I. Cloning and DNA Material: Constructing the Foundation for Protein Analysis
The groundwork for our protein interaction studies began with precise cloning techniques and the careful manipulation of DNA material. Key to our investigation were two phytochrome proteins: DrBphP from Deinococcus radiodurans strain R1, and Agp1 from Agrobacterium fabrum strain C58. These two phytochromes, while both belonging to the phytochrome family, represent a Comparable Opposite in terms of their source organisms and subtle differences in their functional domains, offering a rich platform for comparative analysis.
DrBphP, sourced from a bacterium known for its extreme radiation resistance, was provided as a kind gift in the pET21b(+) plasmid. Agp1, originating from a plant-associated bacterium, was supplied in the pQE12 vector. The initial availability of these plasmids significantly streamlined the research process, allowing us to focus on subsequent modifications and analyses.
A notable feature of the Agp1 sample was a spontaneous R603C mutation, located on the surface of its CA domain. This pre-existing mutation provided an interesting point of comparison against the wild-type DrBphP and further motivated the introduction of specific mutations to DrBphP. Using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit, we systematically introduced mutations into both DrBphP (H533D, E536A, E536T, E536N, E536D, E536Q, and R539A) and Agp1 (D529H and A532E). These targeted mutations were designed to probe the functional roles of specific amino acid residues and to explore the comparable opposite effects of these alterations in the two different phytochrome backgrounds.
To further investigate the interplay between these phytochromes, a chimera construct was created. This involved replacing residues 513–755 of DrBphP with the corresponding residues 509–745 from Agp1. This domain swapping strategy allowed us to examine the influence of the C-terminal region of Agp1 on the DrBphP scaffold, again highlighting the comparable opposite domain contributions to overall protein function. The process began by introducing an XhoI restriction site after DrBphP residue 512, followed by the ligation of the C-terminal Agp1 fragment. Site-directed mutagenesis was subsequently used to refine the junction site, ensuring seamless integration of the Agp1 fragment.
In parallel, response regulators from Deinococcus radiodurans strain R1 (DrRR) and Agrobacterium fabrum strain C58 (AtRR1), the natural partners of DrBphP and Agp1 respectively, were produced. These were crucial for studying the downstream signaling events mediated by the phytochromes. Cloned into pET21b(+) vectors, these response regulators were prepared as a service by Invitrogen, ensuring high quality and consistency.
To visualize and potentially track protein interactions in vivo, EGFP-RR constructs were generated using Gibson assembly cloning. This involved replacing the N-terminal T7 tag of pET21b(+) with an EGFP-C1 sequence, effectively tagging the response regulators with enhanced green fluorescent protein. A ten-residue linker (DSAGSAGSAG) was incorporated between the RR and EGFP sequences to provide flexibility and minimize steric hindrance, allowing for optimal protein folding and interaction.
Alt Text: Plasmids pET21b(+) and pQE12 containing DrBphP and Agp1 genes, respectively, illustrating DNA constructs used for cloning and mutagenesis in bacteriophytochrome research, highlighting the comparable genetic materials used for studying protein function.
II. Sample Expression and Purification: Isolating Proteins for In-depth Analysis
With the DNA constructs prepared, the next critical step was the efficient expression and purification of the proteins. This process aimed to yield high-quality, isolated protein samples necessary for subsequent biochemical and biophysical characterization, allowing us to directly compare the properties of DrBphP and Agp1 and their variants, revealing their comparable opposite behaviors in isolation.
All DrBphP variants and the response regulators were expressed in Escherichia coli strain BL21 (DE3). This strain is commonly used for protein expression due to its genetic background optimized for high-level protein production. Cultures were grown overnight at a controlled temperature of 20–24 °C to promote proper protein folding and minimize the formation of inclusion bodies.
Cell lysis was achieved using EmulsiFlex®, a high-pressure homogenizer that effectively disrupts bacterial cells, releasing the expressed proteins. For the phytochrome samples, a molar excess of biliverdin hydrochloride, the chromophore essential for phytochrome light sensing, was added to the cell lysate. This step is crucial as phytochromes require biliverdin to be functionally active. The mixture was then incubated overnight on ice to allow for efficient chromophore incorporation into the apoprotein. Notably, no external biliverdin was added during the purification of response regulators, as they do not require this chromophore for their function. This difference in cofactor requirement underscores a comparable opposite aspect of phytochromes and their response regulators.
The purification of His6-tagged proteins relied on NiNTA affinity chromatography using HisTrap™ columns. This method selectively binds proteins with a hexahistidine tag, allowing for efficient separation from other cellular components. Following affinity purification, size-exclusion chromatography (SEC) was performed using HiLoad™ 26/600 Superdex™ 200 pg columns. SEC further purifies the proteins based on their size, ensuring homogeneity and removing any aggregates or contaminants. The SEC buffer consisted of 30 mM Tris, pH 8.0, maintaining a consistent environment for protein stability.
Agp1 and its D529H mutant were expressed in NEB Express® Iq E. coli strain. While the purification protocol was largely identical to that used for other samples, a few modifications were implemented. A protease inhibitor mix (ROCHE) and 0.5 mM TCEP (Tris(2-carboxyethyl)phosphine) were included in the sample before lysis. Protease inhibitors prevent protein degradation during lysis, while TCEP is a reducing agent that maintains cysteine residues in their reduced state, preventing disulfide bond formation and aggregation. Furthermore, affinity purification for Agp1 was conducted in a slightly modified buffer (30 mM Tris/HCl, 150 mM NaCl, 1 mM TCEP) with varying imidazole concentrations (5–500 mM). The inclusion of NaCl and the wider range of imidazole concentrations were likely optimized for the specific binding properties of Agp1 to the NiNTA resin.
Finally, all purified protein samples were concentrated to 25–30 mg/ml in (30 mM Tris/HCl, pH 8.0) and flash-frozen in liquid nitrogen. Concentration ensures sufficient protein amounts for downstream experiments, while flash-freezing preserves protein integrity for long-term storage.
Alt Text: SDS-PAGE gel showing purified DrBphP protein samples, demonstrating protein purity achieved through affinity and size-exclusion chromatography, highlighting the comparable quality of protein preparations for biophysical studies.
III. Absorption Spectroscopy: Probing Light-Induced Conformational Changes
Absorption spectroscopy is a powerful technique to investigate the light-responsive nature of phytochromes. By measuring the absorption of light at different wavelengths, we can characterize the different photo states of phytochromes and monitor their transitions. This method allows us to observe the comparable opposite spectral properties of the Pr and Pfr states and how these states are influenced by protein modifications and interactions.
The dark reversion of phytochromes, the spontaneous thermal relaxation from the Pfr (far-red absorbing) to the Pr (red-absorbing) state in the dark, was specifically measured using an Agilent Cary 8454 UV-Visible spectrophotometer. Absorption spectra were recorded in the wavelength range of 690–850 nm, encompassing the absorption maxima of both Pr and Pfr states. Measurements were performed on mixtures of response regulator and Pfr-populated BphP samples to assess the impact of response regulator binding on dark reversion kinetics.
Before measurement, BphP samples were diluted to 1.0 µM in a specific buffer (25 mm Tris/HCl, pH 7.8, 5 mM MgCl2, 4 mM 2-mercaptoethanol, 5% ethylene glycol) to achieve a consistent starting absorbance value. Ten times the concentration of cognate response regulator (100 µM) was then added to the BphP sample to ensure saturation of binding. To generate the Pfr state, phytochromes were illuminated with a 665 nm LED for 3 minutes, a duration sufficient to drive the majority of the phytochrome population into the Pfr state. Data acquisition in the dark commenced immediately following illumination.
Dark reversion data were recorded over a period of two hours, with more frequent measurements taken initially (1 min intervals for the first 10 min) to capture the faster reversion kinetics, and less frequent measurements at later time points (10 min intervals towards the end). All measurements were conducted in complete darkness at ambient room temperature to eliminate any light-induced artifacts.
Steady-state spectra of Pr- and Pfr-state samples were also measured, both in the presence and absence of cognate response regulator. Pr state spectra were obtained from dark-adapted samples, while Pfr spectra were recorded after 3 minutes of 665 nm LED illumination. These steady-state spectra provide a static snapshot of the absorption properties of each state, allowing for a comparable opposite view of their spectral characteristics.
The exponential decay curves from dark reversion data were analyzed using Matlab R2019b and fitted using Eq. (1). This equation describes a multi-exponential decay, allowing for the decomposition of the dark reversion kinetics into distinct components. For DrBphP samples, a three-component fit was necessary, indicating a more complex reversion process, whereas two components were sufficient for the rest of the samples, suggesting a comparable opposite in the dark reversion complexity between different phytochromes.
IV. Size-Exclusion Chromatography (SEC): Analyzing Protein Complex Formation
Size-exclusion chromatography (SEC) was employed not only for protein purification but also as a tool to investigate protein-protein interactions, specifically the formation of complexes between phytochromes and response regulators. By comparing the elution profiles of phytochromes and their mixtures with response regulators, we can gain insights into whether they form stable complexes and how light influences these interactions, revealing a comparable opposite in complex formation under different light conditions.
SEC experiments were performed using a Superdex-200 Increase 3.2/300 column in a buffer (25 mM Tris/HCl pH 7.8, 5 mM MgCl2, 4 mM 2-mercaptoethanol, 5% ethylene glycol) consistent with the buffers used in other assays. Protein absorption was detected at both 489 nm (sensitive to the biliverdin chromophore) and 280 nm (sensitive to protein in general), providing comprehensive detection of protein elution.
To investigate light-dependent interactions, samples were prepared in both dark-adapted (Pr state) and illuminated (Pfr state) conditions. Illuminated samples (designated as R for red-light illuminated, representing the Pfr state) were pre-illuminated with 655 nm LED light for 5 minutes prior to injection to ensure a stable Pfr population. For each SEC run, a small volume (24 µl) of sample mixture (5 mg/ml each of phytochrome and response regulator) was injected, and elution was performed at a flow rate of 70 µl/min.
Molecular weight estimates were determined by calibrating the SEC column using a standard curve of marker proteins with known molecular weights (Vitamin B12, myoglobin, ovalbumin, γ-globulin, and thyroglobulin). By comparing the elution volumes of the protein samples to the standard curve, we could estimate the molecular weight of the eluting species, providing information about the oligomeric state of the proteins and the formation of complexes.
V. Surface Plasmon Resonance (SPR): Quantifying Binding Affinities
Surface plasmon resonance (SPR) is a highly sensitive technique used to study biomolecular interactions in real-time without the need for labels. It allows for the determination of binding kinetics and affinities between interacting molecules. In our study, SPR was used to quantify the binding affinity between phytochromes and response regulators, specifically comparing the interaction strengths of DrBphP and Agp1 with their cognate response regulators in both Pr and Pfr states, highlighting a comparable opposite in their binding behaviors.
For SPR measurements, phytochrome samples were dialyzed overnight into a specific running buffer (20 mM HEPES, 300 mM NaCl2, 5 mM MgCl2, 0.10% (v/v) Tween20, pH 7.5). Dialysis removes any buffer components that might interfere with SPR measurements and ensures the proteins are in the correct buffer environment. Measurements were performed using a Biacore X instrument.
Response regulators were immobilized onto carboxymethyldextran hydrogel-coated SPR Sensorchips. This was achieved by coupling each response regulator to the chip surface at a concentration of 3 mg/mL (150 µM) in an acetate buffer (20 mM sodium acetate, pH 4.2) using EDC/NHS coupling chemistry. This method covalently attaches the response regulator to the sensor chip surface. After coupling, remaining activated groups on the sensor chip were quenched with ethanolamine to prevent non-specific binding.
SPR measurements were conducted by injecting 40 µL of phytochrome sample at a flow rate of 20 µL/min, followed by a wash step with the running buffer. Phytochrome samples were either pre-illuminated with far-red (785 nm, to enrich the Pr state) or red (655 nm, to enrich the Pfr state) LED light before injection, allowing for the assessment of binding in both photo states. All measurements were performed in darkness at room temperature to prevent light-induced state changes during the measurement.
Sensorgrams, which represent the change in refractive index at the sensor chip surface over time, were analyzed using BIAevaluation-software version 4.1. Sharp peaks corresponding to injection start and stop were excluded from analysis as they are artifacts of the injection process. For kinetic analysis, a simple 1:1 interaction model was applied, and kinetic parameters (ka, association rate constant; kd, dissociation rate constant) were determined by simultaneous fitting. Steady-state binding levels (Req) were obtained by fitting a horizontal straight line to the sensorgrams and averaging the response. Req values were then plotted against phytochrome concentrations, and a nonlinear simple fit was performed using Eq. (2) to determine the dissociation constant (KD), a measure of binding affinity.
Alt Text: Surface Plasmon Resonance (SPR) sensorgram illustrating the real-time binding interaction between phytochrome and response regulator proteins, showcasing the kinetic analysis used to determine binding affinities and comparable interaction strengths.
VI. Isothermal Titration Calorimetry (ITC): Measuring Thermodynamic Binding Parameters
Isothermal titration calorimetry (ITC) is a thermodynamic technique that directly measures the heat released or absorbed upon binding between two molecules. ITC provides comprehensive thermodynamic parameters, including binding affinity (KD), enthalpy change (ΔH), and stoichiometry (n). We used ITC to further characterize the interaction between phytochromes and response regulators, comparing thermodynamic profiles for different phytochrome-response regulator pairs to understand the energetic basis of their interactions, revealing comparable opposite thermodynamic signatures of binding.
ITC measurements were conducted using a MicroCal PEAQ-ITC instrument. Purified protein samples, in 30 mM Tris/HCl pH 8.0, were diluted 1:1 with a more complex buffer (2×: 50 mM Tris/HCl pH 7.8, 10 mM MgCl2, 8 mM 2-mercaptoethanol, 10% ethylene glycol) to optimize buffer conditions for ITC. BphP (30–50 µM, 300 µL) was loaded into the sample cell, and RR (750–800 µM, 75 µL) was loaded into the injection syringe. To ensure the BphP sample was in the Pr state, it was briefly illuminated with 785 nm LED light just before application to the cell. The system was equilibrated to 25 °C with a stirring speed of 750 rpm in complete darkness.
The ITC injection scheme began with a small 0.4 µL response regulator injection, followed by 2 µL injections every 150 seconds. Each injection of response regulator into the BphP sample cell results in heat release or absorption if binding occurs. As a control, background signals were measured by injecting response regulator into buffer alone and buffer into phytochrome alone, using identical parameters.
For measurements using a Micro-200 ITC, slightly different concentrations were used (BphP: 170–250 µM; RR: 750–800 µM). The injection scheme also varied slightly, starting with a 0.2 µL injection followed by 2 µL injections every 180 seconds.
All data from triplicate experiments were analyzed using ORIGIN 7-based MicroCal PEAQ-ITC Analysis Software version 1.21. The integrated heat signals from each injection were plotted against the molar ratio of response regulator to phytochrome, and the resulting binding isotherms were fitted to a single-site binding model, excluding the first injection. The KD value, representing the binding affinity, was reported as ±SD from three independent repeats, providing statistically robust binding affinity measurements.
VII. Radiolabeled Kinase Assay: Assessing Phosphorylation Activity
The kinase activity of phytochromes, specifically their ability to phosphorylate response regulators, is a crucial aspect of their signaling mechanism. To assess this activity, a radiolabeled kinase assay was performed using [γ‐32P]ATP. This assay directly measures the transfer of the γ-phosphate from ATP to the response regulator, allowing us to quantify kinase activity and compare the activity of different phytochromes and their variants, revealing a comparable opposite in their phosphorylation efficacies.
Purified BphPs and RRs were diluted to approximate concentrations of 3.5 µM (0.3 mg/ml) and 9 µM (1.7 mg/ml), respectively, in a buffer (25 mM Tris/HCl pH 7.8, 5 mM MgCl2, 4 mM 2-mercaptoethanol, 5% ethylene glycol). Phytochromes were pre-illuminated briefly with saturating 785 nm LED light to ensure they were predominantly in the Pr state before the assay. The kinase reaction was initiated by adding 3.7 kBq of [γ‐32P]ATP in a total reaction volume of 10 µL.
Samples were incubated at 25 °C for 20 minutes either in complete darkness or under constant 655 nm LED illumination (5 mW/cm2), representing the Pr and Pfr states, respectively. The reaction was terminated by adding SDS sample buffer, which denatures the proteins and stops enzymatic activity. Samples were then separated by 12% SDS-PAGE. After electrophoresis, gels were stained with Serva Blue to visualize all proteins, followed by drying in a vacuum drier. The dried gels were then photographed, and their radioactivity was monitored using an X-ray film. The intensity of the radioactive bands corresponding to the response regulator reflects the extent of phosphorylation. The experiment was repeated three times to ensure reproducibility and allow for quantitative comparison of kinase activities.
VIII. Protein Phosphorylation by Acetyl Phosphate and Phos-Tag Detection: Analyzing Phosphorylation States
To further investigate protein phosphorylation, particularly in the context of mobility shifts upon phosphorylation, we employed acetyl phosphate as a phosphoryl donor and Phos-Tag SDS-PAGE for detection. This method allows us to visualize and compare the phosphorylation states of response regulators under different conditions, offering a comparable opposite view of their phosphorylation status in kinase and phosphatase reactions.
Response regulators (2–3 µg) were incubated with 50 mM acetyl phosphate for 30 minutes at 37 °C in a buffer (25 mM Tris/HCl pH 7.8, 5 mM MgCl2, 4 mM 2-mercaptoethanol, 5% ethylene glycol). Acetyl phosphate is a small molecule that can non-enzymatically phosphorylate response regulators, mimicking phosphorylation by a kinase. Following incubation, samples underwent buffer exchange to (30 mM Tris/HCl, pH 8.0) using Vivaspin centrifugal concentrators to remove excess acetyl phosphate. The final phosphoprotein concentrations were adjusted to 1.5 mg/ml (80 µM).
Kinase and phosphatase reactions were conducted in (25 mM Tris/HCl pH 7.8, 5 mM MgCl2, 4 mM 2-mercaptoethanol, 5% ethylene glycol). Desired proteins (2–4 µg each) were incubated in 10 µl total volume at 25 °C, with or without 1 mM ATP. Reactions were initiated by adding ATP to the mixture and incubated either in darkness or under saturating 657 nm red light. After 20–30 minutes, reactions were stopped by adding 5× SDS loading buffer.
To detect phosphorylated RR proteins based on mobility shift, Zn2+-Phos-tag® SDS-PAGE assay was used. 9% SDS-PAGE gels containing 25-µM Phos-tag acrylamide were prepared. Phos-tag binds specifically to phosphorylated proteins, causing them to migrate slower in the gel and resulting in a mobility shift. 10 µl of each reaction were run at 40 mA/gel at room temperature according to manufacturer instructions. The gels were then analyzed to visualize the mobility shifts of phosphorylated response regulators, revealing the extent of phosphorylation under different conditions.
IX. Crystallography: Determining the 3D Structure of Response Regulator
X-ray crystallography is a powerful technique to determine the three-dimensional structure of proteins at atomic resolution. Knowing the 3D structure provides invaluable insights into protein function and interactions. We crystallized DrRR to gain a structural understanding of its domain organization and potential interaction interfaces, providing a comparable opposite structural perspective to complement our biochemical data.
DrRR was crystallized using the hanging drop vapor diffusion method. Protein solution at 10 mg/ml concentration was mixed in a 1:1 ratio with reservoir solution (0.1 M HEPES pH 7.5, 0.3 M CaCl2, 25% PEG400). Crystals formed within a few days and were cryoprotected by flash-freezing in reservoir solution containing 15% glycerol.
Diffraction data were collected at beamline ID23-2 at the European Synchrotron Radiation Facility (ESRF) using 0.873 nm wavelength X-rays. Data processing was performed using the XDS program package. The crystals belonged to space group P41212, with two dimers in the asymmetric unit.
Initial phases were solved by molecular replacement using Phaser version 2.5.7. A DrRR homology model, generated using SWISS-MODEL workspace and a crystal structure of a cyanobacterial response regulator RcpA as a template, served as the search model. The structure was further refined using REFMAC version 5.8.0135 with automatic weighting and NCS restraints. Model building was performed using Coot 0.8.2. For final refinement cycles, TLS regions were implemented using the TLS Motion Determination web server. The final structure achieved Rwork/Rfree of 0.181/0.218. Data collection and refinement statistics are summarized in Table 1, and representative electron density is shown in Supplementary Figure 9. Figures of crystal structures were generated using PyMOL.
Alt Text: Crystal structure of DrRR response regulator protein determined by X-ray crystallography, illustrating the three-dimensional fold and domain arrangement, providing a structural basis for understanding protein function and comparable domain architectures.
X. Computational Modeling: Simulating Protein Interactions at the Molecular Level
Computational modeling, specifically molecular dynamics simulations, provides a complementary approach to experimental techniques, allowing us to simulate protein interactions at the atomic level and explore dynamic aspects of protein behavior. We used computational modeling to investigate the interaction interfaces between phytochromes and response regulators, comparing DrBphP/DrRR and Agp1/AtRR1 complexes and exploring the comparable opposite interaction modes predicted by simulations.
For computational simulations, DrBphP/DrRR and Agp1/AtRR1 complexes were constructed based on the crystal structure of a sensor histidine kinase HK853 and its response regulator RR468 from Thermotoga maritima. Homology models of the dimeric DHp bundle of DrBphP and Agp1 were generated using SWISS-MODEL workspace, using the corresponding DHp part of the T. maritima histidine kinase as a template. For response regulators, the crystal structures of AtRR1 and DrRR were used as dimers. Waters clashing with the interface and phosphates at active sites were removed, while Ca2+ and Mg2+ ion positions were retained and modeled with Mg2+ ions.
To assess the influence of the starting structure, we repeated the modeling procedure using a crystal structure of a T. maritima ThkA/TrrA complex as an alternative template.
Molecular dynamics simulations were performed using Gromacs 2018.8. Both Agp1/AtRR1 and DrBphP/DrRR complexes were converted into Gromacs topology, solvated in a water box, and neutralized with counterions. For DrBphP/DrRR complex, Ca2+ ions were replaced with Mg2+ ions. NaCl ions were added to achieve 0.1 M salt concentration. Amber03 forcefield was used for proteins, and TIP3P for water.
Classical molecular dynamics simulations were performed with minimization and 200 ns of NPT ensemble simulation at 300 K and 1 atm. Bond lengths were constrained using LINCS method, allowing for a 2 fs time-step. PME method was used for electrostatic interactions, and Lennard-Jones interactions were treated with a cutoff. RMSD and RMSF were calculated from the trajectories.
Starting at 100 ns, snapshots were extracted every 10 ns for interaction analysis using PISA service at the European Bioinformatics Institute. Prominent contacts were analyzed by plotting contact distances throughout the MD simulation. Models and simulation parameters are available online at GitHub repository.
XI. Sequence Analysis: Investigating Evolutionary Relationships and Covariance
Sequence analysis, including conservation and covariance analysis, provides evolutionary context and can reveal functionally important residues and interactions within proteins. By analyzing large sequence datasets of phytochromes and response regulators, we can identify conserved regions and covariating residue pairs that may be crucial for protein function and interaction specificity, highlighting comparable opposite evolutionary pressures on different protein families.
To analyze sequence conservation and covariance in sensor histidine kinases, we conducted a BLASTP search for the DHp and CA domains of DrBphP against the non-redundant protein sequence database. Using Biopython and custom Python scripts, we retrieved the top 250,000 sequence hits and clustered them at 30% identity using UCLUST. The 11,994 cluster centroid sequences, along with the original search sequence, were aligned using MUSCLE. The consensus sequence was plotted using WebLogo. Covariance analysis was performed using PSICOV, and pairwise scores above a cutoff of 0.6 were mapped onto a homology model of the DHp and CA domains of DrBphP.
Sequence analysis of response regulator proteins was performed similarly, using a BLAST search for the sequence of D. radiodurans RR. 50,000 sequences with an E-value cutoff of 2.5 × 10−10 were retrieved and clustered at 50% identity using UCLUST. The 4,338 centroid sequences, along with DrRR and AtRR1 sequences, were aligned and analyzed.
For covariance analysis between histidine kinase and response regulator, BLAST hits were scanned for proteins containing consecutive DHp, CA, and RR domains. 6,805 sequences meeting criteria were clustered at 50% identity, resulting in 5,386 centroid sequences. DrBphP and DrRR sequences were concatenated and added. All sequences were aligned and analyzed by PSICOV, and scores above 0.6 were plotted onto a structural model of the DrBphP/DrRR complex. In a control run, sequences were scrambled before PSICOV analysis to verify covariance specificity.
XII. Conclusion: Integrated Methodological Approach to Understand Protein Interactions
This comprehensive methodological overview details the diverse techniques employed to investigate the interactions between bacteriophytochromes and their cognate response regulators. From molecular cloning and protein purification to advanced biophysical and computational methods, this study exemplifies an integrated approach to unraveling the complexities of protein signaling. The use of comparable opposite protein pairs, DrBphP and Agp1, further enriches the analysis, allowing for the identification of both conserved and divergent features in phytochrome signaling mechanisms. These methods provide a robust framework for future studies aimed at dissecting the intricate world of protein-protein interactions in various biological systems.
