Unfolding diminishes fluorescence resonance energy transfer (FRET) of lysine modified β-lactoglobulin: Relevance towards anti-HIV binding.

In accordance with the recent reports by Ng et al. (2001) [12] and Pellengrini et al. (2001) [13], that acetylated and succinylated β-lg has a potent HIV-I and HIV-II type enzyme inhibitory activity, a spectro-fluoremetric approach has been made to understand the mode of interactions playing the key role in inhibition process. In this article, interactions between lysine modified bovine β-lactoglobulin (β-lg) and a hydrophobic fluorescence probe, 1-anilinonapthalene-8-sulfonate (ANS), have been studied with the help of fluorescence resonance energy transfer (FRET) process. Lysine residues of β-lg were modified by acetylation and succinylation. Tryptophan-19 of intact β-lg efficiently transfers energy to ANS, whereas in derivatives, it unexpectedly failed to promote energy transfer in spite of being more solvent exposed with an appreciable overlap integral. Efficient fluorescence resonance energy transfer (FRET) is a consequence of good overlap between emission and absorption spectra of donor and acceptor respectively. Therefore, linearity of this relationship becomes questionable in case of modified bio-molecules. Furthermore, time resolved studies showed that in the derivatives, hydrophobic cavities of β-lg were collapsed so that ANS failed to recognize the deep interior pockets leading to the loss of longer lifetime component. Modifications also prohibited the ionic association through surface leading to the loss of shorter lifetime component. Hence, chemical modification destabilizes β-lg conformations that affect FRET and interactions are strictly electrostatic.