Our Part Contributions: --cp157 Venus, LuxB: cp157 Venus, EL Linker--
LuxB:cp157 Venus
To optimize the spatial arrangement of the donor acceptor Kaku, T., Sugiura, K., Entani, T. et al. used circularly permuted Venus variants such ad (cp50Venus,cp157Venus,cp173Venus, cp195Venus, and cp229Venus)18,19. The highest BRET efficiency was observed when cp157Venus was fused to the C-terminus of luxB (Fig.a). The brightness of luxB:cp157Venus + luxA was about ten times higher than that of luxA + luxB (Fig.b).
Bacterial luciferase has a quantum yield of about 0.1-0.16, while Venus has a quantum yield of around 0.618. Since cp157Venus is similarly bright to the regular Venus, a maximum of six times enhancement in brightness through BRET was expected. However, when the researchers fused luciferase to cp157Venus, they observed a ten times enhancement, which was more than anticipated. This unexpected increase might be due to changes in luciferase's properties from the fusion with cp157Venus. To understand why this happened and how to improve brightness even more, mathematical modeling like Michaelis-Menten kinetics should be applied to determine a Michaelis constant, and docking should be conducted.
cp157 Venus
Circular Permutation involves ligating a protein sequence at the selected points to create two new ends. The two original ends will be attached to create a new linear sequence, containing the original protein domains in a different order. For instance, if the original sequence is A-B-C-D and the cut points are between A and B and between C and D, the new sequence might become C-D-A-B.
New termini were introduced into surface-exposed loop regions of the beta-barrel. cp49Venus, cp157Venus, cp173Venus, cp195Venus, and cp229Venus were given new N termini at Thr-49, Gln-157, Asp-173, Leu-195, and Ile-229, respectively. These circulatory permutated proteins have used cyan and yellow fluorescent proteins (CFP and YFP) as FRET donors and acceptors so it makes sense that luciferase and cp157 would work well together as BRET.
YCs, or yellow cameleon sensors, are categorized into different groups based on their calcium (Ca²⁺) sensing parts. For instance, YC2 has a complete calcium-binding molecule called CaM, which means it can grab calcium well. In contrast, YC3 and YC4 don’t bind calcium as strongly because they have some mutations in the parts of CaM that usually grab onto calcium. Despite calcium not being of interest, making a cpFP from Venus has resulted in improved fluorescence within BRET.
A more thorough approach to improving calcium was taken by Nagai et. Al through the use of a circularly permuted GFP (which we call cpGFP).CYFPs were chosen as they withstand acidic conditions well and mature quickly. The investigators' goal was to change the way the two parts of the protein are oriented, specifically how their transition dipoles (which are their light-absorbing parts) relate to each other.
To make these sensors better at working in acidic environments, scientists have swapped the original yellow fluorescent protein (YFP) with a more stable version called EYFP. This has led to improved sensors, like YC2.1 and YC3.1. Plus, some YCs have been tweaked to mature faster by using brighter versions of YFP, such as citrine or Venus.
Special forwards and backward primers are required to go through the assembly process to make this happen. There is also a GGSGG linker that is added in between the ligated protein domains.
cpFP's
Circularly permuted fluorescent proteins (cpFPs) have been used to develop biosensors that can monitor various intracellular events. When the N-terminus and C-terminus, are connected via a peptide linker new ends are created in a different part of the protein, close to the chromophore, the part that makes it glow. This enhanced the mobility and spectral properties of cpFPs. Mobility refers to the flexibility or movement of certain parts of the protein, particularly around the chromophore [1]. Increased mobility can allow the protein to change shape more easily, which can affect how it interacts with light or other molecules. Spectral properties describe how the protein absorbs and emits light. This includes the wavelengths of light it absorbs (excitation) and the wavelengths it emits as fluorescence (emission).
Autofluorescent proteins or aFPs are proteins that can spontaneously form fluorescent chromophores without the need for additional cofactors, enzymes, or external substances. An example of this is the commonly used reporter protein GFP from Adquoera Victoria. aFPs are widely applicable because they can auto-catalytically form chromophores without additional factors, enabling their use in modern analytical techniques to visualize structures and processes in living cells.
Genetically encoded fluorescent indicators (GEFIs) based on aFPs are a promising tool for visualizing and quantifying enzymatic activity, protein conformational changes, and molecular concentrations in vivo. These chimeric constructs rely on at least one FP whose optical properties are influenced by a cellular parameter of interest, converting biochemical events into visible signals detectable by standard optical equipment. However, GEFIs face limitations due to the rigid structure of aFPs, which shields the chromophore from environmental changes, leading to small response amplitudes when the sensory units show limited conformational shifts.
Circularly permuted fluorescent protein (cpFP)-based sensors offer a solution to these issues. This approach improves sensor performance, and the probes are classified by the analytes measured and their color. GFP-like proteins have a rigid β-barrel structure with 11 β-sheets that house an internal distorted helix, resistant to conformational changes. Circular permutation involves joining the natural termini of a protein with a peptide linker and creating new termini elsewhere in the sequence, initially used to study protein folding.
Biosensors are often created by inserting cpFPs into flexible regions of sensory domains or between interacting domains. Conformational changes in the sensory domain, triggered by ligand binding or cellular changes, are transmitted to the cpFP, altering the chromophore's environment and fluorescence. This is the case through BRET within the Lux B: Venus mutation.
These studies revealed that protein folding depends more on the amino acid sequence than the natural termini's location. Despite the rigid β-barrel of FPs, studies demonstrated that circularly permuted fluorescent proteins (cpFPs) can retain their optical properties. Permutants often exhibit greater pH sensitivity, and longer maturation times, and require lower folding temperatures due to increased structural flexibility and chromophore accessibility. One study showed that ECFP, EGFP, and EYFP can accommodate calmodulin insertion near the chromophore, enhancing their functionality.
Venus Mutations
General
The maturation process of fluorescent proteins is approximately ~3 h at room temperature and its efficiency further decreases at 37 °C, hindering the use of GFP in some biological applications. To improve maturation and performance at 37 °C, mutations such as F99S, M153T, and V163A have been introduced, creating valuable GFP variants. In the case of our project, if using a eukaryotic chassis, a double confirmation system for the presence of Fusarium Oxysporum spp. Cubense is in place with the condition that the temperature of the ecosystem reaches 37 °C as that is the peak in growth of the fungus. If the protein denatures at this temperature, a positive report of the fungus would not occur within the conditions that it is most likely to be there.
Specific
| Mutation | Original Venus Composition | Replacement | Importance | Location |
|---|---|---|---|---|
| F46L | Composed of F46 and F64, two phenyl rings, stacked together; its structure is tense, making it harder to fold | Replaces phenylalanine F46 with leucine L46 | Accelerates protein maturation and facilitates protein folding as rigidity is released. Accelerates the oxidation of the Ca-Cβ bond of Tyr66 during chromophore formation | Position 46 (affects interactions between 42, 44, 65) |
| F64L | Composed of F46 and F64, two phenyl rings, stacked together; its structure is tense, making it harder to fold | Phenylalanine F64 with leucine L64 | Improves stability and reduces halide ion sensitivity. Causes a shift in the central helix and chromophore | Position 64 (affects neighboring residues: Tyrosine 145, Tyrosine 203, Leucine 220, Glutamic Acid 222) |
| M153T | N/A | Methionine 153 with Threonine 153 | Encourages protein maturation and folding; reduces the protein’s sensitivity to chlorine ions | Position 153, in the loop region between strands G and H |
| V163A | N/A | Valine 163 with Alanine 163 | Same as M153T | Position 163, in the loop region between strands G and H |
| S175G | Forms a three-centered hydrogen through bonds between the side chain carboxyl oxygen atom of Asp173 and the amide nitrogens of Gly174 and Ser175 | Serine 175 with glycine 175 | Significantly alters the structure of the backbone in residues 173 to 175 | Position 175 between strands H and I, and causes conformational changes in residues 173 to 175 |
These mutations were then used to engineer a brand-new version of the cp157 protein.
cp157 protein engineering
The cp157 part was made using a reference from the Addgene LuxB: Venus bacterial luciferase expression plasmid. The sequence map was downloaded and cp157 was identified and the circularly permuted protein was broken down into its domain and placed in its original order so it lined up the the mutated Venus sequence. The mutations were then changed on an amino acid level. The protein domains were once again circularly permuted at AA157 and the order of the protein domains was changed to the advantageous configuration. This was then turned into a nucleotide sequence and plugged into the circuit on benchling.
Key Considerations for Lux B: Venus Linker Design
The linker should be of optimal length to maintain the donor and acceptor within the critical proximity range (less than 10 nm) necessary for BRET to occur. Typically, linkers of 10-15 amino acids are used, but this can vary depending on the specific proteins involved.
Glutamic acid-leucine linkers are specific sequences of amino acids used in protein engineering and biosensor design. One of the key benefits of these linkers is their flexibility. The combination of glutamic acid, which is negatively charged, and leucine, which is hydrophobic, creates a linker that allows connected parts of a protein to move freely. This flexibility is crucial because it helps proteins change shape when needed, which is essential for their function. When proteins can adjust their shape easily, they can interact more effectively with other molecules in their environment.
Another advantage of glutamic acid-leucine linkers is their ability to improve the stability of the proteins they connect. Glutamic acid can help stabilize the linker through hydrogen bonds and ionic interactions, while leucine contributes to the protein's overall hydrophobic stability. This stability is important because it ensures that the proteins remain intact and functional in various biological conditions. Additionally, the presence of glutamic acid, which is hydrophilic, can enhance the solubility of the linker in watery environments. This means the proteins can remain dissolved and active in the biological systems where they are used.
Finally, glutamic acid-leucine linkers can facilitate interactions between different protein domains, enhancing the overall function of the engineered protein. Their flexible nature helps reduce steric hindrance, allowing for better access to binding sites and improved interactions with target molecules.
The EL linker improves energy transfer by optimizing the spatial orientation between the donor (luciferase) and acceptor (fluorophore), resulting in a stronger BRET signal that enables the detection of protein interactions at lower concentrations. Additionally, it enhances the signal-to-background ratio by reducing background noise, leading to clearer and longer-lasting signals. This combination significantly increases the sensitivity of the assay.
References
Kaku, Tomomi, et al. “Enhanced Brightness of Bacterial Luciferase by Bioluminescence Resonance Energy Transfer.” Scientific Reports, vol. 11, no. 1, 22 July 2021,https://doi.org/10.1038/s41598-021-94551-4
Samant, Shalaka. “Tech Note: The Latest on Linkers for Recombinant Fusion Proteins.” Kbdna.com, 8 May 2020,www.kbdna.com/publishinglab/lnkr