title: Interactions among the three structural motifs of the C-terminal region of human thrombospondin-2.
abstract: The C-terminal regions of thrombospondins (TSPs) contain three elements, EGF-like modules (E), a series of Ca(2+)-binding repeats (Ca), and a C-terminal sequence (G). We have looked for interactions among these elements in four recombinant proteins based on human TSP-2: E3CaG-2, CaG-2, E3Ca-2, and Ca-2. When bound Ca(2+) was assayed by atomic absorption spectroscopy or an equilibrium dialysis protocol in which Ca(2+) was removed from the proteins prior to equilibrium dialysis, E3CaG-2 bound 22-27 Ca(2+), CaG-2 bound 17-20 Ca(2+), and E3Ca-2 and Ca-2 bound 14-20 Ca(2+). Approximately 10 of the bound Ca(2+) in E3CaG-2 were exchangeable. The far UV circular dichroism (CD) spectrum of Ca(2+)-replete E3CaG-2 contained a strong negative band at 203 nm attributable to Ca and a less intense negative band at 218 nm attributable to Ca and G. Chelation of Ca(2+) with EDTA shifted the 203 nm band of all four proteins and the 218 nm band of E3CaG-2 and CaG-2 to less negative positions. The apparent EC50 for the far UV CD transition was 0.22 mM Ca(2+) for all proteins, indicating that Ca(2+) binding to Ca is primarily responsible for the CD change. Near UV CD and intrinsic fluorescence revealed that the tryptophan residues in G are sensitive to changes in Ca(2+). Differential scanning calorimetry of the proteins in 2 mM Ca(2+) showed that E3CaG-2 melts with two transitions, 44-51 degrees C and 75-83 degrees C. The lower transition required G, while the higher transition required Ca. Both transitions were stabilized in constructs containing E3. These results indicate that E3, Ca, and G function as a complex structural unit, and that the structures of both Ca and G are influenced by the presence or absence of Ca(2+).
authors.affiliation: Department of Medicine, University of Wisconsin-Madison, 53706, USA. tmmisenh@facstaff.wisc.edu
authors.forename: Tina M
authors.initials: TM
authors.lastname: Misenheimer
authors.forename: Blue-leaf A
authors.initials: BL
authors.lastname: Hannah
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Annis
authors.forename: Deane F
authors.initials: DF
authors.lastname: Mosher
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002942:Circular Dichroism; D016384:Consensus Sequence; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D019699:Thrombospondins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099262:Thrombospondin 2
chemical_list: D002118:Calcium; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; D019699:Thrombospondins; D000099262:Thrombospondin 2