Solution conformation of thymosin .beta.4: a nuclear magnetic resonance and simulated annealing study

Abstract

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)

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

“…This is difficult to understand because DNase I has been reported to bind to subdomains 2 and 4 of the actin monomer [19], distant from the carboxy-terminus. Possible explanations for this inconsistency are that either the binding sites of DNase I and T/I9 overlap, despite the large distance, which would be in line with the stretched conformation of the peptide suggested on the basis of NMR studies [20]. Alternatively binding of T/I9 may induce a conformational change in the actin monomer that excludes simultaneous binding of DNase I.…”

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

“…Sequence alignment demonstrated regions of high identity and conservation within the N-and C-terminal regions between the different b-thymosins. Threedimensional structure determination by NMR-techniques demonstrated that in aqueous solutions thymosin b4 (Tb4) is unstructured; however, a defined 3D-structure was obtained when fluorinated alcohols were added to the aqueous solution [Zarbock et al, 1990]. The data obtained under these conditions indicated that Tb4 is a rather extended molecule of almost 5 nm length.…”

Assembly of polypeptide and protein backbone conformations from low energy ensembles of short fragments: Development of strategies and construction of models for myoglobin, lysozyme, and thymosin β4

“…Comparison of the calculated thymosin B4 model with structures derived from NMR measurements Zarbock et al (1990) have investigated the conformation of thymosin p4 in alcoholic solution. 4 dihedral angles identified on the basis of short-range nuclear Overhauser effects (NOEs).…”

“…With respect to the short-range energies, the calculated model oc- discussed in the main text. B4TC02, B4TC04, B4TC15, B4TC22, and a Conformation "construct" was calculated from the mean field as B4TC28 were derived from distance constraints (Zarbock et al, 1990); the remaining codes are identical to the codes used by the Brookhaven Protein Data Bank (Bernstein et al, 1977). The numbers refer to the first residue in the respective fragment (i.e., 1PP2-R-80 corresponds to residues 80-122 of lPP2-R).…”