Reversible unfolding of the gelatin-binding domain of fibronectin: structural stability in relation to function.
Fibronectin, a large multidomain glycoprotein, binds denatured collagen (gelatin) and mediates cell attachment and spreading on collagen-coated surfaces. Despite the high affinity, binding to gelatin is disrupted by relatively mild conditions. We have examined the effects of denaturants on the structure and function of a 42-kDa gelatin-binding fragment (GBF) isolated from chymotryptic and thermolytic digests of the parent protein. Application of linear gradients to GBF-loaded gelatin-agarose columns resulted in peak elution of the fragment at pH 5.2 or 10.2, at 0.4 M dimethylformamide, 0.9 M GdmCl, or 2.0 M urea, conditions far short of those required to induce structural changes detectable by fluorescence or circular dichroism. Solvent perturbation, fluorescence quenching, and chemical modification experiments indicate that about half of the 8 tryptophans, one-third of the 21 tyrosines, and all of the 9 lysine residues are solvent-exposed in the native protein and that 1 or more of the latter are directly involved in binding to gelatin, most likely through a hydrogen-bonding mechanism. Titration with GdmCl produced a single unfolding transition centered near 2.5 M GdmCl as monitored by changes in fluorescence and circular dichroism. This transition was fully reversible with complete recovery of structural parameters and gelatin binding. Treatment with disulfide reducing agents caused rapid irreversible changes in structure similar to those produced by GdmCl with concomitant loss of gelatin binding. Thus, tertiary and secondary structures are important for binding, but binding can be disrupted without perturbing those structures.
Isaacs
B S
BS
Biochemistry Laboratory, American Red Cross Biomedical Research & Development, Rockville, Maryland 20855.
Brew
S A
SA
Ingham
K C
KC
D000818:Animals; D001665:Binding Sites; D005353:Fibronectins / Q000097:blood* / Q000302:isolation & purification; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012867:Skin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D005353:Fibronectins; D010446:Peptide Fragments; D005780:Gelatin