Detection Of Conformational Changes Research Articles

Time-Resolved Diffusion Method to Detect and Reveal Protein Reactions Leading to Their Functions

Abstract Proteins are unique natural macromolecules with high selectivity and efficiency for their functions. Understanding the chemical reaction mechanisms of proteins has long been an important and attractive research subject. To study the reactions, various experimental techniques have been developed. However, the time-resolved detection of conformational changes and intermolecular interactions remains a difficult problem, although dynamics are crucial for elucidating the reaction mechanism at the molecular level. Here, time-resolved diffusion (TRD) methods developed to monitor hidden dynamics are introduced. After describing the principle, this review mainly focuses on some applications demonstrating the importance of the time-resolved detection of diffusion-sensitive conformational change (DSCC), which can reveal spectrally silent reaction processes. Correlation between DSCC and enzymatic function showed that conformation changes detected by DSCC are closely related to biological functions such as enzymatic reactions and DNA recognition. Furthermore, the TRD method can detect signal transduction processes of photosensor proteins. Hence, the time-resolved detection of DSCC is appropriate to study mechanisms of protein functions. Since this method can detect intermolecular interactions between enzymes and substrates in time-domain, one of interesting applications is a time-resolved biosensor for transient species. Advantages of the method and future perspective are discussed.

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Detection of molecular transitions with nitrogen-vacancy centers and electron-spin labels

We present a protocol that detects molecular conformational changes with two nitroxide electron-spin labels and a nitrogen-vacancy (NV) center in diamond. More specifically, we demonstrate that the NV can detect energy shifts induced by the coupling between electron-spin labels. The protocol relies on the judicious application of microwave and radiofrequency pulses in a range of parameters that ensures stable nitroxide resonances. Furthermore, we demonstrate that our scheme is optimized by using nitroxides with distinct nitrogen isotopes. We develop a simple theoretical model that we combine with Bayesian inference techniques to demonstrate that our method enables the detection of conformational changes in ambient conditions including strong NV dephasing rates as a consequence of the diamond surface proximity and nitroxide thermalization mechanisms. Finally, we counter-intuitively show that with our method the small residual effect of random molecular tumbling becomes a resource that can be exploited to extract inter-label distances.

Optimising expression of the large dynamic range FRET pair mNeonGreen and superfolder mTurquoise2ox for use in the Escherichia coli cytoplasm

The fluorescent proteins superfolder mTurquoise2ox (sfTq2ox) and mNeonGreen function excellently in mammalian cells, but are not well expressed in E. coli when forming the N-terminus of constructs. Expression was increased by decreasing structures at the start of their coding sequences in the mRNA. Unfortunately, the expression of mNeonGreen started from methionine at position ten as optimisation introduced an alternative RBS in the GFP N-terminus of mNeonGreen. The original start-codon was not deleted, which caused the expression of isomers starting at the original start-codon and at the start-codon at the beginning of the GFP N-terminus. By omitting the GFP N-terminus of mNeonGreen and optimising the structure of its mRNA, the expression of a mixture of isomers was avoided, and up to ~ 50-fold higher expression rates were achieved for mNeonGreen. Both fluorescent proteins can now be expressed at readily detectable levels in E. coli and can be used for various purposes. One explored application is the detection of in-cell protein interactions by FRET. mNeonGreen and sfTq2ox form a FRET pair with a larger dynamic range than commonly used donor–acceptor pairs, allowing for an excellent signal-to-noise ratio, which supports the detection of conformational changes that affect the distance between the interacting proteins.

Ultrasensitive detection of conformational changes in single lipid vesicles

Encapsulation of physicochemical indicators and incorporation of FRET probes within model membrane vesicles has enabled valuable insights into membrane interactions, including pore formation, solution exchange, changes to membrane morphology and fusion dynamics. To characterize and quantify membrane interactions induced by a wide-variety of disruptive agents, including detergents and protein aggregates linked with neurodegenerative diseases, we have implemented high throughput fluorescence assays based on measuring the extent of Ca2+ entry into, and lipid mixing within, single surface-tethered vesicles.

Nanopore Analysis on the Drug-Induced Conformation Change of a p53-Linker-Mouse Double Minute 2 Protein Complex.

Detection of conformational changes in proteins by protein-protein interaction (PPI) is a key issue in developing drug screening platforms. In order to effectively investigate the conformational change in a protein at a single-molecule level, we propose the use of nanopore detection to identify protein conformational changes resulting from protein-protein interactions and their inhibition by Nutlin-3. We designed a protein complex comprising a p53 peptide and a mouse double minute 2 (MDM2) linked by 6 amino acids, transforming its shape from globular to dumbbell structure by inhibition of interaction between p53 peptide and MDM2. In the NMR experiment, no distinguished crosspeaks were observed upon Nutlin-3 addition. However, the nanopore experiment clearly showed double-peak signals with the addition of Nutlin-3. The observed fraction of the double-peak among single-peak signals increased from 8.77% to 22.03% with a concurrent increase in the Nutlin-3 concentration from a molar ratio of 1 to 10-fold. From the nanopore data, we estimated the dwell time for the elongated form of Nutlin-3-bound protein, which traverses for a longer duration (∼2 times) than the globular form. Finally, the hydrodynamic diameter of the local peak of the double-peak signal was calculated and compared with the X-ray crystallography results. This approach shows feasibility of the nanopore detection to verify the protein conformational change by inhibition of protein-protein interaction at a single-molecule level.

Identification of a New Inhibitor That Stabilizes Interleukin-2-Inducible T-Cell Kinase in Its Inactive Conformation

Interleukin-2-inducible T-cell kinase (ITK) plays an important role in T-cell signaling and is considered a promising drug target. As the ATP binding sites of protein kinases are highly conserved, the design of selective kinase inhibitors remains a challenge. Targeting inactive kinase conformations can address the issue of kinase inhibitor selectivity. It is important for selectivity considerations to identify compounds that stabilize inactive conformations from the primary screen hits. Here we screened a library of 390,000 compounds with an ADP-Glo assay using dephosphorylated ITK. After a surface plasmon resonance (SPR) assay was used to filter out promiscuous inhibitors, 105 hits were confirmed. Next, we used a fluorescent biosensor to enable the detection of conformational changes to identify inactive conformation inhibitors. A single-cysteine-substituted ITK mutant was labeled with acrylodan, and fluorescence emission was monitored. Using a fluorescent biosensor assay, we identified 34 inactive conformation inhibitors from SPR hits. Among them, one compound was bound to a site other than the ATP pocket and exhibited excellent selectivity against a kinase panel. Overall, (1) biochemical screening using dephosphorylated kinase, (2) hit confirmation by SPR assay, and (3) fluorescent biosensor assay that can distinguish inactive compounds provide a useful platform and offer opportunities to identify selective kinase inhibitors.

Label-free detection of conformational changes in switchable DNA nanostructures with microwave microfluidics

Detection of conformational changes in biomolecular assemblies provides critical information into biological and self-assembly processes. State-of-the-art in situ biomolecular conformation detection techniques rely on fluorescent labels or protein-specific binding agents to signal conformational changes. Here, we present an on-chip, label-free technique to detect conformational changes in a DNA nanomechanical tweezer structure with microwave microfluidics. We measure the electromagnetic properties of suspended DNA tweezer solutions from 50 kHz to 110 GHz and directly detect two distinct conformations of the structures. We develop a physical model to describe the electrical properties of the tweezers, and correlate model parameters to conformational changes. The strongest indicator for conformational changes in DNA tweezers are the ionic conductivity, while shifts in the magnitude of the cooperative water relaxation indicate the addition of fuel strands used to open the tweezer. Microwave microfluidic detection of conformational changes is a generalizable, non-destructive technique, making it attractive for high-throughput measurements.

Proteins' Dynamics, Hydration and Conformational Changes Studied by EPR

Site-directed spin labeling EPR enables detection of conformational changes in proteins with almost no restriction in the environmental conditions. Key information for structural analysis is provided by changes in the dynamics of spin-labeled sites and by interspin distances between selected pairs of labels. Dynamics are detected by continuous wave EPR, interspin distances by pulse dipolar techniques (DEER or PELDOR being the most common). The long-range EPR distance constraints, combined with existing structural data at atomic level for one state of the protein, enable the creation of coarse-grained models of complex protein rearrangements in their physiological milieu. Each protein conformational transition due to oligomerization, ligand binding, transfer from a water to a membrane environment, etc. is also tightly coupled to rearrangements in the hydration water surrounding the different protein interfaces. Changes in local hydration dynamics and water accessibility can be monitored directly and with high precision at physiological temperature using Overhauser dynamic nuclear polarization (ODNP), which has several advantages with respect to other EPR techniques which provide water accessibility data. We will show examples of structural studies in which the changes in the protein and its surrounding water environment are observed. Examples will be given on ABC transporters, Bcl-2 proteins, and light receptors.

Maleimido-proxyl as an EPR spin label for the evaluation of conformational changes of albumin.

Albumin is the most abundant plasma protein and as such has been the subject of many studies using a variety of techniques. One of them, capable of monitoring the conformational changes and the binding capacity of proteins, is electron paramagnetic resonance spectroscopy (EPR) spin labeling. To date, albumin has been investigated using a number of different spin labels, mostly spin-labeled fatty acids (SLFAs). However, albumin can bind up to seven equivalents of fatty acids, making it difficult to determine which parts of the molecule undergo conformational changes. To obtain information from a specific site on a protein, spin labels that bind to free cysteine residues may be used. In this work, the applicability of such a label, 3-maleimido proxyl (5-MSL), was evaluated for monitoring conformational changes of bovine serum albumin (BSA) at different temperatures and pH values. Also, the effect of ethanol, reactive oxygen species (hydrogen peroxide and superoxide radical), and the binding of ligands specific for albumin, namely fatty acids, and several drugs were evaluated. The results indicate that the labeling of albumin at its free cysteine residue (Cys-34) using 5-MSL may successfully be used for the detection of conformational changes, even in the case of the subtle alterations induced by ligand binding.

Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food.

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.

Application of Hydrogen/Deuterium Exchange-Mass Spectrometry to Biopharmaceutical Development Requirements: Improved Sensitivity to Detection of Conformational Changes.

The usefulness of the higher-order structure information provided by hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the protein therapeutic field is undisputed; however, its applicability as a method for critical quality and comparability assessment has until now not been demonstrated. Here we present results demonstrating for the first time the applicability of the HDX-MS technique to monitor structural changes due to methionine oxidation at sensitivity levels realistic to the requirements of biopharmaceutical research and development. For the analyzed heavy chain marker peptides deuterium uptake differences due to oxidation at the conserved methionine in position 254 were significantly verifiable at the lowest increase (1%) through spiked oxidized IgG1.

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.

Perrin and Förster unified: Dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond

Dual laser flow cytometric energy transfer (FCET) – elaborated by Trón et al. in 1984 – is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (κ2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the “polarized FRET indices” – quantities composed from FRET efficiency and anisotropies – are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of “hetero-FRET induced homo-FRET relief”, i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of κ2 has been extended.

A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function.

Dynamin, the paradigmatic membrane fission catalyst, assembles as helical scaffolds that hydrolyse GTP to sever the tubular necks of clathrin-coated pits. Using a facile assay system of supported membrane tubes (SMrT) engineered to mimic the dimensions of necks of clathrin-coated pits, we monitor the dynamics of a dynamin-catalysed tube-severing reaction in real time using fluorescence microscopy. We find that GTP hydrolysis by an intact helical scaffold causes progressive constriction of the underlying membrane tube. On reaching a critical dimension of 7.3 nm in radius, the tube undergoes scission and concomitant splitting of the scaffold. In a constant GTP turnover scenario, scaffold assembly and GTP hydrolysis-induced tube constriction are kinetically inseparable events leading to tube-severing reactions occurring at timescales similar to the characteristic fission times seen in vivo. We anticipate SMrT templates to allow dynamic fluorescence-based detection of conformational changes occurring in self-assembling proteins that remodel membranes.

Probing the nanostructure, interfacial interaction, and dynamics of chitosan-based nanoparticles by multiscale solid-state NMR.

Chitosan-based nanoparticles (NPs) are widely used in drug and gene delivery, therapy, and medical imaging, but a molecular-level understanding of the internal morphology and nanostructure size, interface, and dynamics, which is critical for building fundamental knowledge for the precise design and efficient biological application of the NPs, remains a great challenge. Therefore, the availability of a multiscale (0.1-100 nm) and nondestructive analytical technique for examining such NPs is of great importance for nanotechnology. Herein, we present a new multiscale solid-state NMR approach to achieve this goal for the investigation of chitosan-poly(N-3-acrylamidophenylboronic acid) NPs. First, a recently developed (13)C multiple cross-polarization magic-angle spinning (MAS) method enabled fast quantitative determination of the NPs' composition and detection of conformational changes in chitosan. Then, using an improved (1)H spin-diffusion method with (13)C detection and theoretical simulations, the internal morphology and nanostructure size were quantitatively determined. The interfacial coordinated interaction between chitosan and phenylboronic acid was revealed by one-dimensional MAS and two-dimensional (2D) triple-quantum MAS (11)B NMR. Finally, dynamic-editing (13)C MAS and 2D (13)C-(1)H wide-line separation experiments provided details regarding the componential dynamics of the NPs in the solid and swollen states. On the basis of these NMR results, a model of the unique nanostructure, interfacial interaction, and componential dynamics of the NPs was proposed.

Circular dichroism spectroscopy as a tool for monitoring aggregation in monoclonal antibody therapeutics.

Aggregation continues to be a critical quality attribute for a monoclonal antibody therapeutic product due to its perceived significant impact on immunogenicity. This paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding protein aggregation. The second application uses thermal scanning CD as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. For establishing far-UV CD as an orthogonal technique, a mAb was incubated in different downstream processing buffers and another mAb in formulation buffers, and they were analyzed by SEC and far-UV CD for aggregate content and conformational stability, respectively. To examine thermal scanning as a high throughput screening tool, ellipticity as a function of the temperature was measured at 218 nm from 20 to 90 °C. Far-UV CD was found to display high sensitivity toward early detection of conformational changes in mAb. CD measurements were also able to elucidate the different aggregation mechanisms. Furthermore, thermal stability scan allowed us to estimate T(onset) which has been found to correlate with aggregation induced by salt, low pH, and buffer species. T(onset) temperature from thermal scanning at 218 nm using CD was correlated successfully to aggregate content measured by SEC. Results from both the studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.

Ca2+-controlled assembly for visualized detection of conformation changes of calmodulin.

A new strategy has been designed for visualized detection of the conformation changes of calmodulin bound to target peptide (CaM-M13) based on the conformation sensitive property of a water-soluble conjugated polythiophene derivative (PMNT) and the electrostatic interactions of PMNT/CaM-M13. Interestingly, the direct visualized PMNT color changes under UV irradiation and the turbidity changes of samples in aqueous medium can be applied to detect the conformation changes as well as the controllable assembly of PMNT/CaM-M13 with Ca(2+) in aqueous medium. Because of the specific binding of Ca(2+), the assembly of PMNT/CaM-M13 can be applied to sense calcium as well.

3P168 金ナノロッドを用いた高速配向イメージングシステムの開発とF1-ATPase の構造変化検出への応用(分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

3P168 金ナノロッドを用いた高速配向イメージングシステムの開発とF1-ATPase の構造変化検出への応用(分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

3P166 全反射型蛍光顕微鏡における受像偏向と偏光変調 : F_1-ATPase の構造変化検出への応用(分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))

3P166 全反射型蛍光顕微鏡における受像偏向と偏光変調 : F_1-ATPase の構造変化検出への応用(分子モーター,ポスター,第52回日本生物物理学会年会(2014年度))