Methods for Analyzing Biological Macromolecular Complexes and use Thereof

摘要

In a first aspect, the present invention relates to a method of determining assembly, homogeneity and/or thermodynamic stability of biological macromolecular complexes. In particular, the method allows determining maximum stability of said complexes based on fluorescence changes at an appropriate wavelength as a function of temperature whereby said fluorescence changes reflect the status of assembly, homogeneity and/or thermodynamic stability of the biological macromolecular complexes. Said method is based on identifying two state and multistate unfolding fluorescence curves as a function of temperature by fitting said curves according to a multistate unfolding model. In addition, a computer program and a computer program storage medium as well as an apparatus and a system is provided for determining the assembly, homogeneity and/or thermodynamic stability of biological macromolecular complexes.

主权项

1. A method of determining the assembly, homogeneity, and/or thermodynamic stability of biological macromolecular complexes comprising the steps of
providing complexes of one or more biological macromolecules in a sample containing a buffer system and a fluorescence dye; stepwise increase of the temperature of said sample at a rate suitable for measuring fluorescence of said sample after thermal equilibration, thereby determining the fluorescence at an appropriate wavelength as a function of temperature whereby said fluorescence reflects the status of assembly, homogeneity and/or thermodynamic stability of said complex; calculating the assembly, homogeneity, and/or thermodynamic stability of said complex based on:
a) identifying apparent two state unfolding fluorescence curves of the sample as a function of temperature by fitting said curves according to a multistate unfolding model from two state to six state model, e.g. by determining the unfolding state based on the Levenberg-Marquardt Algorithm for obtaining transitional curves and X2 minimization and comparing said curves to perfect two-state curves;b) calculating the slope of said transitional curves at the inflection point in a two-state model;c) calculating the melting temperature Tm, and/or the enthalpy of unfolding ΔH.