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