Structure and rotational dynamics of fluorescently labeled insulin in aqueous solution and at the amphiphile-water interface of reversed micelles.

Insulin is a proteohormone with amphipathic three-dimensional structure and the ligand of a receptor, which itself spans the plasma membrane of glucose-metabolizing cells. In this study, the possible impact of amphiphiles on structural and dynamic properties of the hormone was investigated in reversed micelles mimicking the amphipathic nature of biological membranes. To make insulin susceptible to fluorescence measurements, two derivatives labeled with 2-aminobenzoic acid (Abz), N epsilon B29-Abz-insulin and [AbzB1]insulin, were prepared. First, the Abz-labeled insulins were shown by CD spectroscopy to exhibit conformational properties and self-association as well as the T-->R transition similar to the native hormone. By means of time-resolved fluorescence measurements, not only metal-ion induced hexamerization was observable in aqueous solution: The T-->R allosteric transition of the hexamer was shown to be accompanied by a diminution of its hydrodynamic radius. Second, structure and rotational dynamics of the labeled insulins were investigated in reversed micelles. In sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles, the main-chain conformation is similar to that in aqueous solution according to CD spectroscopy in the far-UV, whereas the weak circular dichroism in the near-UV is indicative of reduced aromatic contacts as well as of the absence of quaternary structure, and the CD spectra show the same shape as found for proteins in an intermediate state of folding referred to as the "molten globule". Fluorescence anisotropy decay measurements of N epsilon B29-Abz-insulin in reversed micelles of AOT, cetyltrimethylammonium bromide, and alpha-L-1,2-dioctanoylphosphatidylcholine showed that the internal mobility of the solubilizate is reduced compared to that in aqueous solution and that the rotational mobility of the labeled insulin decreases with decreasing micellar size. With respect to the immobilization, insulin interacts in a stronger way with the anionic than with the cationic or zwitterionic amphiphile; an integration into the amphiphile monolayer, however, could be ruled out in all cases. In conclusion, the results reveal an evident influence of amphiphiles on the structure and rotational dynamics of insulin. Further investigations should be focused on this finding also with regard to the possible importance of lipid-insulin interactions in vivo.