Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates

Recent advances in attempts to unravel the protein folding mechanism have i ndicated the need to identify the folding intermediates. Despite their tran sient nature, in a number of cases it has been possible to detect and chara cterize some of the equilibrium intermediates, for example, the molten glob ule (MG) state. The key features of the MG state are retention of substanti al secondary structure of the native slate, considerable loss of tertiary s tructure leading to increased hydrophobic exposure, and a compact structure . NMR, circular dichroism, and fluorescence spectroscopies have been most u seful in characterizing such intermediates. We report here a new method for structural characterization of the MG slate that involves probing the expo sed hydrophobic sites with a hydrophobic photoactivable reagent-2[H-3]diazo fluorene. This carbene-based reagent binds to hydrophobic sites, and on pho tolysis covalently attaches itself to the neighboring amino acid side chain s. The reagent photolabels cu-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and p roteolytic fragmentation of the photolabeled protein followed by peptide se quencing permitted identification of the labeled residues. The results obta ined indicate that the sequence corresponding to B (23-34) and C (86-98) he lix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Va142 being the only residue that is significant ly labeled. Our data, like NMR data, indicate that in the MG state of alpha -lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain, However, unlike the NMR method, the photolabeling me thod is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in fol ding intermediates as the reagent binds and on photolysis covalently links to these regions.