Modulation of cutinase stability and structure by phospholipid detergents.
Fusarium solani pisi cutinase hydrolyses triglycerides of different lengths. Here we show that micelle-forming short-chain (C6-C9) phospholipids significantly reduce cutinase stability (both below and above the critical micelle concentration cmc) and rates of folding (only above cmc), trapping cutinase in an inactive state which only regains activity over hours to days, rather than the few seconds required for refolding in the absence of detergent. Destabilization decreases with increasing chain length, and increases with cmc, indicating that monomers and micelles cooperate in destabilizing cutinase. Detergents have little effect on enzymatic activity and confer no changes in secondary structure. Some changes in chemical shift occur around the enzyme active site, although distant regions are also affected. To our knowledge, this is the first example of marked destabilization of a water-soluble protein by zwitterionic detergents, highlighting the multitude of different detergent interactions with enzymes that target amphiphilic substrates and providing means of trapping a protein in a metastable state. We propose a model for destabilization where monomers via various binding sites on the native state prime it for interacting with micelles in a destabilizing fashion, whereas only micelles halt refolding due to the absence of these monomer-binding sites in the denatured state.
Sehgal
Pankaj
P
Center for Insoluble Protein Structures (inSPIN), Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Bang Nielsen
Søren
S
Pedersen
Shona
S
Wimmer
Reinhard
R
Otzen
Daniel E
DE
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D005670:Fusarium / Q000201:enzymology; D008954:Models, Biological; D008958:Models, Molecular; D010743:Phospholipids / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D013696:Temperature
D003902:Detergents; D010743:Phospholipids; D002265:Carboxylic Ester Hydrolases; C019952:cutinase