Intrinsic Fluorescence Measurements Research Articles

The ALS drug riluzole binds to the C-terminal domain of SARS-CoV-2 nucleocapsid protein and has antiviral activity.

Nucleoproteins (N)play an essential role in virus assembly and are less prone to mutation than other viral structural proteins, making them attractive targets for drug discovery. Using an NMR fragment-based drug discovery approach, we identified the 1,3-benzothiazol-2-amine (BZT) group as a scaffold to develop potential antivirals for SARS-CoV-2 nucleocapsid (N)protein. A thorough characterization of BZT derivatives using NMR, X-ray crystallography, antiviral activity assays, and intrinsic fluorescence measurements revealed their binding in the C-terminal domain (CTD) domain of the N protein, to residues Arg 259, Trp 330, and Lys 338, coinciding with the nucleotide binding site. Our most effective compound exhibits a slightly better affinity than GTP and the ALS drug riluzole, also identified during the screening, and displays notable viral inhibition activity. A virtual screening of 218 BZT-based compounds revealed a potential extended binding site that could be exploited for the future development of new SARS-CoV-2 antivirals.

Elucidating the Binding Interaction of a Highly Efficient Phytochemical-Sinapic Acid with Human Serum Albumin: A Spectroscopic and Calorimetric Approach

Polyphenolic compounds have received much attention due to their major function as antioxidants. Sinapic acid (SA) is an orally bioavailable phytochemical with excellent free radical scavenging property transported in the blood by human serum albumin (HSA) which is the chief carrier protein found abundantly in human plasma. SA contains a single phenolic group. This study investigates the binding and interaction mechanism between SA and HSA by multiple spectroscopic and calorimetric techniques, such as UV/visible absorption spectroscopy, intrinsic fluorescence studies, Fourier transform infrared (FTIR) spectroscopy and isothermal titration calorimetry (ITC). UV/visible absorption spectroscopy showed hyperchromicity in the absorption spectra, suggestive of structural change due to complex formation between HSA and SA. Intrinsic fluorescence measurements, performed at three different temperatures, showed quenching of the fluorescence spectra and the mechanism of quenching was static in nature which occurred due to ground state complex formation. The FTIR results demonstrated a 16% decrease in α-helical content of the secondary structure of HSA in the presence of SA. Hydrogen bonds and hydrophobic forces were the main forces of interaction, according to the thermodynamic characteristics found through ITC. Moreover, the data obtained through ITC indicated strong binding affinity between HSA and SA and supported the exothermic and spontaneous nature of the interaction.

Mechanistic soft-sensor design for protein refolding processes based on intrinsic fluorescence measurements

In protein refolding processes the scarce availability of online measurements hampers effective process monitoring. In this work we developed a mechanistic soft-sensor for protein refolding based on online intrinsic fluorescence measurements of tryptophan and tyrosine. In validation experiments using two model proteins, lactate dehydrogenase (LDH) and galactose oxidase, the soft-sensor showed accurate estimates for the prediction of the total sum of folding products (NRMSE <6.1%) by calculating the changing rate of the average emission wavelength. For refolding of the enzyme LDH it was possible to obtain separate predictions of native protein and insoluble aggregates. The soft-sensor design was further extended by a model-based observer approach using particle filtering to incorporate kinetic formulations as well as physical constraints.The novel approach enabled the analysis of kinetic mechanisms during rapid reaction dynamics and can therefore be seen as an enabler to achieve a better understanding of kinetic refolding mechanisms.

Insights into the interaction and inhibitory action of palmatine on lysozyme fibrillogenesis: Spectroscopic and computational studies

Interaction under amyloidogenic condition between naturally occurring protoberberine alkaloid palmatine and hen egg white lysozyme was executed by adopting spectrofluorometric and theoretical molecular docking and dynamic simulation analysis. In spetrofluorometric method, different types of experiments were performed to explore the overall mode and mechanism of interaction. Intrinsic fluorescence quenching of lysozyme (Trp residues) by palmatine showed effective binding interaction and also yielded different binding parameters like binding constant, quenching constant and number of binding sites. Synchronous fluorescence quenching and 3D fluorescence map revealed that palmatine was able to change the microenvironment of the interacting site. Fluorescence life time measurements strongly suggested that this interaction was basically static in nature. Molecular docking result matched with fluorimetric experimental data. Efficient drug like interaction of palmatine with lysozyme at low pH and high salt concentration prompted us to analyze its antifibrillation potential. Different assays and microscopic techniques were employed for detailed analysis of lysozyme amyloidosis.Thioflavin T(ThT) assay, Congo Red (CR) assay, 8-anilino-1-naphthalenesulfonic acid (ANS) assay, Nile Red (NR) assay, anisotropy and intrinsic fluorescence measurements confirmed that palmatine successfully retarded and reduced lysozyme fibrillation. Dynamic light scattering (DLS) and atomic force microscopy (AFM) further reiterated the excellent antiamyloidogenic potency of palmatine.

Probing the interaction mechanisms between sunset yellow dye and trypsin protein leading to amorphous aggregation under low pH conditions

Protein aggregation poses a significant concern in the field of food sciences, and various factors, such as synthetic food dyes, can contribute to protein aggregation. One such dye, Sunset Yellow (SY), is commonly employed in the food industry. Trypsin was used as a model protein to assess the impact of SY. We employed several biophysical techniques to examine the binding and aggregation mechanisms between SY and trypsin at different pHs. Results from intrinsic fluorescence measurements indicate a stronger interaction between SY and trypsin at pH 2.0 compared to pH 6.0. Turbidity data reveal trypsin aggregation in the presence of 0.05–3.0 mM SY at pH 2.0, while no aggregation was observed at pH 6.0. Kinetic data demonstrate a rapid, lag-phase-free SY-induced aggregation of trypsin. Circular dichroism analysis reveals that trypsin adopts a secondary structure in the presence of SY at pH 6.0, whereas at pH 2.0, the secondary structure was nearly lost with increasing SY concentrations. Furthermore, turbidity and kinetics data suggest that trypsin aggregation depends on trypsin concentrations and pH. Our study highlights potential health risks associated with the consumption of SY, providing insights into its impact on human health and emphasizing the necessity for further research in this field.

Comparison of the self-assembly and cytocompatibility of conjugates of Fmoc (9-fluorenylmethoxycarbonyl) with hydrophobic, aromatic, or charged amino acids.

The self-assembly in aqueous solution of three Fmoc-amino acids with hydrophobic (aliphatic or aromatic, alanine or phenylalanine) or hydrophilic cationic residues (arginine) is compared. The critical aggregation concentrations were obtained using intrinsic fluorescence or fluorescence probe measurements, and conformation was probed using circular dichroism spectroscopy. Self-assembled nanostructures were imaged using cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Fmoc-Ala is found to form remarkable structures comprising extended fibril-like objects nucleating from spherical cores. In contrast, Fmoc-Arg self-assembles into plate-like crystals. Fmoc-Phe forms extended structures, in a mixture of straight and twisted fibrils coexisting with nanotapes. Spontaneous flow alignment of solutions of Fmoc-Phe assemblies is observed by SAXS. The cytocompatibility of the three Fmoc-amino acids was also compared via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] mitochondrial activity assays. All three Fmoc-amino acids are cytocompatible with L929 fibroblasts at low concentration, and Fmoc-Arg shows cell viability up to comparatively high concentration (0.63 mM).

Elucidating the structure-function attributes of a trypanosomal arginyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

The effects of biliverdin on pressure-induced unfolding of apomyoglobin: The specific role of Zn2+ ions

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In situ visible absorption study of BV chromophore confirmed the ability of Zn2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ∼100 MPa in presence of Zn2+. By combining HP-fluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket.

Antifouling Properties of Pluronic and Tetronic Surfactants in Digital Microfluidics.

Fouling at liquid-solid interfaces is a pernicious problem for a wide range of applications, including those that are implemented by digital microfluidics (DMF). There are several strategies that have been used to combat surface fouling in DMF, the most common being inclusion of amphiphilic surfactant additives in the droplets to be manipulated. Initial studies relied on Pluronic additives, and more recently, Tetronic additives have been used, which has allowed manipulation of complex samples like serum and whole blood. Here, we report our evaluation of 19 different Pluronic and Tetronic additives, with attempts to determine (1) the difference in antifouling performance between the two families, (2) the structural similarities that predict exceptional antifouling performance, and (3) the mechanism of the antifouling behavior. Our analysis shows that both Pluronic and Tetronic additives with modest molar mass, poly(propylene oxide) (PPO) ≥50 units, poly(ethylene oxide) (PEO) mass percentage ≤50%, and hydrophilic-lipophilic balance (HLB) ca. 13-15 allow for exceptional antifouling performance in DMF. The most promising candidates, P104, P105, and T904, were able to support continuous movement of droplets of serum for more than 2 h, a result (for devices operating in air) previously thought to be out of reach for this technique. Additional results generated using device longevity assays, intrinsic fluorescence measurements, dynamic light scattering, asymmetric flow field flow fractionation, supercritical angle fluorescence microscopy, atomic force microscopy, and quartz crystal microbalance measurements suggest that the best-performing surfactants are more likely to operate by forming a protective layer at the liquid-solid interface than by complexation with proteins. We propose that these results and their implications are an important step forward for the growing community of users of this technique, which may provide guidance in selecting surfactants for manipulating biological matrices for a wide range of applications.

Laser-Induced Fluorescence (LIF) Spectroscopy of Trapped Molecular Ions in the Gas Phase.

This review focuses on the laser-induced fluorescence (LIF) spectroscopy of trapped gas-phase molecular ions, a developing field of research. Following a brief description of the theory and experimental approaches employed in general for fluorescence spectroscopy, the review summarizes the current state-of-the-art intrinsic fluorescence measurement techniques employed for gas-phase ions. Whereas the LIF spectroscopy of condensed matter systems is a well-developed area of research, the instrumentation used for such studies is not directly applicable to gas-phase ions. However, some measurement schemes employed in condensed-phase experiments could be highly beneficial for gas-phase investigations. We have included a brief discussion on some of these techniques as well. Quadrupole ion traps are commonly used for spatial confinement of ions in the ion-trap-based LIF. One of the main challenges involved in such experiments is the poor signal-to-noise ratio (SNR) arising due to weak gas-phase fluorescence emission, high background noise, and small solid angle for the fluorescence collection optics. The experimental approaches based on the integrated high-finesse optical cavities employed for the condensed-phase measurements provide a better (typically an order of magnitude more) SNR in the detected fluorescence than the single-pass detection schemes. Another key to improving the SNR is to exploit the maximum solid angle of light collection by choosing high numerical aperture (NA) collection optics. A combination of these two approaches integrated with ion traps could transmogrify this field, allowing one to study even weak fluorescence emission from gas-phase molecular ions. The review concludes by discussing the scope of the advances in the LIF instrumentation for detailed spectral characterization of fluorophores of weak gas-phase fluorescence emission, considering fluorescein as one example.

Influence of molecular structure and interface behavior on foam properties of rice bran protein nano-particles

According to our previous research, thermal-acidic treatment was an effective way to improve foaming properties, especially foam stability, of rice bran protein (RBP) samples. In this article, the molecular structure and interface behavior of native RBP and rice bran protein nano-particles (RBPNs) were characterized to illustrate the relevant mechanisms affecting foaming properties. The circular dichroism (CD) and intrinsic fluorescence measurements suggested that thermal-acidic treatment could increase the flexible conformation of RBPNs corresponding to a higher surface activity at the interface. The decrease in apparent viscosity of RBPNs also illustrated the improvement in foam capacity of RBPNs. Moreover, the studies of air-water interfacial behavior demonstrated that the interfacial interaction of RBPNs-1 behaved stronger resulting in forming a stronger and more stretchable interfacial viscoelastic film compared to native RBP, which was mainly related to the different intermolecular interactions at the interface between the RBPNs-1 and native RBP. Further, combined with the analysis of the foam microstructure, it could indicate that in addition to the effects of the interface film on foam stability, the RBPNs trapped in Plateau borders provided the jamming effect by impeding the flow and reducing the rate of drainage. These findings are helpful to understand the relationship between molecular structure, interface behavior, and foaming properties.

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.

The drip loss inhibitory mechanism of nanowarming in jumbo squid (Dosidicus gigas) mantles: protein structure and molecular dynamics simulation.

The magnetic nanoparticles plus microwave thawing (MNPMT), a new rewarming technology entitled 'nanowarming', can serve as an effective method to achieve rapid and uniform thawing, thus reducing drip loss. The purpose of this study was to decipher the drip loss inhibitory mechanism of MNPMT in jumbo squid (Dosidicus gigas) from the perspectives of protein structure and ice crystal recrystallization. A number of different techniques such as dynamic rheology, Raman spectra, intrinsic fluorescence measurement, and ultraviolet (UV) absorption spectra were conducted to analyze myofibrillar protein conformation and stability of jumbo squid. Scanning electron microscopy (SEM) and myofibrillar fragmentation index (MFI) were used to observe the growth of ice crystals. The interaction between magnetic nanoparticles (MNPs) and ice crystals was studied by using molecular dynamic (MD) simulation. MNPMT exhibited the highest storage modulus (G') value at 90 °C, suggesting the protein conformation was more stable. The increase in α-helices, fluorescence intensity and characteristic absorption peak of MNPMT illustrated that MNPMT can effectively maintain the secondary and tertiary structure of the protein. Compared with cold storage thawing (CST) and microwave thawing (MT), the MFI value of MNPMT was significantly decreased (P < 0.01). The result of MD simulation showed that MNPs displayed a tendency to gradually approach the surface of ice crystals, and induced a certain degree of damage to the ice crystal surface, thereby markedly inhibiting ice crystal recrystallization. MNPMT can reduce the drip loss by keeping the protein conformation stable and inhibiting the recrystallization of ice crystals during the thawing process. © 2022 Society of Chemical Industry.

Heat treatment as a food-grade strategy to increase the stability of whey protein particles under food system relevant conditions

Whey proteins are well-suited for the production of protein nanoparticles (NPs). However, whey protein NPs are not stable in aqueous media unless they undergo some type of hardening. Heat pretreatment of NPs was investigated as a hardening strategy. The highest stability against redissolution in water, as evaluated by particle size distribution measurements and quantification of the soluble protein level, was achieved when NPs in 80.8 v/v% aqueous ethanol were treated for 30 min at 60 °C. Size exclusion chromatography under non-reducing and reducing conditions showed that intermolecular disulfide bridges were formed during this treatment while conformational changes of proteins, as assessed via intrinsic fluorescence and vibrational spectroscopy measurements, in the particle matrix seemed minimal. Properties of whey protein NPs were largely driven by interactions and reactions between β-lactoglobulins. Hardening also extended the stability of NPs in 8.1% v/v aqueous ethanol against disintegration, and aggregation upon thermal incubation and/or isothermal long-term storage. As untreated and heat pretreated NPs displayed distinct behavior upon resuspension in water and exposure to different environmental conditions, these insights are valuable for developing controlled release NPs displaying specific release kinetics.

Estimation of the Phospholipase A2 Selectivity on POPC/POPG Membranes Using the Interaction Map

The regulation of the activity and selectivity of phospholipase A2 (PLA2), which is capable of cleaving fatty acid in the second position (sn-2) of the phospholipid, is carried out through the membrane-binding and catalytic sites of the enzyme. For hydrolytic activity, PLA2 must first bind to the phospholipid membrane, and the binding efficiency depends on the composition of the membrane. The membrane-binding site of PLA2 is formed by several tens of amino acids and its composition differs from enzyme to enzyme; hydrophobic and positively charged amino acids play a key role in the interaction. In this work, we investigated the interaction of PLA2 from bee venom with phospholipid bilayers of palmitoyl oleoylphosphatidylcholine (POPC) containing different amounts of palmitoyloleoylphosphatidylglycerol (POPG). On the basis of the measurements of the protein intrinsic fluorescence and the anisotropy of the fluorescence of the lipid probe we propose the construction of lipid–protein interaction maps, which reflect both the efficiency of protein binding and changes in the structure of the membrane. These changes cause alterations in the fluorescence anisotropy of the label, which in turn is a measure of the mobility of the lipid environment of the fluorescent probe. Analysis of interaction maps showed that there is a relationship between lipid mobility and enzyme binding efficiency: the optimum interaction of PLA2 with membranes from a POPC/POPG mixture lies in the region of the highest lipid mobility, and not in the region of the highest negative charge. This dependence complements the existing understanding of the process of recognition of the membrane surface by the enzyme and the selection of lipids by the enzyme already bound to the membrane. The proposed mapping method can be extended to other membrane-active proteins.

Exploration of the binding between cuminol and bovine serum albumin through spectroscopic, molecular docking and molecular dynamics methods

Cuminol (4-Isopropylbenzyl alcohol), found in the essential oils of several plant sources, is an important constituent of several cosmetics formulations. The interaction of cuminol with model plasma protein bovine serum albumin was studied in this paper. The experimental studies were mainly carried out using fluorescence spectrophotometry aided with UV visible and CD spectroscopies. Intrinsic fluorescence measurements showed that there was a weak binding between cuminol and BSA. The mechanism of binding involved static quenching with around 1:1 binding. The binding was chiefly supported by hydrophobic forces although a little contribution of hydrogen bonding was also found in the interaction and the values of enthalpy change were negative with positive entropy change. The secondary structure of BSA didn’t change significantly in presence of low concentrations of cuminol, however, partial unfolding of the former taken place when the concentration of the latter increased. Molecular docking analyses showed cuminol binds at the intersection of subdomains IIA and IIIA, i.e. its binding site is in between Sudlow sites I and II. Molecular dynamics simulations results have shown that BSA forms a stable complex with cuminol and the structure of the former didn’t change much in presence of later. Communicated by Ramaswamy H. Sarma

Spontaneous Fibrillation of Bovine Serum Albumin at Physiological Temperatures Promoted by Hydrolysis-Prone Ionic Liquids.

This study highlights the role of time-dependent hydrolysis of ionic liquid anion, [BF4]-, of ionic liquid (IL), 1-ethyl-3-methylimidazolium tetrafluoroborate, [C2mim][BF4], which results in ever-changing pH conditions. Such pH changes along with the ionic interactions bring conformational changes in bovine serum albumin (BSA), leading to the formation of amyloid fibers at 37 °C without external control of pH or addition of electrolyte. The fibrillation of BSA occurs spontaneously with the addition of IL; however, the highest growth rate has been observed in aqueous solution of 10% IL (v/v %) among investigated systems. Thioflavin T (ThT) fluorescence emission has been employed to monitor the growth and development of β-sheet content in amyloid fibrils. The structural alterations in BSA have also been investigated using intrinsic fluorescence measurements. Circular dichroism (CD) measurements confirmed the formation of amyloid fibrils. Transmission electron microscopy (TEM) has been explored to establish the morphologies of BSA fibrils at different intervals of time, whereas atomic force microscopy (AFM) has established the helically twisted nature of grown amyloid fibrils. The docking studies have been utilized to understand the insertion of IL ions in different domains of BSA, which along with decreased pH cause the unfolding and growth of BSA into amyloid fibrils. It is expected that the results obtained from this study would help to understand the impact of IL containing [BF4]- anion on protein stability and aggregation along with providing a new platform to control the formation of amyloid fibrils and other biomaterials driven via ionic interactions and alterations in pH.

Performance study of a sterilization box using a combination of heat and ultraviolet light irradiation for the prevention of COVID-19

SARS-CoV-2 virus and other pathogenic microbes are transmitted to the environment through contacting surfaces, which need to be sterilized for the prevention of COVID-19 and related diseases. In this study, a prototype of a cost-effective sterilization box is developed to disinfect small items. The box utilizes ultra violet (UV) radiation with heat. For performance assessment, two studies were performed. First, IgG (glycoprotein, a model protein similar to that of spike glycoprotein of SARS-COV-2) was incubated under UV and heat sterilization. An incubation with UV at 70 °C for 15 min was found to be effective in unfolding and aggregation of the protein. At optimized condition, the hydrodynamic size of the protein increased to ~171 nm from ~5 nm of the native protein. Similarly, the OD280 values also increased from 0.17 to 0.78 indicating the exposure of more aromatic moieties and unfolding of the protein. The unfolding and aggregation of the protein were further confirmed by the intrinsic fluorescence measurement and FTIR studies, showing a 70% increase in the β-sheets and a 22% decrease in the α-helixes of the protein. The designed box was effective in damaging the protein's native structure indicating the effective inactivation of the SARS-COV-2. Furthermore, the incubation at 70 °C for 15 min inside the chamber resulted in 100% antibacterial efficacy for the clinically relevant E.coli bacteria as well as for bacteria collected from daily use items. It is the first detailed performance study on the efficacy of using UV irradiation and heat together for disinfection from virus and bacteria.

Structural information and membrane binding of truncated RGS9-1 Anchor Protein and its C-terminal hydrophobic segment

Visual phototransduction takes place in photoreceptor cells. Light absorption by rhodopsin leads to the activation of transducin as a result of the exchange of its GDP for GTP. The GTP-bound ⍺-subunit of transducin then activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP leading to photoreceptor hyperpolarization. Photoreceptors return to the dark state upon inactivation of these proteins. In particular, PDE is inactivated by the protein complex R9AP/RGS9-1/Gβ5. R9AP (RGS9-1 anchor protein) is responsible for the membrane anchoring of this protein complex to photoreceptor outer segment disk membranes most likely by the combined involvement of its C-terminal hydrophobic domain as well as other types of interactions. This study thus aimed to gather information on the structure and membrane binding of the C-terminal hydrophobic segment of R9AP as well as of truncated R9AP (without its C-terminal domain, R9AP∆TM). Circular dichroism and infrared spectroscopic measurements revealed that the secondary structure of R9AP∆TM mainly includes ⍺-helical structural elements. Moreover, intrinsic fluorescence measurements of native R9AP∆TM and individual mutants lacking one tryptophan demonstrated that W79 is more buried than W173 but that they are both located in a hydrophobic environment. This method also revealed that membrane binding of R9AP∆TM does not involve regions near its tryptophan residues, while infrared spectroscopy validated its binding to lipid vesicles. Additional fluorescence measurements showed that the C-terminal segment of R9AP is membrane embedded. Maximum insertion pressure and synergy data using Langmuir monolayers suggest that interactions with specific phospholipids could be involved in the membrane binding of R9AP∆TM.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm, should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X‐ray crystallography and cryo‐electron microscopy, screening for small‐molecule binding partners and comparison of effects of point mutations.