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Studies of the oligomerisation mechanism of a cystatin-based engineered protein scaffold

DOI: 10.1038/s41598-019-45565-6 DOI Help

Authors: Matja Zalar (University of Manchester) , Sowmya Indrakumar (Technical University of Denmark) , Colin W. Levy (University of Manchester) , Richard B. Tunnicliffe (University of Manchester) , G√ľnther H. J. Peters (Technical University of Denmark) , Alexander P. Golovanov (University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Scientific Reports , VOL 9 , PAGES 9067

State: Published (Approved)
Published: June 2019
Diamond Proposal Number(s): 17773

Open Access Open Access

Abstract: Engineered protein scaffolds are an alternative to monoclonal antibodies in research and drug design due to their small size, ease of production, versatility, and specificity for chosen targets. One key consideration when engineering such proteins is retaining the original scaffold structure and stability upon insertion of target-binding loops. SQT is a stefin A derived scaffold protein that was used as a model to study possible problems associated with solution behaviour of such aptamers. We used an SQT variant with AU1 and Myc insertion peptides (SQT-1C) to study the effect of peptide insertions on protein structure and oligomerisation. The X-ray structure of monomeric SQT-1C revealed a cystatin-like fold. Furthermore, we show that SQT-1C readily forms dimers and tetramers in solution. NMR revealed that these oligomers are symmetrical, with inserted loops comprising the interaction interface. Two possible mechanisms of oligomerisation are compared using molecular dynamics simulations, with domain swap oligomerisation being thermodynamically favoured. We show that retained secondary structure upon peptide insertion is not indicative of unaltered 3D structure and solution behaviour. Therefore, additional methods should be employed to comprehensively assess the consequences of peptide insertions in all aptamers, particularly as uncharacterized oligomerisation may alter binding epitope presentation and affect functional efficiency.

Journal Keywords: Protein engineering; Proteins; Recombinant protein therapy; Solution-state NMR; X-ray crystallography

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Added On: 25/06/2019 14:27

Documents:
s41598-019-45565-6.pdf

Discipline Tags:

Life Sciences & Biotech Structural biology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)