Organization of the MADS Box from Human SRF Revealed by Tyrosine Perturbation

详细信息    查看全文

• 作者：Barbora Profantov谩 ; Yves-Marie Co茂c ; V谩clav Profant ; Josef 艩t臎p谩nek ; Vladim铆r Kopeck媒 ; Jr. ; Pierre-Yves Turpin ; Bernard Alpert ; Christian Zentz
• 刊名：Journal of Physical Chemistry B
• 出版年：2015
• 出版时间：February 5, 2015
• 年：2015
• 卷：119
• 期：5
• 页码：1793-1801
• 全文大小：411K
• ISSN：1520-5207

文摘

MADS box family transcription factors are involved in signal transduction and development control through DNA specific sequence recognition. The DNA binding domain of these proteins contains a conservative 55鈥?0 amino acid sequence which defines the membership of this large family. Here we present a thorough study of the MADS segment of serum response factor (MADS^SRF). Fluorescence, UV-absorption, and Raman spectroscopy studies were performed in order to disclose its behavior and basic functional properties in an aqueous environment. The secondary structure of MADS^SRF estimated by analysis of Raman spectra and supported by CD has revealed only the C-terminal part as homologous with those of free core-SRF, while the N-terminal part has lost the stable 伪-helical structure found in both the free core-SRF and its specific complex with DNA. The three tyrosine residues of the MADS^SRF were used as spectroscopic inner probes. The effect of environmental conditions, especially pH variations and addition of variously charged quenchers, on their spectra was examined. Two-component fluorescence quenching was revealed using factor analysis and corresponding Stern鈥揤olmer constants determined. Factor analysis of absorbance and fluorescence pH titration led to determination of three dissociation constants pK_a1 = 6.4 卤 0.2, pK_a2 = 7.3 卤 0.2, and pK_a3 = 9.6 卤 0.6. Critical comparison of all experiments identified the deprotonation of His193 hydrogen bonded to Tyr195 as a candidate for pK_a1 (and that of Tyr158 as a candidate for pK_a2). Within MADS^SRF, His193 is a key intermediary between the N-terminal primary DNA binding element and the hydrophobic C-terminal protein dimerization element.