeling Strategy Reveals a Range of Aromatic Side Chain Motion in Calmodulin

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• 作者：Vignesh Kasinath ; Kathleen G. Valentine ; A. Joshua Wand
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：July 3, 2013
• 年：2013
• 卷：135
• 期：26
• 页码：9560-9563
• 全文大小：220K
• 年卷期：v.135,no.26(July 3, 2013)
• ISSN：1520-5126

文摘

NMR relaxation experiments often require site-specific isotopic enrichment schemes in order to allow for quantitative interpretation. Here we describe a new labeling scheme for site-specific ¹³C鈥?sup>1H enrichment of a single ortho position of aromatic amino acid side chains in an otherwise perdeuterated background by employing a combination of [4-¹³C]erythrose and deuterated pyruvate during growth on deuterium oxide. This labeling scheme largely eliminates undesired contributions to ¹³C relaxation and greatly simplifies the fitting of relaxation data using the Lipari鈥揝zabo model-free formalism. This approach is illustrated with calcium-saturated vertebrate calmodulin and oxidized flavodoxin from Cyanobacterium anabaena. Analysis of ¹³C relaxation in the aromatic groups of calcium-saturated calmodulin indicates a wide range of motion in the subnanosecond time regime.