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 mit ogenic protein containing 50 amino acids and 3 disulfide bonds. Homo- and heteronuclear NMR spectra were used to determine nearly complete s equence-specific H-1 and N-15 resonance assignments for hTGFalpha unde r three conditions: pH 6.5 and a temperature of 10-degrees-C, pH 6.5 a nd a temperature of 30-degrees-C, and pH 3.5 and a temperature of 30-d egrees-C. The N-15-enriched samples of hTGFalpha allowed determination of many 3J(H(N)-H(alpha)) vicinal coupling constants. Solution struct ures of human type-alpha transforming growth factor (hTGFalpha) at pH 6.5 and a temperature of 10-degrees-C were determined from NMR data us ing molecular structure generation calculations and restrained energy minimization. These structures are based on 425 conformational constra ints, including 357 NOE-derived upper-bound distance constraints, cons traints on the ranges of 26 dihedral angles based on measurements of v icinal coupling constants, 42 upper- and lower-bound constraints assoc iated with 6 hydrogen bonds and 3 disulfide bonds, and several stereos pecific 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 ne utral and acidic pH. At pH 3.5 the protein structure is partially diso rdered, with most of the hydrogen-bonded backbone structure still inta ct. The hTGFalpha structure is also compared with that of murine epide rmal growth factor. Coordinates for the set of hTGFalpha structures de scribed in this paper have been deposited in the Protein Data Bank.