Biology subjects

Matulis, D.

Publications and source records attributed to Matulis, D..

3 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2024

Targeted anticancer pre-vinylsulfone covalent inhibitors of carbonic anhydrase IX

We designed novel pre-drug compounds that transform into an active form that covalently modifies particular His residue in the active site, a difficult task to achieve, and applied to carbonic anhydrase (CAIX), a transmembrane protein, highly overexpressed in hypoxic solid tumors, important for cancer cell survival and proliferation because it acidifies tumor microenvironment helping invasion and metastases processes. The designed compounds have several functionalities: 1) primary sulfonamide group recognizing carbonic anhydrases (CA), 2) high-affinity moieties specifically recognizing CAIX among all CA isozymes, and 3) forming a covalent bond with the His64 residue. Such targeted covalent compounds possess both high initial affinity and selectivity for the disease target protein followed by complete irreversible inactivation of the protein via covalent modification. Our designed prodrug candidates bearing moderately active pre-vinyl sulfone esters or weakly active carbamates optimized for mild covalent modification activity to avoid toxic non-specific modifications and selectively target CAIX. The lead inhibitors reached 2 pM affinity, highest among known CAIX inhibitors. The strategy could be used for any disease drug target protein bearing a His residue in the vicinity of the active site.

biophysics↗

bioRxiv2021

POT1 Stability and Binding Measured by Fluorescence Thermal Shift Assays

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 - DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

biophysics↗