Post on 16-Jul-2015
Chemical modifications:
An application to interleukin-1β, a structural and functional approach.
Reactive amino groups in IL-1β (purple regions) by NHS-biotinylation studies
Lysines 93,94
Lysine 103*
Alanine 1
Journal of Biological chemistry (1989) 264:17691-17697
Reactive amino acid side chains that react with Acrylodan
(A cystiene specific and environment sensitive reagent)
Journal of Biological Chemistry (1992) 267:3122-3128
All chemical modifications introduced do not have any effects on IL-1β
bioactivity.
(Thymocytes proliferation assays & receptor binding measurements)
Isolation of EDC (water soluble carbodiimide) modified rIL-1β using TSKSP5PW
Modified protein retains native pIModified protein retains native pI
EKC peptides from rIL-1β
Native proteolysis of IL-1β using Endo-Lys-C (Native IL-1β Vs X-
linked IL-1β)
1-88, NYPK and 93-150
Properties of EDC modified interleukin-1beta
• Sensitivity toward Endo-Lys-C digestion (under native condition) is lost.
• Two large peptide fragments and a small peptide is generated instead.
• Chemical modification by EDC affords protection against Endo-Lys-C degradation
• What is this modification and where is it located within the molecule?
EKC treatment post denaturation
Mass Spec (FAB) application
• Definition of sites of cross-linkage
Definition of site of carbodiimide reaction by Mass Spec. (FAB) after V8 (Endo-Glu-C) treatment
Location of salt bridge where carbodiimide crosslinkage was induced
Sole release of NYPK from crosslinked rIL-1β when treated with Endo Lys C under native condition
Bioactivity measurement of various forms of crosslinked rIL-1β
Crosslinked IL-1β, without the NYPK region is NOT active.
Biophysical characteristic of X-linked IL-1β
• Circular Dichroism
• Fluorescence Spectroscopy
• Tyrosine hydroxyl titration study
Crosslinked and EKC treated IL-1β is almost identical to native untreated IL-1β
Subtle conformational change detected between native and crosslinked IL-1β by fluorescence spec.
TryptophanTryptophan
Ex 295Ex 295
Tryptophan & Tryptophan & TyrosineTyrosine
Ex 275Ex 275
CrosslinkedCrosslinked
NativeNative
Hydroxyl titration studies of tyrosine residues in native and crosslinked IL-1β
NativeNative
CrosslinkedCrosslinked
Acknowledgements• Mass Spec (FAB) - David Guido
• Bioassays - Karen Richards, Nigel Staite
• Fluorescence Spec – Dennis Epps
• CD measurements – Mark Prairie, William Krueger
• Crystallography – Barry Finzel
• Guidance & Mentoring – Martin Deibel