Sepúlveda-Becerra M A, Ferreira S T, Strasser R J, Garzón-Rodríguez W, Beltrán C, Gómez-Puyou A, Darszon A
Départamento Genética y Fisiología Molecular, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
Biochemistry. 1996 Dec 10;35(49):15915-22. doi: 10.1021/bi961548e.
The refolding and reassociation of rabbit muscle triosephosphate isomerase (TPI) monomers unfolded with guanidine hydrochloride (GdnHCl) was studied in aqueous media and in reverse micelles (RM) formed with hexadecyltrimethylammonium bromide and n-octane/hexanol. Fluorescence resonance energy transfer (FRET) studies were carried out using TPI labeled at Cys-217 with 5-((2-((iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) and TPI labeled at one of the free amino groups with fluorescein 5'-isothiocyanate (FITC). Efficient FRET between monomers of AEDANS-TPI and FITC-TPI in aqueous media was detected 2-3 min after denaturant dilution and remained constant for hours. The distance between AEDANS and FITC in a labeled, renatured hetero-TPI dimer calculated from FRET results was 48 A, in reasonable agreement with estimates based on the crystal structure of TPI. In RM, recovery of enzyme activity during renaturation correlates with the appearance of a high-intrinsic fluorescence intermediate believed to be a partially folded monomer (Fernández-Velasco et al., 1995). Nevertheless, when AEDANS- and FITC-labeled monomers were mixed in RM, FRET occurred as soon as GdnHCl was diluted (FRET efficiency = 0.36), preceding the changes in TPI intrinsic fluorescence and reactivation. Thereafter, the efficiency of FRET increased during the next hour up to approximately 0.50, where it remained after 24 h, when 80% of the enzyme activity was recovered. The high initial FRET seen in RM could indicate the formation of an inactive dimer within the first minutes after denaturant dilution. The further increase in FRET observed over the next hour could reflect conformational rearrangements of the protein and transition from the inactive to the active dimer.
研究了用盐酸胍（GdnHCl）展开的兔肌肉磷酸丙糖异构酶（TPI）单体在水性介质和由十六烷基三甲基溴化铵与正辛烷/己醇形成的反胶束（RM）中的重折叠和重缔合。使用在Cys-217处用5-（（2-（（碘乙酰基）-氨基）乙基）氨基）萘-1-磺酸（1,5-IAEDANS）标记的TPI和在一个游离氨基处用异硫氰酸荧光素（FITC）标记的TPI进行荧光共振能量转移（FRET）研究。在变性剂稀释后2 - 3分钟检测到水性介质中AEDANS-TPI和FITC-TPI单体之间的高效FRET，并持续数小时保持恒定。根据FRET结果计算的标记、复性的杂合TPI二聚体中AEDANS和FITC之间的距离为48 Å，与基于TPI晶体结构的估计值合理一致。在RM中，复性过程中酶活性的恢复与一种高本征荧光中间体的出现相关，该中间体被认为是部分折叠的单体（Fernández-Velasco等人，1995年）。然而，当AEDANS和FITC标记的单体在RM中混合时，一旦GdnHCl被稀释就发生FRET（FRET效率 = 0.36），早于TPI本征荧光和再活化的变化。此后，FRET效率在接下来的一小时内增加到约0.50，并在24小时后保持在该水平，此时80%的酶活性得以恢复。在RM中最初观察到的高FRET可能表明在变性剂稀释后的最初几分钟内形成了无活性的二聚体。在接下来的一小时内观察到的FRET进一步增加可能反映了蛋白质的构象重排以及从无活性二聚体到活性二聚体的转变。
