title: Biophysical characterization of the signature domains of thrombospondin-4 and thrombospondin-2.
abstract: The signature domain of thrombospondins consists of tandem epidermal growth factor-like modules, 13 calcium-binding repeats, and a lectin-like module. Although very similar, the signature domains of thrombospondin-1 and -2 differ in several potentially important ways from the domains of thrombospondin-3, -4, and -5. We have compared matching recombinant segments representing the signature domains of thrombospondin-2 and -4. In the presence of 2 mM CaCl2, the far UV circular dichroism spectra of thrombospondin-2 and -4 constructs contain a strong negative band at 202 nm, but only the thrombospondin-2 construct has a band at 216 nm. Chelation of calcium shifted the negative bands to lower magnitudes. Titrations of the spectra demonstrated lower cooperativity and affinity for binding of calcium to thrombospondin-4 compared with thrombospondin-2. Atomic absorption spectroscopy demonstrated that the thrombospondin-4 constructs bind seven less calcium than the thrombospondin-2 construct at 0.6 mM CaCl2. In 2 mM CaCl2, the near UV circular dichroism spectra of thrombospondin-2, but not thrombospondin-4, contain a positive band at 292 nm that disappears upon calcium chelation. Intrinsic fluorescence spectra for both proteins were also sensitive to calcium, but the changes were simpler and more marked for thrombospondin-2 than for thrombospondin-4. In differential scanning calorimetry, the thrombospondin-2 construct melted in two distinct transitions at 53.5 and 81.8 degrees C, whereas the first transition for thrombospondin-4 constructs was observed at 63.5 degrees C. Thus, the studies revealed significant differences between the signature domains of thrombospondin-2 and thrombospondin-4 in calcium binding, fine structure, and inter-modular interactions.
authors.affiliation: Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. tmmisenh@wisc.edu
authors.forename: Tina M
authors.initials: TM
authors.lastname: Misenheimer
authors.forename: Deane F
authors.initials: DF
authors.lastname: Mosher
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002152:Calorimetry, Differential Scanning; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D007313:Insecta; D007477:Ions; D037102:Lectins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013054:Spectrophotometry, Atomic; D013696:Temperature; D019699:Thrombospondins / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays; D000099262:Thrombospondin 2
chemical_list: D002118:Calcium; D002122:Calcium Chloride; D002614:Chelating Agents; D004815:Epidermal Growth Factor; D007477:Ions; D037102:Lectins; D011994:Recombinant Proteins; D019699:Thrombospondins; D014364:Tryptophan; D000099262:Thrombospondin 2; C101864:thrombospondin 4