Direct biophysical characterization of human apolipoprotein A-1 in ISCOMs.
Human apolipoprotein A-1 was formulated in "Immune Stimulating Complexes" (ISCOMs). The structure of the protein in ISCOMs was examined directly using several biophysical techniques including Fourier transform infrared (FTIR) spectroscopy, near UV circular dichroism (CD), and fluorescence spectroscopy. Amide I FTIR data indicate that human apolipoprotein A-1 displays a slightly increased alpha-helical content after its incorporation into ISCOMs. Near UV CD and tryptophan fluorescence data suggest that association with ISCOMs results in the tryptophan residues of the protein experiencing a relatively hydrophobic environment, motional restriction, and local electrostatic interactions. These observations are consistent with an increased order in the protein structure upon incorporation in ISCOMs. In addition, biomolecular interaction analysis (BIA), based on surface plasmon resonance (SPR) measurements, suggests that the binding affinity of human apolipoprotein A-1 to a monoclonal anti-human apolipoprotein A-1 antibody is moderately decreased (by 20%) after its incorporation into ISCOMs. This study demonstrates that these biophysical techniques can be used to noninvasively monitor integrity of or changes in secondary and tertiary structure of proteins within the ISCOM particles without the need for protein extraction.
Chen
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H
AstraZeneca Research and Development Boston, 128 Sidney Street, Cambridge, Massachusetts 02139, USA. hongming.chen@arch.us.astra.com
Sanyal
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G
D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000648:ultrastructure; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D006801:Humans; D016882:ISCOMs / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D020349:Surface Plasmon Resonance
D016632:Apolipoprotein A-I; D016882:ISCOMs