SOLUTION STRUCTURE OF HUMAN TYPE-ALPHA TRANSFORMING GROWTH-FACTOR DETERMINED BY HETERONUCLEAR NMR-SPECTROSCOPY AND REFINED BY ENERGY MINIMIZATION WITH RESTRAINTS

Human type-alpha transforming growth factor (hTGFalpha) is a small mitogenic protein containing 50 amino acids and 3 disulfide bonds. Homo- and heteronuclear NMR spectra were used to determine nearly complete sequence-specific H-1 and N-15 resonance assignments for hTGFalpha under three conditions: pH 6.5 and a temperature of 10-degrees-C, pH 6.5 and a temperature of 30-degrees-C, and pH 3.5 and a temperature of 30-degrees-C. The N-15-enriched samples of hTGFalpha allowed determination of many 3J(H(N)-H(alpha)) vicinal coupling constants. Solution structures of human type-alpha transforming growth factor (hTGFalpha) at pH 6.5 and a temperature of 10-degrees-C were determined from NMR data using molecular structure generation calculations and restrained energy minimization. These structures are based on 425 conformational constraints, including 357 NOE-derived upper-bound distance constraints, constraints on the ranges of 26 dihedral angles based on measurements of vicinal coupling constants, 42 upper- and lower-bound constraints associated with 6 hydrogen bonds and 3 disulfide bonds, and several stereospecific H-1 resonance assignments. The overall structure is similar to that described recently for hTGFalpha by other groups [Kline et al. (1990) Biochemistry 29, 7805-7813; Harvey et al. (1991) Eur. J. Biochem. 198, 555-562], but there are differences in some structural details. The resonance frequencies, vicinal coupling constants, and NOEs form the basis for comparisons of the solution structure of hTGFalpha at neutral and acidic pH. At pH 3.5 the protein structure is partially disordered, with most of the hydrogen-bonded backbone structure still intact. The hTGFalpha structure is also compared with that of murine epidermal growth factor. Coordinates for the set of hTGFalpha structures described in this paper have been deposited in the Protein Data Bank.