Use of fluorescently labeled proteins in quantitative sedimentation velocity studies of heterogeneous biomolecular interactions.

摘要

The recent development of a fluorescence optical system for the Beckman XL-I analytical ultracentrifuge by Laue and co-workers (Kroe and Laue, Anal. Biochem. 2009, in press) enables the detection of labeled species at picomolar concentrations and against a background of nonlabeled cosolutes. This thesis describes methods for the quantitative global analysis of fluorescence and absorbance sedimentation velocity data from labeled proteins.;To develop these methods, the reaction of fluorescently-labeled tubulin and stathmin N-terminally labeled with green fluorescence protein (eGFP) was used as a model system. Stathmin binds two heterodimers of tubulin in a kinetically sensitive two-step cooperative reaction (K1 ≈ 104 M-1, K2 ≈ 108 M-1) whose strength is such that meaningful analysis is possible with absorbance optics, but analysis over the reaction's complete dissociation curve requires fluorescence optics.;Stathmin has been observed to promote microtubule dissociation in vitro through mass-action sequestration of tubulin. In vivo, it sits at a confluence of regulatory pathways that modulate its activity by phosphorylation, an event that is required for the G2/M transition. Combined with the observed upregulation of stathmin in many neoplasias, it is evident that the stathmin-tubulin reaction plays an important role in the regulation of the cell cycle.;The direct boundary fitter Sedanal (Stafford and Sherwood, Biophys. Chem. 108, 231-43) was used to fit multiwavelength sedimentation velocity data from mixtures of stathmin-eGFP and tubulin at different conditions of nucleotide (GTP or GDP) and pH (6.9 or 7.5) to global values of K 1 and K2. The unique absorbance spectrum of stathmin-eGFP permits selective monitoring of its sedimentation. This also allowed direct observation of the interaction of stathmin-eGFP with vinca-induced tubulin spirals.;Fitting fluorescence data from a serial dilution of stathmin-eGFP and fluorescently labeled tubulin gave robust estimates of binding constants and unexpectedly demonstrated the importance of intermediate species in kinetically controlled reactions.;The methods described here can, in principle, be applied to any tightly associating heterocomplex and demonstrate the utility of labeled components and fluorescence optics in the investigation of macromolecular association reactions.