Low‐field NMR: A tool for studying protein aggregation

Abstract

Low-field nuclear magnetic resonance (NMR) spin-spin relaxation (T 2 ) measurements were used to study the denaturation and aggregation of β-lactoglobulin (β-LG) solutions of varying concentrations (1-80 g L −1 ) as they were heated at temperatures ranging from ambient up to 90• C. For concentrations of 1-10 g L −1 , the T 2 of β-LG solutions did not change, even after heating to 90• C. A decrease in T 2 was only observed when solutions having higher concentrations (20-80 g L −1 ) were heated. Circular dichroism (CD) spectroscopy and fluorescence tests using the dye 1-anilino-8-naphthalene sulfonate (ANS) on 0.2 and 1 g L −1 solutions, respectively, indicated there were changes in the protein's secondary and tertiary conformations when the β-LG solutions reached 70• C and above. In addition, dynamic light scattering (DLS) showed that protein aggregation occurred only at concentrations above 10 g L −1 and for heating at 70 • C and above. The hydrodynamic radius increased as T 2 decreased. When excess 2-mercaptoethanol was added, the changes in both T 2 and the hydrodynamic radius followed the same trend for all β-LG protein concentrations between 1 and 40 g L −1 . These observations led to the conclusion that the changes in T 2 were due to protein aggregation, not protein unfolding.

Spectroscopic studies of the interactions between β-lactoglobulin and bovine submaxillary mucin

“…Moreover, according to the DLS results, the interaction between the two proteins caused a more compact molecular configuration, which might also affect the protein and bulk water exchange rates due to changes in the availability of charged groups on the protein surface. This is in agreement with the observations of Indrawati et al (2007), which stated that the transverse relaxation time was inversely correlated to the hydrodynamic radius of the BLG solutions studied.…”

“…The obtained relaxation times for BLG were in the range from 1918 to 2143 ms, which is similar to the values obtained by Indrawati, Stroshine, and Narsimhan (2007) and Kumosinski and Pessen (1982) for similar concentrations of BLG. A significantly shorter T 2 relaxation time was obtained for the BSM, indicating a stronger protein-water interaction and more "restricted" water structure in the BSM solutions compared to the BLG solutions.…”

Determination of the Gelation Mechanism of Freeze–Thawed Hen Egg Yolk

“…Though the structure of β-LG and yolk LDL are different, the aggregation is similar so the effect on T2 is comparable between the two food components. With high concentrations of β-LG as well as application of heat and prolonged heating, β-LG form aggregates possessing a fractal pattern, and these fractal aggregates form a gel (Aymard, Gimel, Nicolai, & Durand, 1996;Indrawati, Stroshine, & Narsimhan, 2007). Based on the hypothesis regarding the gelation mechanism of yolk, gelation induced by freeze-thawing is a result of the formation of lipoprotein aggregates.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…It should be noted that a variety of other approaches exist for the identification of protein aggregation, including SEC coupled to multi-angle light scattering (MALS) [341] and NMR [342]. However, for SEC-MALS, detailed characterization of subunit composition may be not achieved.…”