A Fluorescent, Reagentless Biosensor for ADP Based on Tetramethylrhodamine-Labeled ParM

Fluorescence assays for ADP detection are of considerable current interest, both in basic research and in drug discovery, as they provide a generic method for measuring the activity of ATPases and kinases. The development of a novel fluorescent biosensor is described that is based on a tetramethylrhodamine-labeled, bacterial actin homologue, ParM. The design of the biosensor takes advantage of the large conformational change of ParM on ADP binding and the strong quenching of the tetramethylrhodamine fluorescence by stacking of the dye. ParM was labeled with two tetramethylrhodamines in close proximity, whereby the fluorophores are able to interact with each other. ADP binding alters the distance and relative orientation of the tetramethylrhodamines, which leads to a change in this stacking interaction and so in the fluorescence intensity. The final ADP biosensor shows ∼15-fold fluorescence increase in response to ADP binding. It has relatively weak affinity for ADP (<i>K</i>_d = 30 μM), enabling it to be used at substoichiometric concentrations relative to ADP, while reporting ADP concentration changes in a wide range around the <i>K</i>_d value, namely, submicromolar to tens of micromolar. The biosensor strongly discriminates against ATP (>100-fold), allowing ADP detection against a background of millimolar ATP. At 20 °C, the labeled ParM binds ADP with a rate constant of 9.5 × 10⁴ M⁻¹ s⁻¹ and the complex dissociates at 2.9 s⁻¹. Thus, the biosensor is suitable for real-time measurements, and its performance in such assays is demonstrated using a sugar kinase and a mammalian protein kinase

Development of a range of fluorescent reagentless biosensors for ATP, based on malonyl-coenzyme A synthetase

The range of ATP concentrations that can be measured with a fluorescent reagentless biosensor for ATP has been increased by modulating its affinity for this analyte. The ATP biosensor is an adduct of two tetramethylrhodamines with MatB from Rhodopseudomonas palustris. Mutations were introduced into the binding site to modify ATP binding affinity, while aiming to maintain the concomitant fluorescence signal. Using this signal, the effect of mutations in different parts of the binding site was measured. This mutational analysis revealed three variants in particular, each with a single mutation in the phosphate-binding loop, which had potentially beneficial changes in ATP binding properties but preserving a fluorescence change of ~3-fold on ATP binding. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent's 6 μM to 123 μM and 42 μM, respectively. Kinetic measurements showed that the effect of these mutations on affinity was by an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >100 μM. In contrast, a third variant, S170A, decreased the dissociation constant of ATP to 3.8 μM and has a fluorescence change of 4.2 on binding ATP. This variant has increased selectivity for ATP over ADP of >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurements in which ADP is present

Detecting Intramolecular Conformational Dynamics of Single Molecules in Short Distance Range with Subnanometer Sensitivity

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nanoparticles and nanodevices with nanometer spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3–8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to subnanometer distance changes in a FRET-insensitive short-range at the single molecule level. A ParM protein mutant labeled with two rhodamines works as a single molecule adenosine 5′-diphosphate (ADP) sensor that has 20 times brighter fluorescence signal in the ADP bound state than the unbound state. Single molecule time trajectories show discrete transitions between fluorescence on and off states that can be directly ascribed to ADP binding and dissociation events. The conformational changes observed with 20:1 contrast are only 0.5 nm in magnitude and are between crystallographic distances of 1.6 and 2.1 nm, demonstrating exquisite sensitivity to short distance scale changes. The systems also allowed us to gain information on the photophysics of self-quenching induced by rhodamine stacking: (1) photobleaching of either of the two rhodamines eliminates quenching of the other rhodamine fluorophore and (2) photobleaching from the highly quenched, stacked state is only 2-fold slower than from the unstacked state

Domain organisation and expression screen of human NLRP1.

(A) Human NLRP1 domain organisation; black lines indicate some of the key residues reported to be important for protein function. K340 and E414 belong to the Walker A and Walker B motifs, respectively and are important for ATP processing. H623 is a conserved residue across all the NLRs, the correspondent residues in NLRC4 and in Apaf1 are involved in stabilising the ADP-bound conformation. H1190, F1216 and S1217 are reported to be important for the auto-proteolysis of the FIIND domain. (B) Schematic representation of the soluble constructs produced in insect cells. The boundaries of each construct are indicated on the left. (C) SDS-gels of the recombinant proteins from insect cells after the first metal affinity purification step. A black star indicates the protein of interest and a black arrow heads indicates proteolytic degradation products.</p

SAXS-derived envelope of NLRP1(227–990) and comparison to the open and closed conformations of NLRC4.

(A) SAXS-derived envelope for NLRP1(227–990) calculated from the SAXS data obtained at 2 mg/mL, the maximum dimension (Dmax) and the width of the upper and lower lobes are also reported. (B and C) Structural fitting of the structures of NLRC4 spanning residues 93 to 793 in an open conformation (PDB 3jbl, χ2 of 1.37, in magenta) and in a closed conformation (PDB 4kxf, χ2 of 1.97, in yellow) into the NLRP1 envelope.</p

ATP hydrolysis and oligomerization assay of NLRP1(227–990).

<p>(A) HPLC chromatogram showing the profile of a solution of ADP and ATP standards run on an ion exchange Partisil SAX column (grey solid line), retention times for ADP 2.81 minutes and ATP 6.12 minutes. The sample prepared by unfolding of NLRP1(227–990) contained mainly ATP (>85%) (black solid line). The peak with a retention time of about 2 minutes is background from the buffer. (B) Malachite green assay performed on a solution of NLRP1(227–990) in the presence of ATP 1mM (black squares) and in the presence of ATP 1mM and MDP 0.1mg/mL (black triangles). In both cases the concentration of protein was 0.1 mg/mL and the experiment was performed at room temperature for 1 hour. A blank experiment, without NLRP1 and MDP (black diamonds) and a positive control with DnaK (black stars) were also performed. (C) Size exclusion chromatography (Superose 6 10/300 column) of NLRP1(227–990) in absence (black solid line), in presence of MDP and ATP (grey dotted line) and in presence of MDP and a non-hydrolysable ATP analogue (grey solid line). Black arrow heads indicate the retention volumes of molecular weight standards.</p

The Biophysical Characterisation and SAXS Analysis of Human NLRP1 Uncover a New Level of Complexity of NLR Proteins

<div><p>NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs.</p></div

SEC-SAXS analysis of NLRP1(227–990).

(A) Scattering data obtained on two protein samples with concentrations of 2 mg/mL (open circles) and 6 mg/mL (open squares) respectively. (B) The linear low-q regions of the scattering curves used for the Guinier analysis. (C) Kratky analysis performed on the data sets at two different concentrations. (D) Normalized inter-atomic pairwise distribution function, P(r), calculated for the two concentrations used in the analysis, showing a maximum particle size of about 115 Å. (E and F) Fitting of the experimental SAXS curve at 2 mg/mL (open circle) with the structure of NLRC4 in two different conformations, open conformation (derived from PDB 3jbl) and closed conformation (derived from PDB 4kxf); the residual difference between the experimental and the calculated values of Log[I(q)] are reported for both fits. In both cases the fitting structures were truncated to residues 93–793 which represent the region that is homologous to the NLRP1 construct used for the SAXS analysis.</p

The biophysical characterisation and SAXS analysis of human NLRP1 uncover a new level of complexity of NLR proteins

NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs

Preliminary biophysical characterisation of recombinant NLRP1(227–990).

<p>(A) Size exclusion chromatography profile of the construct NLRP1(227–990) on a S200 XK16/60 column. Black arrow heads indicate the retention volumes of molecular weight standards. The sample migrates as a single species with an apparent molecular weight between 44–158 kDa. (B) Far-UV circular dichroism spectrum of NLRP1(227–990). (C) Thermal unfolding of NLRP1(227–990) obtained by following the CD signal at 222 nm as a function of the temperature increased at 2°C/min, the value of the mid-point transition is 54.3±0.5°C.</p