Protocol for performing and optimizing differential scanning fluorimetry experiments

Wu, Taiasean; Hornsby, Michael; Zhu, Lawrence; Yu, Joshua C.; Shokat, Kevan M.; Gestwicki, Jason E.

doi:10.1016/j.xpro.2023.102688

Cited by 19 publications

(13 citation statements)

References 39 publications

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“…DSF is a widely known high‐throughput stability analysis method for proteins that enables T m determination of samples prepared in a 96‐well plate within 2 h using a real‐time qPCR machine. Despite the fast and easy‐to‐handle methodology, its application using many samples on a plate has been primarily limited to buffer (Houser et al, 2021 ; Wu et al, 2023 ) or low‐molecular‐weight ligand (Huynh & Partch, 2015 ; Li & Zhang, 2021 ) screening for the same protein. This is partly because, although melting curve assays require the absence of impurities, sample preparation of various proteins on a plate scale is technically difficult because of the laborious nature of conventional purification methods.…”

Section: Discussionmentioning

confidence: 99%

High‐throughput system for the thermostability analysis of proteins

Ito,

Matsunaga,

Nakakido

et al. 2024

Protein Science

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Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high‐throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate‐scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per‐plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single‐chain variable fragment of nivolumab, harvesting the melting temperature (Tm) ranging from −9.3 up to +10.8°C compared to the wild‐type sequence. Our findings will allow for data‐driven stabilization in protein design and streamlining the rational approaches.

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Section: Discussionmentioning

confidence: 99%

High‐throughput system for the thermostability analysis of proteins

Ito,

Matsunaga,

Nakakido

et al. 2024

Protein Science

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“…Common DSF applications include the assessment of ligand binding (Huynh & Partch, 2015 ; Ravalin et al, 2019 ) and optimization of buffers for shelf‐stability or structural characterization (Boivin et al, 2013 ; Chari et al, 2015 ; Reinhard et al, 2013 ; Ristic et al, 2015 ). The theory (Gao et al, 2020 ; Wu et al, 2020 ), applications (Garlick & Mapp, 2020 ; Scott et al, 2016 ; Simeonov, 2013 ), and protocols (Huynh & Partch, 2015 ; Wu, Hornsby, et al, Wu, Hornsby, et al, 2023b ) for DSF have all been extensively reviewed.…”

Section: Introductionmentioning

confidence: 99%

“…We named this analysis tool DSFworld, which is freely available online ( https://gestwickilab.shinyapps.io/dsfworld/ ). Since its initial pre‐publication launch (Wu, Hornsby, et al, 2023b ), DSFworld has averaged over 1000 h of use per month.…”

Section: Introductionmentioning

confidence: 99%

DSFworld: A flexible and precise tool to analyze differential scanning fluorimetry data

Wu,

Gale‐Day,

Gestwicki

2024

Protein Science

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Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from which Tma is calculated vary widely in shape and complexity. However, the tools available for calculating Tma are only compatible with the simplest of DSF curves, hindering many otherwise straightforward applications of the technology. To overcome this limitation, we designed new mathematical models for Tma calculation that accommodate common forms of variation in DSF curves, including the number of transitions, the presence of high initial signal, and temperature‐dependent signal decay. When tested these models against DSFbase, an open‐source database of 6235 raw, real‐life DSF curves, these models outperformed the existing standard approaches of sigmoid fitting and maximum of the first derivative. To make these models accessible, we created an open‐source software and website, DSFworld (https://gestwickilab.shinyapps.io/dsfworld/). In addition to these improved fitting capabilities, DSFworld also includes features that overcome the practical limitations of many analysis workflows, including automatic reformatting of raw data exported from common qPCR instruments, labeling of data based on experimental variables, and flexible interactive plotting. We hope that DSFworld will enable more streamlined and accurate calculation of Tma values for DSF experiments.

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“…The melting curve of an uncharacterized protein or complex can be unpredictable, and testing of various dyes will help validate the findings [6]. For folded and globular proteins, hydrophobic residues are typically internal and inaccessible to the solvent, making TSA a useful tool to assess if a batch of proteins is properly folded, with low initial fluorescence readings, or denatured after purification, with high initial fluorescence readings near room temperature [7,8].…”

Section: Introductionmentioning

confidence: 99%

“…For most samples that are properly folded, the thermal profile is sigmoidal, and a melting temperature (Tm) value can be calculated, representing the temperature where 50% of the protein is unfolded [2,7]. The Tm is a useful value to compare the thermal stability of proteins under various conditions, such as choosing storage buffers and salts or for comparing the stability of mutant proteins [9,10].…”

Section: Introductionmentioning

confidence: 99%

Use of TSAR, Thermal Shift Analysis in R, to identify Folic Acid as a Molecule that Interacts with HIV-1 Capsid

Gao,

McFadden,

Wen

et al. 2023

Preprint

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Thermal shift assay (TSA) is a versatile biophysical technique for studying protein interactions. Here, we report a free, open-source software tool TSAR (Thermal Shift Analysis in R) to expedite and automate the analysis of thermal shift data derived either from individual experiments or large screens of chemical libraries. The TSAR package incorporates multiple, dynamic workflows to facilitate the analysis of TSA data and returns publication-ready graphics or processed results. Further, the package includes a graphic user interface (GUI) that enables easy use by non-programmers, aiming to simplify TSA analysis while diversifying visualization. To exemplify the utility of TSAR we screened a chemical library of vitamins to identify molecules that interact with the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1). Our data show that hexameric CA interacts with folic acidin vitro.

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Protocol for performing and optimizing differential scanning fluorimetry experiments

Cited by 19 publications

References 39 publications

High‐throughput system for the thermostability analysis of proteins

High‐throughput system for the thermostability analysis of proteins

DSFworld: A flexible and precise tool to analyze differential scanning fluorimetry data

Use of TSAR, Thermal Shift Analysis in R, to identify Folic Acid as a Molecule that Interacts with HIV-1 Capsid

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