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Published on: August 15, 2014
Direct biophysical characterization of human apolipoprotein A-1 in ISCOMs
1AstraZeneca Research and Development Boston, 128 Sidney Street, Cambridge, Massachusetts 02139, USA. hongming.chen@arch.us.astra.com
Insights
Human apolipoprotein A-1 incorporated into Immune Stimulating Complexes (ISCOMs) shows increased alpha-helical content and structural order. Biophysical techniques confirmed these structural changes and a moderate decrease in antibody binding affinity.
Area of Science:
- Biophysics
- Protein Chemistry
- Immunology
Background:
- Immune Stimulating Complexes (ISCOMs) are potent vaccine adjuvants.
- Understanding protein structure within ISCOMs is crucial for optimizing their immunogenicity.
- Human apolipoprotein A-1 (ApoA-1) is a key component in some ISCOM formulations.
Purpose of the Study:
- To investigate the structural integrity and changes of human apolipoprotein A-1 (ApoA-1) upon incorporation into ISCOMs.
- To evaluate the impact of ISCOM formulation on ApoA-1's tertiary structure and antigenicity.
- To demonstrate the utility of biophysical methods for analyzing proteins within ISCOMs.
Main Methods:
- Fourier transform infrared (FTIR) spectroscopy (Amide I band) to assess secondary structure (alpha-helical content).
- Near UV circular dichroism (CD) and fluorescence spectroscopy to probe tertiary structure and tryptophan residue environment.
- Biomolecular interaction analysis (BIA) using surface plasmon resonance (SPR) to measure antibody binding affinity.
Main Results:
- FTIR data revealed a slight increase in alpha-helical content of ApoA-1 after ISCOM incorporation.
- Near UV CD and fluorescence spectroscopy indicated a more hydrophobic environment, restricted motion, and local electrostatic interactions for tryptophan residues.
- SPR measurements showed a moderate (20%) decrease in the binding affinity of ApoA-1 to its specific monoclonal antibody post-ISCOM formulation.
- These findings suggest increased structural order of ApoA-1 within ISCOMs.
Conclusions:
- Incorporation into ISCOMs induces conformational changes in human apolipoprotein A-1, leading to increased structural order.
- Biophysical techniques provide a non-invasive means to monitor protein structural integrity within ISCOMs without extraction.
- The observed changes in ApoA-1 structure may influence its interaction with antibodies and potentially its biological function.
Abstract:
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.
