CalFitter 2.0: Leveraging the power of singular value decomposition to analyse protein thermostability

Abstract

The importance of the quantitative description of protein unfolding and aggregation for the rational design of stability or understanding the molecular basis of protein misfolding diseases is well established. Protein thermostability is typically assessed by calorimetric or spectroscopic techniques that monitor different complementary signals during unfolding. The CalFitter webserver has already proved integral to deriving invaluable energy parameters by global data analysis. Here, we introduce CalFitter 2.0, which newly incorporates singular value decomposition (SVD) of multi-wavelength spectral datasets into the global fitting pipeline. Processed time- or temperature-evolved SVD components can now be fitted together with other experimental data types. Moreover, deconvoluted basis spectra provide spectral fingerprints of relevant macrostates populated during unfolding, which greatly enriches the information gains of the CalFitter output. The SVD analysis is fully automated in a highly interactive module, providing access to the results to users without any prior knowledge of the underlying mathematics. Additionally, a novel data uploading wizard has been implemented to facilitate rapid and easy uploading of multiple datasets. Together, the newly introduced changes significantly improve the user experience, making this software a unique, robust, and interactive platform for the analysis of protein thermal denaturation data. The webserver is freely accessible at https://loschmidt.chemi.muni.cz/calfitter.

wTSA-CRAFT: an open-access web server for rapid analysis of thermal shift assay experiments

“…MoltenProt also allows the determination of the slope of the thermal unfolding curve, which is equally crucial for protein stability ( Kotov et al 2021 ). Another tool, CalFitter 2.0, implemented on a web server, incorporates an algorithm that allows access to results without prior knowledge of the underlying mathematics ( Kunka et al 2022 ). Finally, TSA-CRAFT , for TSA—Curve Rapid and Automatic Fitting Tool ( Lee et al 2019 ), was developed for a very basic analysis of DSF data.…”

Advancing Enzyme’s Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning

“…In contrast to the thermal denaturation, all four proteins were almost completely and reversibly unfolded by high concentrations of urea, which allowed us to probe their thermodynamic stability at 25 °C (Figure c). We resolved the complex urea-induced effects on the protein intrinsic fluorescence spectra (including bidirectional change of the signal intensity and redshift of the spectral maximum) using the SVD (Figure c) . The three most significant SVD components well represented all spectral datasets; thus, changes in their amplitudes were used for fitting.…”

“…We resolved the complex urea-induced effects on the protein intrinsic fluorescence spectra (including bidirectional change of the signal intensity and redshift of the spectral maximum) using the SVD ( Figure 3 c). 37 The three most significant SVD components well represented all spectral datasets; thus, changes in their amplitudes were used for fitting. The resistance to urea-induced denaturation increased in the order of DhaA115 < DhaA222 < DhaA223 < DhaA231 and the best fit in all four cases was achieved using the three-state model of unfolding ( Figure 3 c, black lines).…”

Investigating the effect of fusion partners on the enzymatic activity and thermodynamic stability of poly(ethylene terephthalate) degrading enzymes

“…Here, we found Spycatcher003 and Spycatcher003:SpyTag003 to be fully reversible and in equilibrium between the native and denaturated state (N = D) with T m of 52 °C without SpyTag003 and 98 °C with SpyTag003, slightly higher than previous reports 22 . In modelling the irreversible unfolding kinetics of IsPETase, TfCut1 and LCC ICCG using the CalFitter server 33,34 , all were found to go directly from their native state to a denatured state, with no reversibility (N -> D); the T m was measured at 53 °C, 78 °C and 95 °C respectively.…”

“…Melting curves were measured at variable scan rates from 24 °C/hour to 192 °C/hour, with low feedback using the MicroCal PEAQ-DSC automated system. Data was integrated using the Malvern software, and fitted to thermal-denaturation models using Calfitter 33,34 to derive all the T m , T act and E act .…”