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The conformation of the polypeptide thymosin beta 4 in solutions of 60% (v/v) trifluoroethanol-d3 and 50% (v/v) hexafluoroisopropyl-d2 alcohol in water is investigated by nuclear magnetic resonance (NMR) spectroscopy. Under these conditions thymosin beta 4 adopts an ordered structure. By use of a combination of two-dimensional NMR techniques, the 1H NMR spectrum of thymosin beta 4 is assigned. A set of 180 approximate interproton distance constraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 33 phi constraints obtained for JNH alpha coupling data and the 23 psi dihedral angles identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Ten independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 30 calculated structures satisfy the experimental constraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 30 converged structures indicates that there are two helical regions extending from residues 4-16 and from residues 30-40, which are well defined both in terms of atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 2 A. The two helices exhibit typical amino acid preferences for specific locations at the ends of helices.(ABSTRACT TRUNCATED AT 250 WORDS)