Part:BBa_K5209001

Cys476 to Gly mutation in HSA for improved NIR-II dye binding

Sequence and Features

Assembly Compatibility:

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  COMPATIBLE WITH RFC[12]
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  COMPATIBLE WITH RFC[21]
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  COMPATIBLE WITH RFC[23]
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  COMPATIBLE WITH RFC[1000]

Brief Description

Optimized NIR-II Fluorescent Probe Based on C476G Mutant Human Serum Albumin for Enhanced Biomedical Imaging.

Description

This part is a mutant form of Human Serum Albumin (HSA) where the cysteine residue at position 476 is mutated to glycine (C476G). This mutation enhances the covalent binding efficiency of HSA with a series of near-infrared II (NIR-II) dyes, including the Cn-1080 family. By disrupting the disulfide bond between Cys476 and Cys487, a reactive thiol group (-SH) near Cys477 is exposed, increasing the accessibility of the binding site. Encapsulating NIR-II dyes using this mutant protein prevents protein corona formation and controls the metabolism of free dyes in vivo, leading to extended circulation time and improved imaging performance.

Part_Fig 1: Schematic diagram of the binding and purification process of HSA mutant protein with CO-1080.

This mutant protein significantly advances NIR-II imaging by enhancing dye stability and imaging capabilities, making it ideal for deep tissue imaging in biological environments.

Function

The C476G mutation disrupts the disulfide bond between Cys476 and Cys487, exposing the thiol group (-SH) near Cys477, which accelerates the covalent binding between HSA and NIR-II dyes (such as CO-1080 and other Cn-1080 dye molecules). At room temperature, co-incubation of the dye and the mutated protein efficiently forms a protein-encapsulated probe complex. This mutation fundamentally improves the binding efficiency between the dye and the protein, making the application of the probe molecules in medical diagnostics or biological imaging in research more efficient and stable.

Computational simulation:

To investigate how site-directed mutations in HSA improve its binding affinity to the CO-1080 dye, we conducted molecular docking simulations. The focus was on evaluating the binding of wild-type HSA and several recombinant HSA mutants, particularly rHSA-C476G, with CO-1080. Theoretical simulations using the Glide docking mode predicted the binding interactions between wild-type HSA and rHSA-C476G with CO-1080. Part_Fig 1A shows these simulation results, highlighting the significant differences in binding between the mutated protein (rHSA-C476G) and CO-1080 compared to the wild-type.

Part_Fig 2: Simulation of wild-type HSA and several rHSAs binding to CO-1080

By comparing docking scores, Part_Fig 1B presents the non-covalent binding interactions between CO-1080 and various HSA mutants. The results showed that rHSA-C476G had a significantly better docking score than wild-type HSA, indicating that the C476G mutation improved the binding affinity with CO-1080. Additionally, as shown in Part_Fig 1C, the binding energy of rHSA-C476G with CO-1080 was significantly higher than that of other mutants, further proving that the enhanced binding was due to the breaking of the disulfide bond and the exposure of free –SH groups.

These findings reveal the key role of protein mutations in improving probe binding, particularly the Cys476 mutation, which increased the number of binding sites by breaking the disulfide bond, making it easier for CO-1080 to bind to the protein. The improvement in binding energy and docking scores provides a solid theoretical foundation for the enhanced NIR-II imaging performance of the probe.

Experimental Validation:

Following the docking simulations, we produced recombinant HSA (rHSA) with the Cys476-to-Gly mutation using site-directed mutagenesis (SDM). Experimental validation confirmed that the C476G mutation led to an expansion of the protein cavity, which facilitated easier access of CO-1080 to the binding site. The mutation also exposed free –SH groups that accelerated the covalent binding reaction between CO-1080 and rHSA, particularly under mild conditions at room temperature. These findings confirmed the success of the C476G mutation in improving both non-covalent and covalent binding interactions.

Part_Fig 3: Gel electrophoresis results of CO-1080@albumin incubated at room temperature for 2 h

Usage

The C476G mutant of Human Serum Albumin (rHSA-C476G) efficiently forms covalent bonds with NIR-II dyes, such as Cn-1080 (including CO-1080), creating stable protein-dye complexes under mild conditions. This incubation process, which can be performed at room temperature, greatly simplifies the application of NIR-II imaging probes in a variety of research and diagnostic settings.

• Simplicity of Protein-Dye Conjugation: Researchers can easily incubate the C476G mutant protein with NIR-II dyes at room temperature, without the need for heating. This streamlined process significantly reduces experimental complexity, improves reproducibility across labs, and facilitates high-throughput screening and large-scale applications.
• Rapid and Efficient Covalent Binding: The C476G mutation significantly accelerates the covalent bonding between HSA and NIR-II dyes, reducing probe preparation time. This feature is particularly beneficial for studies that require rapid probe preparation and long-term stability, improving overall experimental efficiency and the reliability of imaging results.

Protocol Section

To validate the use of recombinant albumin in NIR-II imaging applications, we take the assessment of blood-testis barrier (BTB) integrity as an example. By selecting rHSA-C476G as the carrier protein for the NIR-II dye, we constructed fluorescent probes (FPs) to evaluate BTB disruption. These probes, based on the Cn-1080 family of dyes, were incubated with the recombinant protein at room temperature for 2 hours. Following incubation, NIR-II whole-body imaging and testicular imaging were performed, showing that the CO-1080@rHSA complex demonstrated superior brightness and TSR (testis-skin ratio) in BTB-damaged mice compared to controls. This simple protocol allowed us to rapidly assess BTB integrity using the optimized protein-dye complex, highlighting the ease of preparation and strong imaging performance.

NIR-II FPs based on recombinant albumin under mild conditions for rapidly assessing BTB disruption

Considering both non-covalent affinity and covalent binding ability, we ultimately chose rHSA-C476G, the optimal recombinant HSA, to construct the FPs for assessing BTB integrity (Part_Fig 4A). With the mutation of Cys476 to Gly476, the modified protein cavity acted as an improved microreactor that covalently bound to CO-1080 under mild conditions. Here, BTB-damaged mice injected with CO-1080@rHSA were used as the treated group, while both BTB-damaged and normal mice injected with CO-1080@HSA were classified as the control groups. All the FPs were incubated at room temperature for 2 h. NIR-II whole-body imaging and testicular imaging of the treated and control groups revealed that CO-1080@rHSA outperformed CO-1080@HSA in assessing BTB integrity, both in terms of brightness and TSR (Part_Fig 4, B to E, and Part_Fig. 5). Notably, the brightness of BTB-damaged testes showed a statistically significant difference from that of normal testes at early as 1 h after CO-1080@rHSA injection. Similar to the effect observed with CO-1080@HSA incubated at 60 °C for 2 h in testicular imaging, there was no overlap in the brightness of BTB-damaged and normal testes at 6 h of CO-1080 injection, when the TSR reached 4.3. CO-1080@rHSA retained the advantage of NIR-II FPs based on exogenous albumin for rapidly assessing BTB integrity and had similar brightness and TSR to CO-1080@HSA (incubated at 60 °C for 2 h) for in vivo imaging. Notably, it is unique advantage lies in its ability to be prepared by simple mixing of CO-1080 and recombinant HSA without the need for heating.

Part_Fig4. 1 Optimized recombinant HSAconstructed by site-directed mutagenesis strategy for efficiently assessing BTB integrity. (A) Schematic of the construction of rHSA-C476G based on genetic recombination technology. Comparison of (B) NIR-II whole-body imaging and (C) testicular imaging in BTB-damaged mice injected with CO-1080@rHSA as the treated group, BTB-damaged mice injected with CO-1080@HSA and normal mice injected with CO-1080@HSA as control groups at specific time points post-injection (>1200 nm collection, 200 ms, n=3 for each group). (D) Brightness measurement of testis and skin of the treated and control groups at various time points post-injection (>1200 nm, 200 ms, n=6 for each group). (E) Statistical analysis of quantitative differences on the testis and skin of treated and control groups at various time points post-injection and comparison of significant differences in the effectiveness of CO-1080@rHSA and CO-1080 on identifying BTB disruption at 6 h after injection (mean ± SD, n=6 for each group). *P &lf 0.05, **P &lf 0.01, ***P &lf 0.001, ****P &lf 0.0001. Some schematic diagrams were designed using BioRender software. The protein structure was generated by the Protein Data Bank (PDB). Here, CO-1080@rHSA and CO-1080@HSA for all NIR-II bioimaging was incubated at room temperature for 2 h and the injection dose was 200 μL (200 μM).

Part_Fig 5. Statistical analysis of quantitative differences on the testis and skin at various time points post-injection and comparison of significant differences in the effectiveness of CO-1080@rHSA and CO-1080 on identifying BTB disruption (mean ± SD, n=6 for each group). *P &lf 0.05, **P &lf 0.01, ***P &lf 0.001, ****P &lf 0.0001.

Application

The C476G mutant of HSA (rHSA-C476G) is designed for applications where efficient covalent binding of NIR-II dyes to proteins is required, specifically enhancing the probe's performance in biomedical imaging and diagnostics. By facilitating the formation of stable protein-dye complexes at room temperature, this mutant protein significantly improves the efficiency and reliability of NIR-II dye-based imaging probes in the following areas:

• Biomedical Imaging: The protein-dye complex formed with rHSA-C476G can be used in deep tissue imaging, especially for applications involving the blood-testis barrier (BTB) and blood-brain barrier (BBB). The ability to bind Cn-1080 dye molecules (such as CO-1080) rapidly and covalently under mild conditions extends the practical use of the probe in clinical diagnostics, allowing for enhanced visualization of tissues and organs that are challenging to image with traditional techniques.
• Tumor Surgery Navigation and Rapid Detection: The rHSA-C476G-based NIR-II probes can assist surgeons in identifying tumor boundaries in real-time, facilitating the detection of tumor-infiltrating lymph nodes and helping to ensure complete tumor resection. This technology is valuable in surgical procedures, improving precision and outcomes by providing clear, high-resolution imaging during operations.
• Neurological Disease Diagnosis: The mutant protein can be used to improve diagnostic imaging for neurological conditions such as stroke. By enhancing the performance of NIR-II dyes in imaging the blood-brain barrier (BBB), it enables earlier and more accurate diagnosis of neurological diseases. The probe’s rapid covalent binding capability allows for efficient imaging in animal models, offering insights for clinical applications.
• Lymphatic and Vascular Imaging: This protein-dye complex can enhance the imaging of the lymphatic system and vascular structures, making it valuable for diagnosing cardiovascular and lymphatic system diseases. The long circulation time of the probe enables detailed imaging of these systems, providing critical information for diagnosis and treatment planning.
• Basic Biomedical Research: rHSA-C476G-based probes are suitable for high-resolution imaging in live animal studies. These probes aid researchers in understanding complex biological processes, including disease mechanisms, drug interactions, and treatment effects, by providing real-time, in vivo imaging capabilities.
• Fluorescence-Guided Surgery: The rHSA-C476G-based NIR-II probes are ideal for fluorescence-guided surgery, providing real-time, high-resolution imaging to help surgeons accurately identify tumor margins and resect tissues. These probes also assist in detecting sentinel lymph nodes and small metastatic regions, improving surgical precision and reducing tumor recurrence. The room temperature preparation of the probe makes it easy to use in clinical settings, especially for cancers such as liver, pancreatic, ovarian, and cervical cancers.

These applications were identified through discussions with multiple departments at the First Hospital of Jilin University and other clinical experts from hospitals nationwide.

In all of these applications, the C476G mutation in HSA improves the probe’s long-term stability and effectiveness, offering reliable performance in biomedical imaging, surgical guidance, and diagnostic procedures. Additionally, the protocol for creating the protein-dye complex is straightforward, requiring only room temperature incubation, making it accessible and easy to replicate for future research and clinical uses.