Studies with Site-Specific Labeled HAMLET Figure 2. Tertiary structure studied with near UV CD spectra. (A) The near UV spectra of the native refolded hLA and hLA-ext proteins show positive and negative peaks indicating the CD bands arising from aromatic amino acids and suggest the presence of tertiary structure. When these proteins are treated with a chelating agent to remove Ca2+ ion, these proteins acquire a molten globule state (B). Interestingly the aD-hLA and aD- hLA-ext show similar near UV CD spectra as molten globule hLA. doi:10.1371/journal.pone.0026093.g002 Figure 3. Incorporation of a polypeptide tag to full length hLA and aD-hLA proteins. Schematic representation of the C-terminal extension of 17 amino acids engineered on hLA showing the acceptor Thr residue for the site-specific labeling and the thrombin cleavage site. (A) SDS-PAGE analyses of purified hLA, hLA-ext, with and without thrombin treatment. To confirm the presence of the C-terminal extension peptide, the protein was treated with thrombin. The mobility of hLA-ext treated with thrombin is comparable to wild-type hLA. (B) Since the molecular weight of the released C-terminal polypeptide is only 1337 Da it could not be observed on any SDS-PAGE gel. However, MALDI-TOF spectroscopic analysis of the thrombin treated samples showed a release of a correct molecular weight peptide from the proteins carrying the C-terminal extension peptide. (C) The protein samples aD-hLA and aD-hLA-ext with and without Thrombin treatment were analyzed on SDS-PAGE similar to hLA-ext shown in (A). doi:10.1371/journal.pone.0026093.g003 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26093