Oxidative folding of leech-derived tryptase inhibitor via native disulfide-bonded intermediates.
Leech-derived tryptase inhibitor (LDTI), comprising 46 residues and a fold stabilized by three disulfide bonds, is the only protein known to inhibit human beta-tryptase with high affinity. The present work examines its oxidative folding and reductive unfolding with chromatographic and disulfide analysis of the trapped intermediates. LDTI folds and unfolds through a sequential oxidation of its cysteine residues that give rise to the accumulation of a few one- and two-disulfide intermediates. Three species containing two native disulfide bonds (IIa, IIb, and IIc) are detected in LDTI folding, but only one (IIb) seems to be productive and oxidizes into the native structure. Stop/go experiments indicate that the intermediates IIa and IIc must reduce or rearrange their disulfide bonds to reach the productive route. The acquisition of the native structure is extremely fast and efficient, probably influenced by the low levels of non-native three-disulfide (scrambled) isomers occurring along the reaction. Finally, the Cys14-Cys40 disulfide bond, buried in native LDTI and formed in IIa and IIb intermediates, appears to be a key factor for both the initiation of folding and the stability of this molecule. Together, the derived data provide a molecular basis for development of new LDTI variants with altered properties.
Arolas
Joan L
JL
Institut de Biotecnologia i Biomedicina, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain.
Bronsoms
Sílvia
S
Aviles
Francesc X
FX
Ventura
Salvador
S
Sommerhoff
Christian P
CP
D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D018384:Oxidative Stress; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D004220:Disulfides; D011506:Proteins; D011994:Recombinant Proteins; C090901:leech-derived tryptase inhibitor C