The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V_H22 and V_H92 in the variable heavy chain (V_H) and V_L23, V_L87 and V_L88 in the variable light chain (V_L). The influence of two unusual contiguous Cys at positions V_L87 and V_L88 was studied by considering the wild type fragment and mutant variants: V_L-C88S, V_L-C87S, V_L-C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V_L-C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V_L-C87 by a usual residue at this position like Tyr caused distant structural changes at the V_H region that confers a higher mobility to the V_H-CDR2 and V_H-CDR3 loops improving the scFv binding to the antigen.

RAMOSA1 (RA1) is a Cys2-His2-type (C2H2) zinc finger transcription factor that controls plant meristem fate and identity and has played an important role in maize domestication. Despite its importance, the origin of RA1 is unknown, and the evolution in plants is only partially understood. In this paper, we present a well-resolved phylogeny based on 73 amino acid sequences from 48 embryophyte species. The recovered tree topology indicates that, during grass evolution, RA1 arose from two consecutive SUPERMAN duplications, resulting in three distinct grass sequence lineages: RA1-like A, RA1-like B, and RA1; however, most of these copies have unknown functions. Our findings indicate that RA1 and RA1-like play roles in the nucleus despite lacking a traditional nuclear localization signal. Here, we report that copies diversified their coding region and, with it, their protein structure, suggesting different patterns of DNA binding and protein–protein interaction. In addition, each of the retained copies diversified regulatory elements along their promoter regions, indicating differences in their upstream regulation. Taken together, the evidence indicates that the RA1 and RA1-like gene families in grasses underwent subfunctionalization and neofunctionalization enabled by gene duplication.

NAD-dependent formate dehydrogenases (NAD-FdHs) are essential biocatalysts with distinct metabolic functions depending on their source. These enzymes have a great potential for industrial applications in NADH regeneration and carbon capture and conversion technologies. However, although they catalyze the reversible oxidation of formate to CO2, a primary bottleneck for their application in CO2 conversion is the pronounced reaction rate asymmetry, as the reverse reaction (CO2 reduction) rate is several orders of magnitude slower than the forward reaction (HCOO- oxidation). This asymmetry is fundamentally a kinetic manifestation of the thermodynamic equilibrium favoring formate oxidation, as defined by the Haldane relationship. While structural studies have identified conserved active sites, the role of specific molecular features, such as distinct amino acid residues and loops, in determining substrate binding affinities and why CO2 reduction kinetics is so slow even in absence of formate and NAD+ remains unexplored in the current literature. This work addresses some of these issues through fluorescence spectroscopy and Molecular Dynamics simulations. Results showed that the yeast NAD-FdH has independent binding sites for the NAD(H) and that the affinity for NADH is ~200-fold higher than for NAD+. On the other hand, the bacterial enzyme exhibits negative cooperativity for NAD+ binding and positive cooperativity for NADH binding, and their affinities are roughly of the same order. Based on these findings, we discuss the factors that could explain why some NAD-FdHs are more proficient in CO2 reduction than others.

RAMOSA1 (RA1) is a Cys2-His2-type (C2H2) zinc finger transcription factor that controls plant meristem fate and identity and has played an important role in maize domestication. Despite its importance, the origin of RA1 is unknown and the evolution in plants is partially understood. In this paper, we present a well resolved phylogeny based on 73 amino acid sequences from 48 embryophyte species. The recovered tree topology indicates that, during grass evolution, RA1 arose from two consecutive SUPERMAN duplications resulting in three distinct grass sequence lineages: RA1-like A, RA1-like B, and RA1; however, most of these copies have unknown functions. Our findings indicate that RA1 and RA1-like play roles in the nucleus despite lacking a traditional nuclear localization signal. Here we report that copies diversified their coding region and, with it, their protein structure, suggesting different patterns of DNA binding and protein-protein interaction. In addition, each of the retained copies diversified regulatory elements along their promoter regions, indicating differences in their upstream regulation. Taken together, we propose that the RA1 and RA1-like gene families in grasses may have undergone subfunctionalization and neofunctionalization enabled by gene duplication.