Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts

Jens Madsen, Thomas Rebsdorf Sørensen, Jørn Døvling Kaspersen, Maria Berggård Silow, Jesper Vind, Jan Skov Pedersen, Allan Svendsen, Daniel E Otzen

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Abstract

We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein-protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and <5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small Angle X-ray Scattering. These oligomers account for ~25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.

OriginalsprogEngelsk
TidsskriftBBA Proteins and Proteomics
Vol/bind154
Nummer12
Sider (fra-til)1914-1921
Antal sider8
ISSN1570-9639
DOI
StatusUdgivet - dec. 2015

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