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Biochim Biophys Acta. 2015 Dec;1854(12):1914-1921. doi: 10.1016/j.bbapap.2015.09.007. Epub 2015 Oct 1.

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

Author information

1
iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Zealand Pharma A/S, Smedeland 36, DK-2600 Glostrup, Denmark.
2
iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
3
iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark; Department of Chemistry, Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark.
4
Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
5
Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark. Electronic address: asv@novozymes.com.
6
iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark. Electronic address: dao@inano.au.dk.

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.

KEYWORDS:

Enzyme activity; Gel filtration; Lattice contacts; Lipase; Oligomers; Protein design; Saxs

PMID:
26431886
DOI:
10.1016/j.bbapap.2015.09.007
[Indexed for MEDLINE]

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