Amorphous protein aggregation monitored using fluorescence self‐quenching

• Published in: FEBS letters 2016-10, Vol.590 (20), p.3501-3509
• Main Authors: Jozawa, Hiroki, Kabir, Md. Golam, Zako, Tamotsu, Maeda, Mizuo, Chiba, Kazuhiro, Kuroda, Yutaka
• Format: Article
• Language: English
• Subjects: Amino Acids - chemistry; Animals; Aprotinin - chemistry; Aprotinin - genetics; Cattle; Dynamic Light Scattering; Fluorescein - chemistry; Fluorescence; fluorescence self‐quenching; molecular condensation; Mutation; protein aggregation; Protein Conformation - drug effects; SCP tag; Sodium Chloride - pharmacology; Solubility; Staining and Labeling
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1002/1873-3468.12439

Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt‐induced formation of amorphous aggregation as monitored by fluorescence self‐quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native‐like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS‐Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM‐BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM‐BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein‐protein interaction yielding a ‘molecular condensation’ phase. According to this model, C2G would rapidly turn from ‘condensates’ to aggregates, whereas C5S and C5A tagged FAM‐BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state.