Biophysical Studies of the Type 1 Repeats of Human Thrombospondin-1 to Characterize the Structural Basis of its Angiostatic Effect

Thrombospondin-1 (TSP1) is a disulfide bonded trimer of 450 kD); each monomer contains three type 1 repeats (T1Rs). TSP1 has several documented functions including its role as an angiogenic inhibitor. TSP1 and TSP1 fragments that include the T1Rs cause an endothelial cell-specific inhibition of growth and migration. The T1Rs were expressed using the baculovirus protein expression system. Recombinant baculoviruses were generated that express the three T1Rs in tandem (P123) and the third T1R (P3) as secreted histidine-tagged fusion proteins. N-terminal sequencing of P123 and P3 confirmed that the signal sequence was homogeneously removed. Protein purity, molecular mass determination, and disulfide-bond content was determined using mass spectroscopy. Circular Dichroism (CD) was used to access secondary and tertiary structure. The far-UV CD spectra for P3 and P123 are marked by distinctive positive ellipticity that is lost by thermal denaturation. The near-UV CD spectra are positive and upon heating approach zero ellipticity. Fluorescence spectroscopy revealed that the tryptophans were quenched and the maximum emission wavelength was red-shifted upon treatment with either DTT or guanidine hydrochloride. These results indicate that the T1R encodes an independently folding protein module with spectral properties dominated by the conserved tryptophans.