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Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited., Competing Interests: Declaration of Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Identification of the drug target of lead compounds is an important means for rapid and efficient drug discovery. Protein chips are a high-throughput protein function analysis technology that has been widely used in screening drug protein targets in recent years. However, the verification of the results after high-throughput protein chip screening is still cumbersome. Based on our mature protein chip preparation platform, we prepared a protein chip containing 150 important high-frequency protein targets and used antibodies to prove the availability of the protein chip. To improve the accuracy of target screening, we combined the label-free differential scanning fluorimetry (DSF) with the protein chip, proposing the Chip-DSF strategy. Subsequently, we tested the method with small molecular ginsenoside-Rg2 (Rg2). The Chip-DSF strategy was used to successfully screen the potential target protein KRAS(G12C) of Rg2. Consistently, we found that Rg2 could inhibit NCI-H23 cell proliferation by inducing cell cycle arrest. Also, we found that Rg2 could reduce the amount of KRAS protein and inhibit the phosphorylation of KRAS downstream key signaling protein ERK1, RPS6, and P70S6K in NCI-H23 cells. Collectively, our Chip-DSF strategy could achieve rapid target verification which improved the accuracy and efficiency of target screening of protein chips., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Differential scanning fluorimetry (DSF) can be used to detect the binding of a small molecule ligand to a purified target protein. Upon binding with certain ligands, the protein can be stabilized from thermal denaturation. DSF uses a fluorescent dye and Real-Time PCR instrument to detect the unfolding process of proteins during thermal denaturation. The experiment can be set up and finished in 1 day once the purified protein is available.

Osmolytes are naturally occurring organic compounds that protect cellular proteins and other macromolecules against various forms of stress including temperature extremes. While biological studies have correlated the accumulation of certain classes of osmolytes with specific forms of stress, including thermal stress, it remains unclear whether or not these observations reflect an intrinsic chemical class hierarchy amongst the osmolytes with respect to effects on protein stability. In addition, very little is known in regards to the molecular elements of the osmolytes themselves that are essential for their functions. In this study, we use differential scanning fluorimetry to quantify the thermal stabilizing effects of members from each of the three main classes of protecting osmolytes on two model protein systems, C-reactive protein and tumor necrosis factor alpha. Our data reveals the absence of a strict chemical class hierarchy amongst the osmolytes with respect to protein thermal stabilization, and indicates differential responses of these proteins to certain osmolytes. In the second part of this investigation we dissected the molecular elements of amino acid osmolytes required for thermal stabilization of myoglobin and C-reactive protein. We show that the complete amino acid zwitterion is required for thermal stabilization of myoglobin, whereas removal of the osmolyte amino group does not diminish stabilizing effects on C-reactive protein. These disparate responses of proteins to osmolytes and other small molecules are consistent with previous observations that osmolyte effects on protein stability are protein-specific. Moreover, the data reported in this study support the view that osmolyte effects cannot be fully explained by considering only the solvent accessibility of the polypeptide backbone in the native and denatured states, and corroborate the need for more complex models that take into account the entire protein fabric., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Differential scanning fluorimetry (DSF) is a widely used thermal shift assay for measuring protein stability and protein-ligand interactions that are simple, cheap, and amenable to high throughput. However, data analysis remains a challenge, requiring improved methods. Here, the program SimpleDSFviewer, a user-friendly interface, is described to help the researchers who apply DSF technique in their studies. SimpleDSFviewer integrates melting curve (MC) normalization, smoothing, and melting temperature (Tm) analysis and directly previews analyzed data, providing an efficient analysis tool for DSF. SimpleDSFviewer is developed in Matlab, and it is freely available for all users to use in Matlab workspace or with Matlab Runtime. It is easy to use and an efficient tool for researchers to preview and analyze their data in a very short time., (© 2019 The Protein Society.)

In biotherapeutic protein research, an estimation of the studied protein's thermal stability is one of the important steps that determine developability as a function of solvent conditions. Differential Scanning Fluorimetry (DSF) can be applied to measure thermal stability. Label-free DSF measures amino acid fluorescence as a function of temperature, where conformational changes induce observable peak deformation, yielding apparent melting temperatures. The estimation of the stability parameters can be hindered in the case of multidomain, multimeric or aggregating proteins when multiple transitions partially coincide. These overlapping protein unfolding transitions are hard to evaluate by the conventional methodology, as peak maxima are shifted by convolution. We show how non-linear curve fitting of intrinsic fluorescence DSF can deconvolute highly overlapping transitions in formulation screening in a semi-automated process. The proposed methodology relies on synchronous, constrained fits of the fluorescence intensity, ratio and their derivatives, by combining linear baselines with generalized logistic transition functions. The proposed algorithm is applied to data from three proteins; a single transition, a double separated transition and a double overlapping transition. Extracted thermal stability parameters; apparent melting temperatures T m,1 , T m,2 and melting onset temperature T onset are obtained and compared with reference software analysis. The fits show R 2  = 0.94 for single and R 2  = 0.88 for separated transitions. Obtaining values and trends for T onset in a well-described and automated way, will aid protein scientist to better evaluate the thermal stability of proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Systematic identification of buffer formulations and small molecule chaperones that improve the expression, stability, and storage of proteins with therapeutic interest has gained enormous importance in biochemical research as well as in biotechnology and biomedical applications. In particular, the biochemical characterization of disease-related proteins and their genetic variants that result in misfolding requires systematic determination of protein stability, screening of optimal buffer conditions for biophysical and structural studies, and in some cases, the identification of small molecule chaperones with the potential to ameliorate folding defects. Among the several techniques available, differential scanning fluorimetry (DSF) is currently an extensively employed screening and analysis method for thermal shift and protein stability assays. Here we describe a step-by-step generic protocol for fast characterization of protein thermal stability and analysis of stabilization in thermal-shift assays by additives, ligands and chemical chaperones using β-oxidation mitochondrial dehydrogenases as model. These enzymes are associated to inborn errors of metabolism caused by mutant variants with folding and stability defects for which we previously established folding correction afforded by their cognate cofactors and substrates. With this example we thus illustrate the potential applications of the method in screening small molecule folding correctors among metabolites, ligands, cofactors or candidate drugs with therapeutic potential in protein folding diseases.

Protein-polymer conjugates are increasingly viewed as promising avenues to producing industrial enzymes with high activity capable of withstanding potentially harsh reaction conditions, or to designing novel therapeutics with triggered release, controlled masking, or increased resistance to proteolytic degradation. Common among these applications are the desire to improve the stability of protein-polymer conjugates to unfolding by exposure to chemicals or thermal stress. Thus, assays that allow researchers to robustly and easily characterize protein-polymer conjugates by obtaining thermodynamic parameters for folding stand to play an important role in the development of improved protein-polymer conjugates. Herein, we describe two techniques, differential scanning fluorimetry and intrinsic tryptophan fluorescence, used in our laboratories to obtain thermodynamic parameters of unfolding that allow for direct comparison of protein-polymer conjugates and the myriad effects of variations in attachment site, polymer identity, and polymer length. These two experiments, which are easily amenable to parallelization, are presented as high-throughput replacements for more traditionally employed circular dichroism experiments and as complements to functional chemical stability or functional thermal stability experiments. Each assay is presented in a parallelized format that allows for rapid scaling and high-throughput analysis of protein-polymer conjugate libraries. Descriptions of the assays include a discussion of advantages and disadvantages alongside protocol details and approaches to data analysis., (© 2017 Elsevier Inc. All rights reserved.)

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT-PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL L-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT-PCR machines in a hydrophilic or hydrophobic environment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor(®) provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye., (Copyright © 2015 John Wiley & Sons, Inc.)

The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence-based thermal-shift assay (Thermofluor). Here, a novel 96-condition screen for Thermofluor experiments is presented which consists of buffer and additive parts. The buffer screen comprises 23 different buffers and the additive screen includes small-molecule additives such as salts and nucleotide analogues. The utilization of small-molecule components which increase the thermal stability of a protein sample frequently results in a protein preparation of higher quality and quantity and ultimately also increases the chances of the protein crystallizing.

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT-PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL L-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT-PCR machines in a hydrophilic or hydrophobic environment.

Among various factors, the direct environment (e.g. pH, buffer components, salts, additives, etc....) is known to have a crucial effect on both the stability and activity of proteins. In particular, proper buffer and pH conditions can improve their stability and function significantly during purification, storage and handling, which is highly relevant for both academic and industrial applications. It can also promote data reproducibility, support the interpretation of experimental results and, finally, contribute to our general understanding of the biophysical properties of proteins. In this study, we have developed a high throughput screen of 158 different buffers/pH conditions in which we evaluated: (i) the protein stability, using differential scanning fluorimetry and (ii) the protein function, using either enzymatic assays or binding activity measurements, both in an automated manner. The modular setup of the screen allows for easy implementation of other characterization methods and parameters, as well as additional test conditions. The buffer/pH screen was validated with five different proteins used as models, i.e. two active-site serine β-lactamases, two metallo-β-lactamases (one of which is only active as a tetramer) and a single-domain dromedary antibody fragment (VHH or nanobody). The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins. Besides the determination of the optimum buffer and pH, the collection of pH profiles of many different proteins may also allow to delineate general concepts to understand and predict the relationship between pH and protein properties. [ABSTRACT FROM AUTHOR]

Enzymes are attractive tools for synthetic applications. To be viable for industrial use, enzymes need sufficient stability towards the desired reaction conditions such as high substrate and cosolvent concentration, non‐neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purification by thermal treatment and higher process temperatures but also due to the associated higher stability against other destabilising factors. Therefore, high‐throughput screening (HTS) methods are desirable for the identification of thermostable biocatalysts by discovery from nature or by protein engineering but current methods have low throughput and require time‐demanding purification of protein samples. We found that nanoscale differential scanning fluorimetry (nanoDSF) is a valuable tool to rapidly and reliably determine melting points of native proteins. To avoid intrinsic problems posed by crude protein extracts, hypotonic extraction of overexpressed protein from bacterial host cells resulted in higher sample quality and accurate manual determination of several hundred melting temperatures per day. We have probed the use of nanoDSF for HTS of a phylogenetically diverse aldolase library to identify novel thermostable enzymes from metagenomic sources and for the rapid measurements of variants from saturation mutagenesis. The feasibility of nanoDSF for the screening of synthetic reaction conditions was proved by studies of cosolvent tolerance, which showed protein melting temperature to decrease linearly with increasing cosolvent concentration for all combinations of six enzymes and eight water‐miscible cosolvents investigated, and of substrate affinity, which showed stabilisation of hexokinase by sugars in the absence of ATP cofactor. Enzymes: Alcohol dehydrogenase (NADP+) (EC 1.1.1.2), transketolase (EC 2.2.1.1), hexokinase (EC 2.7.1.1), 2‐deoxyribose‐5‐phosphate aldolase (EC 4.1.2.4), fructose‐6‐phosphate aldolase (EC 4.1.2.n). nanoDSF is a valuable tool to rapidly determine thermal stability of native proteins in crude protein extracts. It can be used for high‐throughput screening of enzyme libraries from metagenomic sources or from saturation mutagenesis, for rapid screening of tolerance for water‐miscible organic cosolvent and of substrate affinity, for example, sugar binding by hexokinase even in the absence of ATP. [ABSTRACT FROM AUTHOR]

Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) were used to measure the transition temperatures of four proteins: RNase A, invertase, rituximab, and the NISTmAb (NIST Reference Material, RM 8671). The proteins were combined with several different fluorescent dyes for the DSF measurements. This study compares the results of DSC and DSF measurements of transition temperatures with different types of proteins, dye combinations, and thermal scan rates. As protein unfolding is often influenced by kinetic effects, we measured the transition temperatures of the proteins using DSC over a range of temperature scan rates and compared them to the data obtained from DSF over comparable temperature scan rates. The results when the proteins were combined with Sypro Orange® and bis-ANS for the DSF measurements had the best correlations with the transition temperatures determined by calorimetry. The scan rate was found to be an important variable when comparing results between DSC and DSF. The van't Hoff enthalpy changes for the transitions were calculated from the DSC data by using a non-two-state model and from the DSF values using a two-state model. The calculated van't Hoff enthalpy changes did not show a good correlation between the two methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:677-686, 2017 [ABSTRACT FROM AUTHOR]

Inhibitors of AKR1B10 belonging to the aldo-keto reductase (AKR) superfamily are considered promising candidates for anti-cancer drugs. AKR1B1, a structurally similar isoform of AKR1B10, is involved in glucose metabolism. Thus, selective inhibition of AKR1B10 is required for the development of anti-cancer drugs. In this study, we first compared correlations between melting temperature and the 50% inhibition concentration obtained from differential scanning fluorimetry (DSF) and an enzyme inhibitory experiment, respectively, and a good correlation was found, except for compounds with low solubility. This result indicates that the DSF method is useful for drug screening for the AKR superfamily. We then evaluated their selectivity as inhibitors against all seven major human AKR1 family proteins and found that C18 is most specific for AKR1B10. [ABSTRACT FROM AUTHOR]

Collapsin response mediator protein-5 ( CRMP-5) is the latest identified member of the CRMP cytosolic phosphoprotein family, which is crucial for neuronal development and repair. CRMPs exist as homo- and/or hetero-tetramers in vivo and participate in signaling transduction, cytoskeleton rearrangements, and endocytosis. CRMP-5 antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners. We determined the crystal structures of a full length and a truncated version of human CRMP-5, both of which form a homo-tetramer similar to those observed in CRMP-1 and CRMP-2. However, solution studies indicate that CRMP-5 and CRMP-1 form weaker homo-tetramers compared with CRMP-2, and that divalent cations, Ca2+ and Mg2+, destabilize oligomers of CRMP-5 and CRMP-1, but promote CRMP-2 oligomerization. On the basis of comparative analysis of the CRMP-5 crystal structure, we identified residues that are crucial for determining the preference for hetero-oligomer or homo-oligomer formation. We also show that in spite of being the CRMP family member most closely related to dihydropyrimidinase, CRMP-5 does not have any detectable amidohydrolase activity. The presented findings provide new detailed insights into the structure, oligomerization, and regulation of CRMPs. [ABSTRACT FROM AUTHOR]

Abstract: Thermal shift methods such as differential scanning fluorimetry and differential static light scattering are widely used to identify stabilizing conditions for proteins that might promote crystallization. Here we report a comparison of the two methods when applied to optimization of buffer conditions for protein–protein complexes. Most of the protein complexes under study were amenable to analysis using these two techniques. Protein complexes behave towards thermal denaturation in a manner similar to single proteins, showing a more or less sharp transition consistent with a two-state model of unfolding. A comparison of the melting and aggregation temperatures for single components and the reconstituted complexes can provide additional evidence for complex formation and can be used to identify buffer conditions in which protein–protein complex formation is favored. [Copyright &y& Elsevier]

Abstract: Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (T m) of 39.3±1.0 and 37.1±0.7°C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35°C over 120min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔT mmax =8°C, half-maximal effective ligand concentration, AC50 =3μM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in T m of ∼16°C, followed by MeCbl at ∼13°C, each evaluated at 50μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi>OHCbl>CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3μM, which compares well with the K d of 6μM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (ΔT mmax =4°C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7μM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease. [Copyright &y& Elsevier]