title: Organization of the MADS box from human SRF revealed by tyrosine perturbation.
abstract: 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 55-60 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 α-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-Volmer constants determined. Factor analysis of absorbance and fluorescence pH titration led to determination of three dissociation constants pKa1 = 6.4 ± 0.2, pKa2 = 7.3 ± 0.2, and pKa3 = 9.6 ± 0.6. Critical comparison of all experiments identified the deprotonation of His193 hydrogen bonded to Tyr195 as a candidate for pKa1 (and that of Tyr158 as a candidate for pKa2). Within MADS(SRF), His193 is a key intermediary between the N-terminal primary DNA binding element and the hydrophobic C-terminal protein dimerization element.
authors.affiliation: Lab. Jean Perrin, CNRS UMR 8237, UPMC Université Paris 6 , 4 place Jussieu, 75252 Paris Cedex 05, France.
authors.forename: Barbora
authors.initials: B
authors.lastname: Profantová
authors.forename: Yves-Marie
authors.initials: YM
authors.lastname: Coïc
authors.forename: Václav
authors.initials: V
authors.lastname: Profant
authors.forename: Josef
authors.initials: J
authors.lastname: Štěpánek
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kopecký
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Turpin
authors.forename: Bernard
authors.initials: B
authors.lastname: Alpert
authors.forename: Christian
authors.initials: C
authors.lastname: Zentz
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D017433:Protein Structure, Secondary; D026362:Serum Response Factor / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010455:Peptides; D026362:Serum Response Factor; D014443:Tyrosine