Molecular Motions and Interactions in Aqueous Solutions of Thymosin‐β₄, Stabilin CTD and Their 1 : 1 Complex, Studied by ¹H‐NMR Spectroscopy

Abstract

Wide‐line 1H NMR measurements were extended and all results were interpreted in a thermodynamics‐based new approach on aqueous solutions of thymosin‐β4 (Tβ4), stabilin cytoplasmic domain (CTD), and their 1 : 1 complex. Energy distributions of potential barriers controlling the motion of protein‐bound water molecules were determined. Heterogeneous and homogeneous regions were found in the protein‐water interface. The measure of heterogeneity of this interface gives quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to ∼20 % of the individual whole proteins. About 40 % of the binding sites of free Tβ4 get involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein‐water interactions. The complex is more disordered than Tβ4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Protein–Protein Connections—Oligomer, Amyloid and Protein Complex—By Wide Line 1H NMR

“…The studied proteins were prepared, and their qualities were checked as in [ 2 , 3 , 4 , 5 ]. Briefly, untagged versions of α-synuclein variants were expressed in E. coli cells and purified using anion exchange chromatography, while stabilin CTD was expressed with an N-terminal His-tag and purified on a Ni-NTA affinity column.…”

“…Melting diagrams ( MD s) are the relative amounts of the proteins’ mobile water measured by intensities of wide-line 1 H NMR signals vs. normalized functional temperature. The mobile hydration differences were calculated from MD s of WT and A53T αS monomers, oligomers and amyloids in [ 3 ], and from MD s of Tβ 4 , stabilin-2 CTD and their 1:1 complex in [ 4 , 5 ]. In this work, potential barriers corresponding to normalized functional temperature are used.…”

“…The selection of the studied proteins represents a system in which the constituents are different in their degree of polymerization and another system where the constituents interact with each other. This work presents as novelty compared to the previous works [2][3][4][5] the differences between α-Ss of different degrees of polymeriza-tion and sheds light on the bonds between the constituting proteins in the Tβ 4 -stabilin CTD complex.…”

“…Detailed structural and preparation information can be found in [ 2 , 3 ]. The other system consists of two proteins—thymosin-β 4 (Tβ 4 ) and cytoplasmic domain of stabilin-2 (stabilin CTD)—which form a 1:1 complex (see [ 4 , 5 ] for structural information and for details of preparation).…”

“…It is capable of mediating its distinct yet specific interactions without adapting distinct folded structures in the different complexes [ 42 ]. Weak binding was confirmed between Tβ 4 and stabilin-2 CTD and it was suggested that the proteins become slightly more disordered in the complex [ 4 , 5 ].…”