Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys¹¹⁹-Cys²⁹⁹) and a unique N-linked glycosylation site (Asn³⁰⁹) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr⁷⁸) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.

Introduction: In pharmaceutical design where future drugs are developed by targeting a specific chosen protein, the evaluation of ligand affinity is crucial. For this very purpose are a multitude of diverse methods which are continuously being improved, which, in turn, makes it difficult to choose which techniques to use in practice. Areas covered: In this review, the authors discuss both experimental and computational approaches for affinity evaluation. Basic principles, general limitations and advantages, as well as main areas of application in drug discovery, are overviewed for some of the most popular ligand binding assays. The authors further provide a guide to affinity predictions, collectively covering several techniques that are used in the first stages of rational drug design. Expert opinion: All affinity estimation methods have limitations and advantages that partially overlap and complement one another. Some of the suggested best practices include cross-verification of data using at least two different techniques and careful data interpretation.

Current Isothermal Titration Calorimetry (ITC) data in the literature have relatively high errors in the measured enthalpies of protein-ligand binding reactions. There is a need for universal validation standards for titration calorimeters. Several inorganic salt co-precipitation and buffer protonation reactions have been suggested as possible enthalpy standards. The performances of several commercial calorimeters, including the VP-ITC, ITC200, and Nano ITC-III, were validated using these suggested standard reactions.

The well-known genotypic and phenotypic diversity of G. vaginalis resulted in its classification into at least four subgroups (clades) with diverse genomic properties. To evaluate the virulence potential of G. vaginalis subgroups, we analyzed the virulence-related phenotypic characteristics of 14 isolates of clade 1, 12 isolates of clade 2, 8 isolates of clade 4 assessing their in vitro ability to grow as a biofilm, produce the toxin vaginolysin, and express sialidase activity. Significant differences in VLY production were found (p = 0.023), but further analysis of clade pairs did not confirm this finding. The amount of biofim did not differ significantly among the clades. Analysis of sialidase activity indicated statistically significant differences among the clades (p < 0.001). Production of active recombinant G. vaginalis sialidase demonstrated the link between the sld gene and enzymatic activity, which may be differentially regulated at the transcriptional level. Statistical classification analysis (random forests algorithm) showed that G. vaginalis clades could be best defined by the profiles of two phenotypic characteristics: sialidase activity and vaginolysin production. The results of principal component analysis and hierarchical clustering suggested that all isolates can be subgrouped into three clusters, the structures of which are determined based on phenotypic characteristics of the isolates. Clade 4 was the most homogenous group, as all isolates were found in the same cluster, which is characterized by low production of all studied virulence factors. Clade 2 isolates were mainly distributed between two clusters, whereas clade 1 isolates were found in all three clusters that were characterized by a distinct profile of phenotypic characteristics. Our findings suggest that G. vaginalis subgroups with different virulence potential might play distinct roles in vaginal microbiota.