Fluorescence-Based Protein Stability Monitoring-A Review.

Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.

Ice crystal recrystallization inhibition of type I antifreeze protein, type III antifreeze protein, and antifreeze glycoprotein: effects of AF(G)Ps concentration and heat treatment.

This study compared ice recrystallization behaviors of frozen dessert model systems containing type I antifreeze protein (AFP I), type III antifreeze protein (AFP III), and antifreeze glycoprotein (AFGP) at -10 °C. Specifically, effects of AF(G)P concentration and heat treatment (95 °C for 10 min) were examined. The concentration dependence of the ice recrystallization rate constant reasonably well fit a sigmoidal function: the fitting procedure was proposed, along with cooperative coefficient α, and a new index of AF(G)P ice recrystallization inhibition (IRI) activity (C50). After 95 °C heat treatment for 10 min, AFP III lost its ice crystal recrystallization inhibitory activity the most: AFP I was less affected; AFGP was almost entirely unaffected. These different thermal treatment effects might reflect a lower degree of protein aggregation because of hydrophobic interaction after heat treatment or might reflect the simplicity and flexibility of the higher order structures of AFP I and AFGP.

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase.

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix.

Substrate specificity and ecological significance of PstS homologs in phosphorus uptake in marine Synechococcus sp. WH8102.

Phosphorus, a vital macronutrient, often limits primary productivity in marine environments. Marine Synechococcus strains, including WH8102, rely on high-affinity phosphate-binding proteins (PstS) to scavenge inorganic phosphate in oligotrophic oceans. However, WH8102 possesses three distinct PstS homologs whose substrate specificity and ecological roles are unclear. The three PstS homologs were heterologously expressed and purified to investigate their substrate specificity and binding kinetics. Our study revealed that all three PstS homologs exhibited a high degree of specificity for phosphate but differed in phosphate binding affinities. Notably, PstS1b displayed nearly 10-fold higher binding affinity (KD = 0.44 µM) compared to PstS1a (KD = 3.3 µM) and PstS2 (KD = 4.3 µM). Structural modeling suggested a single amino acid variation in the binding pocket of PstS1b (threonine instead of serine in PstS1a and PstS2) likely contributed to its higher Pi affinity. Genome context data, together with the protein biophysical data, suggest distinct ecological roles for the three PstS homologs. We propose that PstS1b may be involved in scavenging inorganic phosphorus in oligotrophic conditions and that PstS1a may be involved in transporting recycled phosphate derived from organic phosphate cleavage. The role of PstS2 is less clear, but it may be involved in phosphate uptake when environmental phosphate concentrations are transiently higher. The conservation of three distinct PstS homologs in Synechococcus clade III strains likely reflects distinct adaptations for P acquisition under varying oligotrophic conditions.IMPORTANCEPhosphorus is an essential macronutrient that plays a key role in marine primary productivity and biogeochemistry. However, intense competition for bioavailable phosphorus in the marine environment limits growth and productivity of ecologically important cyanobacteria. In oligotrophic oceans, marine Synechococcus strains, like WH8102, utilize high-affinity phosphate-binding proteins (PstS) to scavenge inorganic phosphate. However, WH8102 possesses three distinct PstS homologs, with unclear substrate specificity and ecological roles, creating a knowledge gap in understanding phosphorus acquisition mechanisms in picocyanobacteria. Through genomic, functional, biophysical, and structural analysis, our study unravels the ecological functions of these homologs. Our findings enhance our understanding of cyanobacterial nutritional uptake strategies and shed light on the crucial role of these conserved nutrient uptake systems in adaptation to specific niches, which ultimately underpins the success of marine Synechococcus across a diverse array of marine ecosystems.

Contribution of Nudt12 enzyme to differentially methylated dinucleotides of 5'RNA cap structure.

Recent findings have substantially broadened our knowledge about the diversity of modifications of the 5'end of RNAs, an issue generally attributed to mRNA cap structure (m7GpppN). Nudt12 is one of the recently described new enzymatic activities involved in cap metabolism. However, in contrast to its roles in metabolite-cap turnover (e.g., NAD-cap) and NADH/NAD metabolite hydrolysis, little is known regarding its hydrolytic activity towards dinucleotide cap structures. In order to gain further insight into this Nudt12 activity, comprehensive analysis with a spectrum of cap-like dinucleotides was performed with respect to different nucleotide types adjacent to the (m7)G moiety and its methylation status. Among the tested compounds, GpppA, GpppAm, and Gpppm6Am were identified as novel potent Nudt12 substrates, with KM values in the same range as that of NADH. Interestingly, substrate inhibition of Nudt12 catalytic activity was detected in the case of the GpppG dinucleotide, a phenomenon not reported to date. Finally, comparison of Nudt12 with DcpS and Nud16, two other enzymes with known activity on dinucleotide cap structures, revealed their overlapping and more specific substrates. Altogether, these findings provide a basis for clarifying the role of Nudt12 in cap-like dinucleotide turnover.

Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins.

Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.

Characterization of P. aeruginosa Glucose 6- Phosphate Isomerase: A Functional Insight via In-Vitro Activity Study.

BACKGROUND: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. METHODS: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. RESULTS: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. CONCLUSION: G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

Selective targeting of the αC and DFG-out pocket in p38 MAPK.

The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.

High-throughput screening of formulations to optimize the thermal stability of a therapeutic monoclonal antibody.

Monoclonal antibodies (mAbs) are an important class of biotherapeutics. Successful development of a mAb depends not only on its biological activity but also on its physicochemical properties, such as homogeneity and stability. mAb stability is affected by its formulation. Among the many techniques used to study the stability of mAbs, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of the protein unfolding transition (Tm) based on the change in fluorescence intensity of the environmentally sensitive dye SYPRO Orange. With DSF adapted to a 96-well plate format, we have shown that low-pH or high-salt concentrations decrease the thermal stability of mAb1, whereas some excipients, such as sucrose, polysorbate 80, and sodium phosphate, increase its stability. The basal fluorescence of SYPRO Orange was enhanced by the presence of detergents, limiting the use of this approach to diluted detergent solutions. Throughput of DSF can be increased further with the use of a 384-well plate. DSF thermograms are in good agreement with the melting profiles obtained by differential scanning calorimetry (DSC). The Tms determined by DSF and DSC were well correlated, with the former being on average lower by 3 °C.

Explanatory chapter: troubleshooting protein expression: what to do when the protein is not soluble.

Production of soluble protein remains a bottleneck in the biochemistry and structural biology fields. Unfortunately, there is no 'magic bullet' that solves all solubility problems. The following is a protocol to test whether a protein expressed recombinantly is soluble, and possible strategies to circumvent insolubility issues.

Discovery of Small-Molecule Glucokinase Regulatory Protein Modulators That Restore Glucokinase Activity.

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.

Experimental validation of FINDSITE(comb) virtual ligand screening results for eight proteins yields novel nanomolar and micromolar binders.

BACKGROUND: Identification of ligand-protein binding interactions is a critical step in drug discovery. Experimental screening of large chemical libraries, in spite of their specific role and importance in drug discovery, suffer from the disadvantages of being random, time-consuming and expensive. To accelerate the process, traditional structure- or ligand-based VLS approaches are combined with experimental high-throughput screening, HTS. Often a single protein or, at most, a protein family is considered. Large scale VLS benchmarking across diverse protein families is rarely done, and the reported success rate is very low. Here, we demonstrate the experimental HTS validation of a novel VLS approach, FINDSITE(comb), across a diverse set of medically-relevant proteins. RESULTS: For eight different proteins belonging to different fold-classes and from diverse organisms, the top 1% of FINDSITE(comb)'s VLS predictions were tested, and depending on the protein target, 4%-47% of the predicted ligands were shown to bind with µM or better affinities. In total, 47 small molecule binders were identified. Low nanomolar (nM) binders for dihydrofolate reductase and protein tyrosine phosphatases (PTPs) and micromolar binders for the other proteins were identified. Six novel molecules had cytotoxic activity (<10 µg/ml) against the HCT-116 colon carcinoma cell line and one novel molecule had potent antibacterial activity. CONCLUSIONS: We show that FINDSITE(comb) is a promising new VLS approach that can assist drug discovery.