Domain structure and domain-domain interactions in the carboxy-terminal heparin binding region of fibronectin.
The domain structures and stabilities of fragments isolated from the so-called 'hep 2' region of plasma fibronectin have been investigated by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The 30 kDa hep-2A fragment contains three type III modules (III12 to III14), whereas the 40 kDa hep-2B fragment contains four such modules (III12 to III15). Melting of these fragments at neutral pH was irreversible and accompanied by rapid aggregation. In contrast, melting was completely reversible in 50 mM-glycine at pH 2.7, where DSC measurements revealed the presence of three independently folded domains in 30kDa hep-2A and four in 40 kDa hep-2B. That each domain represented a single module was confirmed by measurements with four single-module subfragments, all of which melted reversibly, even at neutral pH. At neutral pH in the presence of 6 M-urea, 30 kDa hep-2A melted reversibly in a sharp peak from which only two transitions could be resolved by deconvolution. Only the larger of these was stabilized by heparin and was assigned to modules III13 and III14. Upon isolation, module III13 melted at lower temperature than in the parent fragment where it is stabilized through an interaction with module III14. We conclude that all type III modules in the hep-2 region of fibronectin constitute independently folded domains. Modules III13 and III14 form a highly co-operative structure through functionally significant interactions that can be disrupted with acid or sufficient concentrations of urea or guanidinium chloride.
Novokhatny
V
V
Holland Laboratory, American Red Cross, Rockville, MD 20855.
Schwarz
F
F
Atha
D
D
Ingham
K
K
D000818:Animals; D002152:Calorimetry, Differential Scanning; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D013552:Swine; D013696:Temperature
D005353:Fibronectins; D006493:Heparin