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I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering
Author(s) -
Tsai Amos M.,
van Zanten John H.,
Betenbaugh Michael J.
Publication year - 1998
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19980805)59:3<273::aid-bit2>3.0.co;2-8
Subject(s) - circular dichroism , rnase p , chemistry , denaturation (fissile materials) , protein aggregation , nuclear magnetic resonance spectroscopy , ribonuclease , crystallography , spectroscopy , protein folding , protein structure , dynamic light scattering , protonation , analytical chemistry (journal) , nuclear magnetic resonance , materials science , stereochemistry , chromatography , organic chemistry , biochemistry , nuclear chemistry , ion , rna , physics , quantum mechanics , nanoparticle , gene , nanotechnology
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 heat‐denatured 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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:273–280, 1998.