Biophysical evaluation of the oligomerization and conformational properties of the N-terminal domain of TDP-43.

TDP-43 is an RNA-binding protein that presents four domains comprising an N-terminal region, two RNA recognition motifs and a C-terminal region. The N-terminal domain (NTD) has a relevant role in the oligomerization and splicing activity of TDP-43. In this work, we have expressed, purified and biophysically characterized the region that includes residues 1 to 102 that contains the nuclear localization signal (residues 80-102, NLS). Furthermore, we have evaluated the oligomerization equilibrium for this protein fragment. Also, we have determined changes in the tertiary structure and its stability in a broad range of pH values by means of different spectroscopic methods. Additionally, we compared this fragment with the one that lacks the NLS employing experimental and computational methods. Finally, we evaluated the motion of dimeric forms to get insights into the conformational flexibility of this TDP-43 module in an oligomeric state. Our results suggest that this domain has a conformational plasticity in the vicinity of the single tryptophan of this domain (Trp68), which is enhanced by the presence of the nuclear localization signal. All these results help to understand the molecular features of the NTD of TDP-43.

Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin.

BACKGROUND: The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin. METHODS: We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. RESULTS: γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in γS-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT. CONCLUSIONS: Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site. GENERAL SIGNIFICANCE: Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma.

The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, ß and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the ß subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) µM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03µmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

Assessment and Impacts of Phosphorylation on Protein Flexibility of the α-d-Phosphohexomutases.

Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in the enzyme mechanism, the phosphorylation state of the catalytic phosphoserine has recently been found to have widespread effects on the structural flexibility of enzymes in this superfamily. These effects must be carefully accounted for when assessing other perturbations to these enzymes, such as mutations or ligand binding. In this chapter, we focus on methods for assessing and modulating the phosphorylation state of the catalytic serine, as well as straightforward ways to probe the impacts of this modification on protein structure/flexibility. This knowledge is essential for producing homogeneous and stable samples of these proteins for biophysical studies. The methods described herein should be widely applicable to enzymes across the PHM superfamily and may also be useful in characterizing the effects of posttranslational modifications on other proteins.