Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin: All six modules are independently folded

• Published in: Journal of molecular biology Vol. 217; no. 3; pp. 563 - 575
• Main Authors: Litvinovich, Sergei V., Strickland, Dudley K., Medved', Leonid V., Ingham, Kenneth C.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 05.02.1991; Elsevier
• Subjects: Amino Acid Sequence; Binding Sites; Biological and medical sciences; Calorimetry, Differential Scanning; Fibronectins - metabolism; Fibronectins - ultrastructure; Fundamental and applied biological sciences. Psychology; Gelatin - metabolism; Guanidines - chemistry; Hot Temperature; Humans; Hydrogen-Ion Concentration; Molecular biophysics; Molecular Sequence Data; Peptide Fragments - chemistry; Physico-chemical properties of biomolecules; Protein Conformation; Protein Denaturation; Spectrometry, Fluorescence; Thermodynamics; Human; Differential scanning calorimetry; Gel electrophoresis; Enthalpy; Thermal denaturation; Environmental factor; Glycoproteins; Binding site; Fibronectin; Domain structure; Gelatin; Electrophoretic mobility; Thermodynamic parameter; PH; Intramolecular interaction
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(91)90758-X

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I “finger” modules and two type II “kringle-like” modules arranged in the order I 6-II 1-II 2-I 7-I 8-I 9. This fragment exhibits a single reversible melting transition near 64 °C in TBS buffer (0.02 m-Tris buffer containing 0.15 m-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH 2 terminus (I 6-II 1-II 2-I 7) melts reversibly near 65 °C with ΔH cal ΔH vH = 1.3 , also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I 6-II 1) 16 kDa (II 2-I 7) and 21 kDa (I 8-I 9) according to their apparent M r in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with ΔH cal ΔH vH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 °C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 m-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I 6, I 7 and I 9), one of which (I 6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II 2 and I 7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I 6, I 7 and I 9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.