Conformation and Self-association of Human Recombinant Transforming Growth Factor-β3 in Aqueous Solutions

Abstract

The transforming growth factors-␤ (TGF-␤) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-␤ isoforms TGF-␤3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-␤3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-␤3. In addition, the aggregation of TGF-␤3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-␤3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH ϳ3.9, a monomer i dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8.5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-␤3 dissolved in solution and aggregated TGF-␤3. Aggregated TGF-␤3 showed a higher ␤-sheet content and lower ␤-turn and random coil contributions compared with monomeric TGF-␤3. Both the solution structure and the aggregate structure of TGF-␤3 were different from the crystal structure. This was in contrast to TGF-␤2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-␤3 conformation in solution in the range of 2.3 < pH < 11.0 was reversible. Aggregation of TGF-␤3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, c NaCl ‫؍‬ 164 mM) TGF-␤3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.

A cytokine-neutralizing antibody as a structural mimetic of 2 receptor interactions

“…The observed displacement of these 2 ␤-strands is even more pronounced when compared with the unbound TGF-␤3 (5 Å) and is mainly caused by adaptation to the CDR loops of GC-1008. Furthermore, the observed change in the orientation of ␣-helix 3 of TGF-␤3 was not surprising because this helix, together with its connecting loop segment, is supposed to be a rather flexible region (35,36). It allows a conformational adaptation of the TGF-␤3 homodimer to its corresponding binding partners as observed in the TGF-␤3-T␤RII complex (37), in which this region was even disordered and accompanied by a massive rearrangement of the 2 monomers.…”

Ion‐specific modulation of protein interactions: Anion‐induced, reversible oligomerization of a fusion protein

“…10,24 There also have been concerns about oligomeric forms of a drug presenting an increased immunogenic risk. 25,26 For these reasons, reversible self-association interactions have been studied in almost all classes of protein therapeutics, including peptides, 27 hormones, [28][29][30] growth factors, 31 cytokines, 32 antibodies, 33,34 and recently even in single-chain Fv molecules. 35 This report is focused on characterizing the saltdependent reversible self-association pathway of a fusion protein called peptibody A (PbA).…”

Dynamics-modulated Biological Activity of Transforming Growth Factor β3

“…This is in agreement with our previous NMR study (13), which showed that the secondary structure and the global fold of the TGF-␤3 dimer in solution are close to those obtained for the protein crystal, but some regions are in dynamic equilibrium with an unfolded conformation. Moreover the CD studies in aqueous solution show that the ␣-helical content of TGF-␤3 in solution is less then expected from the known crystal and solution structures of TGF-␤s, including free TGF-␤3 in crystal (12). In contrast, a good agreement between the experimental CD spectrum measured in aqueous solution at pH 3.0 and CD spectrum calculated from crystal structure was found for TGF-␤2 (12).…”

“…X-ray crystallographic (9,10) and NMR (11) analyses have revealed identical folds of the TGF-␤ isoforms. However, recent studies have shown important structural differences of TGF-␤3 in solution with respect to its crystal structure and the structures of other TGF-␤s (12,13). In view of the therapeutic potential of TGF-␤, we present here a dynamic study of TGF-␤3 aimed at explaining its behavior in aqueous solution and characterizing determinants involved in the interaction with receptors.…”

“…NMR measurements were performed with a sample of 1.4 mM 15 N-labeled TGF-␤3 in mixed solvent of 87% H 2 O, 5% D 2 O, 6% dioxane-d 8 and 2% methanol-d 3 at pH 2.9 and 40°C. It was shown that these conditions do not affect the TGF-␤3 structure in solution, but provide optimal resolution in 1 H-15 N correlation spectra of TGF-␤3 (see Supplementary Material) and fully remove aggregation of TGF-␤3, which prevent NMR relaxation analysis (12,13). The backbone 15 N longitudinal R 1 and transverse R 2 relaxation rates and 15 …”