Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2‐trichloroacetic acid (TCA). In this study, we examine the mechanism of the TCA‐induced protein precipitation reaction. TCA‐induced protein precipitation curves are U‐shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. TCA is significantly less effective in precipitating unfolded states of proteins. Results of the 1‐anilino‐8‐napthalene sulfonate (ANS) and size‐exclusion chromatography, obtained using acidic fibroblast growth factor (aFGF), show that a stable “molten globule‐like” partially structured intermediate accumulates maximally in 5% (w/v) of trichloroacetate. Urea‐induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. ¹H‐¹⁵N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the β‐strands at the N‐ and C‐ terminal ends (of aFGF) are disrupted in the trichloroacetate‐induced “MG‐like” state. The results of the study clearly demonstrate that TCA‐induced protein precipitation occurs due to the reversible association of the “MG‐like” partially structured intermediate state(s). In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome.

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, κ-hefutoxin1 and κ-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that κ-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any β−sheets. Based on the presence of the functional diad (Tyr⁵/Lys¹⁹) at a distance (6.0 ± 1.0 Å) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. κ-Hefutoxin 1 not only blocks the voltage-gated K⁺-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of κ-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

Fibroblast growth factors (FGFs) are cell-signaling proteins with diverse functions in cell development, repair, and metabolism. The human FGF family consists of 22 structurally related members, which can be classified into three separate groups based on their action of mechanisms, namely: intracrine, paracrine/autocrine, and endocrine FGF subfamilies. FGF19, FGF21, and FGF23 belong to the hormone-like/endocrine FGF subfamily. These endocrine FGFs are mainly associated with the regulation of cell metabolic activities such as homeostasis of lipids, glucose, energy, bile acids, and minerals (phosphate/active vitamin D). Endocrine FGFs function through a unique protein family called klotho. Two members of this family, α-klotho, or β-klotho, act as main cofactors which can scaffold to tether FGF19/21/23 to their receptor(s) (FGFRs) to form an active complex. There are ongoing studies pertaining to the structure and mechanism of these individual ternary complexes. These studies aim to provide potential insights into the physiological and pathophysiological roles and therapeutic strategies for metabolic diseases. Herein, we provide a comprehensive review of the history, structure–function relationship(s), downstream signaling, physiological roles, and future perspectives on endocrine FGFs.