Two GlnBP double cysteine variants were generated by site-directed mutagenesis, allowing the insertion of two cysteine residues into GlnBP at positions (V111C – G192C) and (T59C – T130C), separately. Escherichia coli BL21-pLysS cells were freshly transformed with the plasmid carrying the coding sequence for GlnBP WT or a GlnBP variant, and grown in 2 L LB medium (100 mg/mL Kanamycin and 50 mg/mL chloramphenicol) at 37 °C under aerobic conditions. At an OD_600nm of 0.6-0.8, overexpression of the proteins of interest was induced upon addition of 1 mM IPTG to the culture media. The cells were further grown for 1.5-2.0 hours after induction and then harvested by centrifugation for 20 minutes at 1,529 g (Beckman, JA10) at 4 °C. All subsequent operations were carried out at 4 °C, and all solutions were stored at 4 °C. Cell pellets from 2 L culture were collected in a 50 mL falcon and resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 1 M KCl, 10 mM imidazole, 10% glycerol) with 1 mM dithiothreitol (DTT). At 4 °C, the falcon was gently shaken overnight.

Cells were disrupted by sonication (Branson tip sonication; amplitude: 25%; 10 min; 0.5 s on-off pulses; temperature was kept low by the use of an ice-water bath). Centrifugation was used to fractionate the cell lysate (at 4 °C for 30 min at 4,416 g, Eppendorf, Centrifuge 5804 R) and at 4 °C for 1 hour for ultracentrifugation (70,658 g, Beckman, Type 70Ti) in vacuum, and the pellet was discarded. The protein was purified by affinity chromatography using the Ni²⁺-Sepharose fast flow resin (GE Healthcare), pre-equilibrated with 10 column volumes of buffer A containing 1 mM DTT and gravity loaded with the supernatant from the preceding ultra-centrifugation step. The resin-bound protein was washed with 10 column volumes of buffer A containing 1 mM DTT, followed by buffer B containing 1 mM DTT (50 mM Tris-HCl, pH 8,0, KCl 50 mM, imidazole 20 mM, glycerol 10%), and finally eluted in buffer C (50 mM Tris-HCl, pH 8.0, KCl 50 mM, imidazole 250 mM, glycerol 10%) with 1 mM DTT. The eluted sample was concentrated (Vivaspin6 columns, 10 kDa MWCO, 6 mg/mL), dialyzed against PBS buffer supplemented with 1 mM DTT, and stirred gently at 4 °C overnight. SDS-PAGE was used to quantify the yield of protein overexpression and purification (Comassie staining). The absorbance at 280 nm was used to estimate the protein concentration (knowing the molar extinction coefficient of GlnBP ∼25,900 M^-1 cm^-1). The protein was then split into aliquots and kept at a temperature of -20 °C. All proteins were further purified using size-exclusion chromatography (ÄKTA pure system, Superdex 75 Increase 10/300 GL, GE Healthcare). The purified protein was split into aliquots and stored at -80 °C prior to the measurements.

The stock concentrations of GlnBP variants were estimated at about 6 mg/mL. Each GlnBP variant was thawed from -80 °C, then the protein was diluted to a final concentration of 3-4 μM (final volume of ∼20 mL) in the unfolding buffer (PBS buffer) containing 6 M guanidine hydrochloride (GndHCl). Subsequently, the solution was incubated for 3 hours under gentle stirring at ambient temperature. Next, the unfolded GlnBP variants were centrifuged (3,046 g, 30 min at 4 °C) to remove insoluble aggregates which could act as nuclei to trigger aggregation during refolding process. A Snakeskin TM dialysis membrane was prepared (pre-cooled at 4 °C and soaked in refolding buffer - PBS buffer with 1 mM DTT, pH 7.4 - for 2 min). The GlnBP variants were transferred into the dialysis tubing, which were sealed tightly afterwards by double-knots and clips at each end. The unfolded GlnBP variant was refolded by a two-step dialysis, in the presence of a total 200-fold excess of refolding buffer. First, each protein was dialyzed against 2 L refolding buffer overnight under gentle stirring at 4 °C. Then, buffer was exchanged with additional 2 L refolding buffer for another day at 4 °C. The refolded protein was then concentrated from 20 mL to final 500 μL (Vivaspin 10 kDa MWCO; 3,000 g × 15 min at 4 °C) and further purified by size-exclusion chromatography (ÄKTA pure system, Superdex-75 Increase 10/300 GL, GE Healthcare). The unfolding and refolding process for GlnBP WT was conducted under the same conditions as described for the GlnBP variants.

The ITC measurements were performed in a MicroCal PEAQ-ITC isothermal titration calorimeter (Malvern Instruments). The prediction ITC software “MicroCal PEAQ-ITC Control” was employed for designing and conducting the experiments. Once the K_d value and the binding stoichiometry (N) were assigned as predefined values, the concentration of both the protein and the titrant (ligand) stock solutions could be calculated by the “design-experiment” function on the software to get an optimal sigmoidal one-site binding curve. GlnBP concentration was assessed using the Nanophotometer (N60 Touch, Implen GmbH) with at least three reading repeats to get accurate determinations of concentration values. For all ITC measurements, the temperature was set at 25 °C with stirring speed at 750 rev / min. The GlnBPs solution (10 μM in PBS buffer pH 7.4, 300 μL) was manually loaded into the sample cell. The titrant (L-Glutamine, 100 μM in PBS buffer, pH 7.4) was automatically loaded into the titration syringe and injected in the sample cell with a titration speed of 2 μL every 150 second and a total of 19 injections. As a control experiment, L-Glutamine was titrated into the sample cell containing PBS buffer without GlnBPs. All the titration data were analyzed using the MicroCal PEAQ-ITC Analysis Software.

SPR assays were performed on a Biacore T200 (Cytiva) using a CM5 Series S carboxymethyl dextran sensor chip coated with His-antibodies from the Biacore His-capture kit (Cytiva). Briefly, the chips were equilibrated with running buffer until the dextran matrix was swollen. Afterwards, two flow cells of the sensor chip were activated with a 1:1 mixture of N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide according to the standard amine coupling protocol. A final concentration of 50 μg/mL anti-histidine antibody in 10 mM acetate buffer pH 4.5 was loaded onto both flow cells using a contact time of 420 s for gaining a density of approximately 10,000 resonance units (RU) on the surface. By injection of 1 M ethanolamine/HCl pH 8.0, free binding sites of the flow cells were saturated. Preparation of chip surfaces was carried out at a flow rate of 10 μL/min. All experiments were carried out at a constant temperature of 25 °C using PBS buffer (0.01 M phosphate buffer, 2.7 mM KCl, 0.137 M NaCl, pH 7.4) supplemented with 0.05 % (v/v) detergent P20 as running buffer.

For interaction analysis, GlnBP-6His (1.5 μM) was captured onto one flow cell using a contact time of 240 s at a constant flow rate of 10 μL/min. This resulted in a capture density of approximately 1,200 RU of GlnBP-6His. Eight different concentrations of glutamine (7.8, 15.6, 31.25, 62.5, 125, 250, 500 and 1,000 nM) were injected onto both flow cells using an association time of 50 s and a dissociation time of 360 s. The flow rate was kept constant at 30 μL/min. As control, running buffer was injected. The chip was regenerated after each cycle by removing GlnBP-6His completely from the surface using 10 mM glycine pH 1.5 for 60 s at a flow rate of 30 μL/min.

Sensorgrams were recorded using the Biacore T200 Control software 2.0.2. The surface of flow cell 1 was not coated with GlnBP-6His and used to obtain blank sensorgrams for subtraction of the bulk refractive index background with the Biacore T200 Evaluation software 3.1. The referenced sensorgrams were normalized to a baseline of 0. Peaks in the sensorgrams at the beginning and the end of the injection are due to the run-time difference between the flow cells for each chip.

In total, 26 SPR sensorgrams in three sets of measurements were recorded. To correct for remaining drift in the sensorgrams, the initial 60 s of the sensorgrams prior to Gln injection and the last 100 s of the dissociation phase where first fitted with an exponential function, which was subtracted from the sensorgrams. The drift-corrected sensorgrams were fitted to the reaction scheme of Eq. (1) based on the differential equations^{68, 101}. where [L]_bulk = [Gln] and [L]_surf are the free glutamine concentrations in the bulk flow and at the sensor surface, [P]_tot is the total concentration of surface-immobilized protein, and [PL] is the concentration of bound protein complexes. Conversion to the SPR binding response r via [PL]=α r and [P]_tot=α r_max leads to fit results for the binding rate constants that are insensitive to the (unknown) conversion factor α, which can be understood from the fact that the quasi-steady-approximation d[L]_surf⁄dt ≈ 0 holds for SPR setups^{68, 101}. The association phases of the sensorgrams were fitted with initial conditions [L]_surf=0 and r=0 and fit parameters k_t and r_max at different values of k_on after substitution of k_off by K_d k_on. Prior to these fits with fixed k_on, a remaining small vertical off-set of the sensorgrams was determined as additional fit parameter in fits with unconstrained, large k_on and subtracted from the sensorgrams. The first 50 s of the dissociation phases were fitted with single fit parameter k_t for the initial conditions [L]_surf=[L]_bulk and r=r_max⁄(1 + K_d⁄[L]_bulk), with r_max determined from fits of the association phase of the sensorgram for unconstrained, large k_on. Background-corrected sensorgrams that do not reach binding equilibrium in the association phase (because of small [Gln]), still show marked drifts in binding equilibrium, or do not resolve the initial increase of the binding signal of the association phase (because of large [Gln]) were discarded, which leads to the 13 sensorgrams of Figures 6B,C and S17 with fit results for α = 1 µM/RU. Fits with e.g., α = 1 mM/RU (not shown) lead to practically identical results. All fits were conducted with Mathematica 13 based on the functions ParametricNDSolveValue to obtain numerical solutions of the differential equations and NonlinearModelFit for fitting parameters of these solutions.

The refolded GlnBP(111C-192C) and GlnBP(59C-130C) variants were labeled with commercial maleimide derivatives of AF555/AF647 or ATTO 532/ATTO 643⁹⁵, and then purified by SEC. The chromatogram of refolded GlnBP(111C-192C) labeled with AF555/AF647 is shown in Figure 2B, and those of all other variants and dye labeling combinations are displayed in Figure S2. First, the His-tagged protein was incubated in 10 mM DTT in PBS buffer for 30 min to reduce all oxidized cysteine residues. Subsequently, the protein was diluted 10 times with PBS buffer and immobilized on a Nickel Sepharose 6 Fast Flow resin (GE Healthcare). The resin was washed extensively with milliQ water followed by PBS buffer pH 7.4. To remove the excess of DTT, the resin was washed with PBS buffer. The protein was left on the resin and incubated overnight at 4 °C with 5-10 times molar dye excess in PBS buffer pH 7.4. Subsequently, the unreacted fluorophores were removed by washing the resin with 6 mL of PBS buffer. Bound proteins were eluted with 800 μL of elution buffer (PBS buffer, pH 7.4 400 mM Imidazole) The labeled protein was further purified by size-exclusion chromatography (ÄKTA pure, Superdex-75 Increase 10/300 GL, GE Healthcare) to eliminate remaining fluorophores and remove other contaminants and soluble aggregates. The selected elution fractions were used without further treatment for smFRET experiments as described below. In general all experiments were carried out at room temperature using 25–50 pM of double-labeled GlnBP protein in PBS buffer (pH7.4). Titration experiments were performed by adding specific concentrations of ligand (glutamine) to the buffer.

Single-molecule μsALEX experiments were carried out at room temperature on a custom-built confocal microscope. In short, alternating excitation light (50 μs period) was provided by two diode lasers operating at 532 nm (OBIS 532-100-LS, Coherent, USA) and 640 nm (OBIS 640-100-LX, Coherent, USA). Both lasers were combined by coupling them into a polarization maintaining single-mode fiber (P3-488PM-FC-2, Thorlabs, USA) and subsequently guided into the microscope objective (UplanSApo 60X/1.20W, Olympus, Germany) via a dual-edge dichroic mirror (ZT532/640rpc, Chroma, USA). In general, the 532 and 640 nm diode lasers operated at 60 and 25 μW, respectively (measured at the back aperture of the objective), unless stated otherwise. Fluorescence light was collected by the same objective, focused onto a 50 μm pinhole and separated into two spectral channels (donor and acceptor fluorescence) by a dichroic beamsplitter (H643 LPXR, AHF, Germany). Fluorescence emission was collected by two avalanche photodiodes (SPCM-AQRH-64, Excelitas) after additional filtering (donor channel: BrightLine HC 582/75 and acceptor channel: Longpass 647 LP Edge Basic, both from Semrock, USA). The detector outputs were recorded via an NI-Card (PCI-6602, National Instruments, USA) using a custom-written LabView program.

Data analysis for μsALEX was performed using an in-house written software package as described in¹⁶. Three relevant photon streams were extracted from the recorded data based on the alternation period, corresponding to donor-based donor emission F(DD), donor-based acceptor emission F(DA) and acceptor-based acceptor emission F(AA). Bursts from single-molecules were identified using published procedures⁶¹ based on an all-photon-burst-search algorithm with a threshold of 15, a time window of 500 μs and a minimum total photon number (F(DD)+D(DA)+F(AA)) of 150, unless stated otherwise in the figure caption.

For each fluorescence burst, the stoichiometries S* and apparent FRET efficiencies E* were calculated and then presented for all bursts yielding a two-dimensional (2D) histogram. Uncorrected apparent FRET efficiency, E*, monitors the proximity between the two fluorophores and is calculated according to E* = F(DA)/(F(DD)+F(DA)). Apparent stoichiometry, S*, is defined as the ratio between the overall fluorescence intensity during the green excitation period over the total fluorescence intensity during both green and red periods and describes the ratio of donor-to-acceptor fluorophores in the sample: S*=(F(DD)+F(DA)/(F(DD)+F(DA)+F(AA)). Collecting the E* and S* values of all detected bursts into a 2D E*-S* histogram yielded subpopulations that can be separated according to their E*- and S*-values. The 2D histograms were fitted using a 2D gaussian function, yielding the mean apparent FRET efficiency and its standard deviation or width of the distribution. μsALEX, assists in sorting single molecules based on their donor/acceptor dye brightness ratio (stoichiometry S*) and uncorrected mean FRET efficiency (apparent FRET E*), which can be related on the mean inter-dye distance^{95, 102}

Analysis with mpH²MM was conducted as described previously by the Lerner lab⁶³. In short, the FRET Bursts software¹⁰³ was used for detecting single-molecule photon bursts using the dual channel burst search⁶¹ AND-gate algorithm with a sliding window of m=10 photons searching for instances with an instantaneous photon rate of at least F=6 times the background rate. Afterwards, bursts of such consecutive photons were filtered to have at least 50 photons originating from donor excitation and at least 50 photons originating from acceptor excitation. In the data analysis, the photon stream was then divided into photon streams of different bursts, and a time shift was applied to acceptor excitation originating photons stream so that their arrival time range overlap with that of donor excitation originating photon streams. Optimizations were conducted with state models of increasing numbers of states, and the Viterbi algorithm was employed for calculating the integrated complete likelihood (ICL). Optimizing for larger numbers of states ceased once the ICL ceased to decrease between successively larger state models. Optimized models were manually examined, and the optimal state model selected considering the ICL and the reasonableness of the model given prior knowledge based on transition rates and the E* and S* values of the states. After selection of the most-likely state model, the corresponding most-likely state-path determined by the Viterbi algorithm was used to segment bursts into dwells and to classify burst by which states were present within each burst.

To support the idea that apo and holo state in solution match with that of the crystal structure, we performed a quantitative comparison of inter-dye distances calculated from dye accessible volumes (AV) on structural models of apo and holo protein, and those derived from the experimental smFRET results. For dye AV calculations we used the FPS method, established by the Seidel lab¹⁰⁴ (Figure S1). The experimental data were corrected for setup-dependent parameters according to refs.^{60, 96} to obtain accurate FRET values from μsALEX data. Using a Förster distance of 5.2 nm for AF555/AF647, we found good agreement, i.e., 0.3-0.5 nm deviations (and 1.0 nm in one case) between the calculated and experimentally derived inter-dye distances for both mutants (Figure S1).

Solution-based smFRET experiments were performed on a home-built dual-color confocal microscope that combines multiparameter fluorescence detection (MFD) with pulsed interleaved excitation (PIE).⁶⁶ MFD-PIE experiments have been described in detail previously.¹⁰⁵ With MFD-PIE, it is possible extract FRET efficiency, stoichiometry, fluorescence lifetime and fluorescence anisotropy information from each single-molecule burst. Correction factors including direct acceptor excitation (α), spectral crosstalk (β) and detection correction factor (γ) are also accounted for reporting accurate the FRET efficiency values.¹⁰⁶ The accurate FRET efficiency (E) can be determined from:

where F_GG, F_GR and F_RR are background-corrected fluorescence signals detected in green/ donor (G), red/acceptor (R) after donor excitation and acceptor channels, respectively.

Alternatively, the use of picosecond pulsed lasers and time-correlated single photon counting (TCSPC) electronics enable calculating FRET efficiencies from the quenching of the donor in presence of acceptor. According to the formula:

τ_D(A) is the fluorescence lifetime of the donor in presence of acceptor and τ_D(0) is the fluorescence lifetime of the donor only species. Static species can be observed on the theoretical static FRET line, which is a linear relation between E and τ_D(A). Sub-ms conformational dynamics can also be identified and judged by observing the right-shifted populations from the static FRET line.

For the measurements here, 100 pM of GlnBP labeled with ATTO 532 and ATTO 643 was placed on a BSA-passivated LabTek chamber and measured for 2 hours. The sample was excited with 532 and 640 nm pulsed lasers with a repetition rate of 26.6 MHz and laser powers of 45 and 23 μW (measured at the back aperture of the objective), respectively.

Burst-wise FCS analysis is an alternative approach to observe sub-ms conformational dynamics. In this approach, donor (DD) and acceptor (AA) signals detected from single-molecule events are cross-correlated. Thus, fluctuations in the FRET efficiencies appear as an anti-correlated signal in the donor-acceptor fluorescence cross-correlation function. Burst with sufficient photons detected in both the donor and acceptor channels were selected. A time window of 50 ms was applied around each burst. If another burst was detected within this time window, both were eliminated to ensure correlation functions that are specific to the selected bursts. All the above mentioned data analysis was done using the PIE analysis with Matlab (PAM) software package¹⁰⁷.

Biotin-streptavidin interaction was used to immobilize tagged proteins and labeled DNA on a PEG-functionalized coverslip for single molecule studies. The protein-his-tag and a biotin-NTA chelated with Ni²⁺ were used to mark GlnBP(111C-192C) labeled with maleimide modified derivatives of ATTO 532/ATTO 643, whilst DNA labeled with Cy3B/ATTO 647N was directly tagged with a biotin. To prepare a functionalized glass surface, cover slides (1.5H Marienfeld Superior) were first sonicated in MQ water for 30 min. The slides were rinsed three times with MQ water, sonicated for 30 min in HPLC-grade acetone, rinsed three times with MQ water again. Then, the slides were sonicated with 1 M KOH for 30 min, rinsed three times with MQ water and dried with a stream of nitrogen air. To remove any organic material left on the surface, the cover slides were plasma-cleaned for 15 min with oxygen. To create a mPEG/biotin–coated surface, the slides were immediately incubated in a 99:1 solution of mPEG3400-silane (abcr, AB111226) and biotin-PEG3400-silane (Laysan Bio Inc) in a Toluene solution overnight at 55 °C. After incubation, the slides were sonicated (10 min in ethanol, 10 min in MQ water), dried under nitrogen stream, and kept under vacuum. Prior to TIRF experiments, each slide was incubated with a 0.2 mg/mL streptavidin in PBS solution for 10 min utilizing Ibidi sticky-slide (18 well) for single molecule studies. PBS buffer pH7.4 was used to wash away the unbound excess of streptavidin. For GlnBP(111C-192C) immobilization, 20 nM biotin-NTA (QIAGEN) was charged with 50 nM Ni²⁺ and incubated on the slide for 10 min before rinsing away the unbound excess biotin-NTA and Ni²⁺ with PBS (this step was omitted for the labeled DNA samples). GlnBP(111C-192C) at 0.8 nM and dsDNA at 0.04 nM were incubated for 5 and 1 min, respectively. For single-molecule data collecting, imaging buffer (PBS, pH 7.4) containing 2 mM Trolox for protein. For dsDNA we used PBS buffer in combination with an oxygen scavenging system (pyranose oxidase at 3 U/mL, catalase at final concentration of 90 U/mL, and 40 mM glucose). After that, the chambers were sealed with Silicone Isolators™ Sheet Material (Grace Bio-labs). All the single-molecule investigations were done at room temperature.

Single-molecule TIRF measurements were conducted on a homebuilt microscope using an Olympus iX71 inverted microscope body. Light from a 532 nm continuous wave laser (532 nm OBIS, Coherent) was transmitted off-axis onto the back-focal plane of a microscope objective (UAPON TIRF 100X 1.49NA, Olympus) via a dualband dichroic beamsplitter (TIRF Dual Line Beamsplitter zt532/640rpc, AHF Analysetechnik) to generate total internal reflection at the glass-water interface. Fluorescent emission was then split spectrally using a Dual View System (DV2, Photometrics) equipped with a dichroic beamsplitter (zt640rdc, AHF Analysetechnik). The two emission channels were then spectrally filtered using emission filters (582/75 Brightline HC and 731/137 BrightLine HC respectively, both AHF Analysetechnik). Image series were acquired using an EMCCD camera (C9100-13, Hamamatsu) in combination with the μManager¹⁰⁸ software. The iSMS¹⁰⁹ software was used to retrieve and calculate traces of the donor and acceptor fluorescence intensity from consecutive fluorescent images.