I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering
✍ Scribed by Amos M. Tsai; John H. van Zanten; Michael J. Betenbaugh
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 220 KB
- Volume
- 59
- Category
- Article
- ISSN
- 0006-3592
No coin nor oath required. For personal study only.
✦ Synopsis
The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, T m , of approximately 65°C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around T m using near-and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55°C and below, while denatured structures appeared at 65°C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65°C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75°C and pH 7.8 were indeed derived from heatdenatured protein. However, aggregation was also detected at 55°C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH.