Bimolecular fluorescence complementation
Encyclopedia
Bimolecular fluorescence complementation (also known as BiFC) is a technology typically used to validate protein
interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein
and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure
and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.
was first reported in subtilisin-cleaved bovine pancreatic ribonuclease
, then expanded using β-galactosidase
mutants that allowed cells to grow on lactose.
Recognition of many proteins' ability to spontaneously assemble into functional complexes as well as the ability of protein fragments to assemble as a consequence of the spontaneous functional complex assembly of interaction partners to which they are fused was later reported for ubiquitin
fragments in yeast protein interactions.
In 2000, Ghosh et al developed a system that allowed a green fluorescent protein
(GFP
) to be reassembled using an anti-parallel
leucine zipper
in E. coli cells. This was achieved by dissecting GFP into C- and N-terminal GFP fragments. As the GFP fragment was attached to each leucine zipper by a linker, the heterodimerisation of the anti-parallel leucine zipper resulted in a reconstituted, or re-formed, GFP protein that could be visualised. The successful fluorescent signal indicated that the separate GFP peptide fragments were able to correctly reassemble and achieve tertiary folding
. It was, therefore, postulated that using this technique, fragmented GFP could be used to study interaction of protein–protein pairs that have their N-C termini in close proximity.
After the demonstration of successful fluorescent protein fragment reconstitution in mammalian cells, Hu et al. described the use of fragmented yellow fluorescent protein
(YFP
) in the investigation of bZIP and Rel family transcription factor
interactions. This was the first report bZIP protein interaction regulation by regions outside of the bZIP domain
, regulation of subnuclear
localization of the bZIP domains Fos and Jun
by their different interacting partners, and modulation of transcriptional activation
of bZIP and Rel proteins through mutual interactions. In addition, this study was the first report of an in vivo
technique, now known as the bimolecular fluorescence complementation (BiFC) assay, to provide insight into the structural basis of protein complex formation through detection of fluorescence caused by the assembly of fluorescent reporter protein fragments tethered to interacting proteins.
activation occurs through an autocatalytic cyclization reaction that occurs after the protein has been folded correctly. This was advanced with the successful reconstitution of the YFP fluorophore from protein fragments that had been fused to interacting proteins within 8 hours of transfection was reported in 2002.
that can be used for the fusion protein created. Transient protein expression is used to identify protein–protein interactions in vivo as well as in subcellular localisation of the BiFC complex. However, one must be cautious against protein over-expression, as this may skew both preferential localisation and the predominant protein complexes formed. Instead, weak promoters, the use of low levels of plasmid DNA in the transfection, and plasmid vectors that do not replicate in mammalian cells should be used to express proteins at or near their endogenous levels to mimic the physiological cellular environment. Also, careful selection of the fluorescent protein is important, as different fluorescent proteins require different cellular environments. For example, GFP can be used in E. coli cells, while YFP is used in mammalian cells.
Stable cell lines with the expression vector integrated into its genome allows more stable protein expression in the cell population, resulting in more consistent results.
wild-type
proteins. Finally, the addition of the fluorescent fragment fusion must not affect the biological function of the protein, preferably verified using assays that evaluate all of the proteins' known functions.
that tethers the fluorescent reporter protein fragment to the protein of interest, forming the fusion protein. When designing a linker sequence, one must ensure that the linker is sufficiently soluble
and long to provide the fluorescent protein fragments with flexibility and freedom of movement so that the fragment and its partner fragment will collide frequently enough to reconstitute during the interaction of their respective fused proteins. Although it is not documented, it is possible that the length or the sequence of the linker may influence complementation of some proteins. Reported linker sequences RSIAT and RPACKIPNDLKQKVMNH (single amino acid code) and AAANSSIDLISVPVDSR (Sigma) have been successfully used in BiFC experiments.
must be able to express proteins that are able to form fusion protein
s with fluorescent protein fragments without disrupting the protein's function. In addition, the expected protein complex must be able to accept stabilisation of the fluorescent protein fragment interaction without affecting the protein complex function or the cell being studied. Many fluorescent protein fragments that combine in several ways can be used in BiFC. Generally, YFP is recommended to serve as the reporter protein, cleaved at residue
155 (N-terminal consisting of residues 1–154 and C-terminal consisting of residues 155–238) or residue 173 in particular, as these sets of fragments are highly efficient in their complementation when fused to many interacting proteins and they produce low levels fluorescence when fused to non-interacting proteins. It is suggested that each target protein is fused to both the N- and C-terminal fragments of the fluorescent reporter protein in turn, and that the fragments are fused at each of the N- and C-terminal ends of the target proteins. This will allow a total of eight different permutations, with interactions being tested:
system of choice, as not all reporter proteins can fluoresce or be visualised in all model systems
.
s to ensure that the fluorescence from fluorescent reporter protein reconstitution is not due to unspecific contact.
Some controls include fluorophore fragments linked to non-interacting proteins, as the presence of these fusions tend to decrease non-specific complementation and false positive results.
Another control is created by linking the fluorescent protein fragment to proteins with mutated interaction faces. So long as the fluorescent fragment is fused to the mutated proteins in the same manner as the wild-type protein, and the protein expression levels and localisation are unaffected by the mutation
, this serves as a strong negative control, as the mutant proteins, and therefore, the fluorescent fragments, should be unable to interact.
Internal controls are also necessary to normalise for differences in transfection efficiencies and protein expression in different cells. This is accomplished by co-transfecting cells with plasmids encoding the fusion proteins of interest as well as a whole (non-fragmented) protein that fluoresces at a different wavelength from the fluorescent reporter protein. During visualisation, one determines the fluorescence intensities of the BiFC complex and the internal control which, after subtracting background signal, becomes a ratio. This ratio represents the BiFC efficiency and can be compared with other ratios to determine the relative efficiencies of the formation of different complexes.
into the cells to be studied. After transfection, one must wait, typically about eight hours, to allow time for the fusion proteins to interact and their linked fluorescent reporter protein fragments to associate and fluoresce.
in the visible range extremely most cells often makes the BiFC signal orders of magnitude higher than background fluorescence.
If fluorescence is detected when the fusion proteins are expressed, but is lacking or significantly reduced after the expression of the mutated negative control, it is likely that a specific interaction occurs between the two target proteins of interest. However, if the fluorescence intensity is not significantly different between the mutated negative control fusion protein and its wild-type counterpart, then the fluorescence is likely caused by non-specific protein interactions, so a different combination of fusion protein conformations should be tested.
If no fluorescence is detected, an interaction may still exist between the proteins of interest, as the creation of the fusion protein may alter the structure or interaction face of the target protein or the fluorescence fragments may be physically unable to associate. To ensure that this result is not a false negative, that there is no interaction, the protein interaction must be tested in a situation where fluorescence complementation and activation requires an external signal. In this case, if the external signal fails to cause fluorescence fragment association, it is likely that the proteins do not interact or there is a physical impediment to fluorescence complementation.
, rather than relying on secondary effects or staining by exogenous molecules that can fail to distribute evenly. This, and the ability to observe the living cells for long periods of time, is made possible by the strong intrinsic fluorescence of the reconstituted reporter protein reduces the chances of an incorrect readout associated with the protein isolation process.
proportions. Instead, BiFC can detect interactions among protein subpopulations
, weak interactions, and low expression proteins due to the stable complementation of the fluorescent reporter protein. In addition, successful fluorescent protein reconstitution has been reported for protein partners over 7 nm apart, so long as the linkers binding the fluorophore fragment to the protein of interest has the flexibility needed to associate with its corresponding fragment.
Furthermore, the strength of the protein interaction can be quantitatively determined by changes in fluorescent signal strength.
of in vivo protein–protein interaction assays.
.
s, which cannot survive in the presence of oxygen. This limits the use of BiFC to aerobic organism
s.
. This range in colours has made the development of multicolour fluorescence complementation analysis possible. This technique allows multiple protein complexes to be visualised simultaneously in the same cell. In addition, proteins typically have a large number of alternative interaction partners. Therefore, by fusing fragments of different fluorescent proteins to candidate proteins, one can study competition between alternative interaction partners for complex formation through the complementation of different fluorescent colour fragments.
of interest. Similar to BiFC, if the mRNA and protein interact, the Venus protein will be reconstituted and fluoresce. Also known as the RNA bridge method, as the fluorophore and other interacting proteins form a bridge between the protein and the RNA of interest, this allows a simple detection and localisation of RNA-protein interactions within a living cell and provides a simple method to detect direct or indirect RNA-protein association (i.e. within a complex) that can be verified through in vitro analysis of purified compounds or RNAi
knockdown
of the bridging molecule(s).
s, or deletion or knockdown of the gene of interest, and the subsequent effects on both the strength of the protein–protein interactions and the location of the interaction can be observed within seconds.
s.Thus, BiFC is an important tool in understanding transcription factor localisation in subcellular compartments.
(BiFC-FC). This allows protein–protein interaction surfaces to be mapped through the introduction of site-directed
or random mutations that affect complex formation.
and mechanisms that occur in the normal cell may provide a misleading picture of intracellular and physiological occurrences. Furthermore, proteins studied in vitro may be studied at concentrations vastly different from their normal abundance levels, may not necessarily be transported efficiently into the cells, or may not be selective enough to function in the host genome. Finally, by studying proteins in vitro, one is unable to determine the influence of specific protein–protein interactions in the cell on the functional or physiological consequences.
Other in vivo assays most commonly used to study protein–protein interactions include fluorescence resonance energy transfer (FRET) and yeast two-hybrid
(Y2H
) assay. Each of these assays has their advantages and disadvantages in comparison to BiFC:
) chromophore
or fluorophore
(if the chromophores are fluorescent) to a nearby acceptor
. In this method, fluorophores are chemically linked or genetically fused to two proteins hypothesised to interact. If the proteins interact, this will bring the fluorophores into close spatial proximity. If the fluorophores are oriented in a manner that exposes the fluorophores to one another, usually ensured when designing and constructing the fluorophore-protein linkage/fusion, then the energy transfer
from the excited donor fluorophore will result in a change in the fluorescent intensities or lifetimes of the fluorophores.
(Y2H
) is a genetic screening technique that can be used to detect physical (binding) protein–protein or protein–DNA
interactions. It tests a 'bait' protein of known function that is fused to the binding domain of the transcription factor GAL4
against potential interacting proteins or a cDNA library that express the GAL4 activation domain (the 'prey').
| Dr. C-D Hu Presentation – Bimolecular Fluorescence Complementation (BiFC): Principles & Applications
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein
Bioreporter
Bioreporters are intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment. Bioreporters contain two essential genetic elements, a promoter gene and a reporter gene. The promoter...
and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.
History
Biochemical complementationComplementation (genetics)
In genetics, complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations that produce the same phenotype . These strains are true breeding for their mutation...
was first reported in subtilisin-cleaved bovine pancreatic ribonuclease
Ribonuclease
Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.-Function:All organisms studied contain...
, then expanded using β-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
mutants that allowed cells to grow on lactose.
Recognition of many proteins' ability to spontaneously assemble into functional complexes as well as the ability of protein fragments to assemble as a consequence of the spontaneous functional complex assembly of interaction partners to which they are fused was later reported for ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
fragments in yeast protein interactions.
In 2000, Ghosh et al developed a system that allowed a green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
) to be reassembled using an anti-parallel
Antiparallel (biochemistry)
In biochemistry, two molecules are antiparallel if they run side-by-side in opposite directions or when both strands are complimentary to each other....
leucine zipper
Leucine zipper
A leucine zipper, aka leucine scissors, is a common three-dimensional structural motif in proteins. These motifs are usually found as part of a DNA-binding domain in various transcription factors, and are therefore involved in regulating gene expression...
in E. coli cells. This was achieved by dissecting GFP into C- and N-terminal GFP fragments. As the GFP fragment was attached to each leucine zipper by a linker, the heterodimerisation of the anti-parallel leucine zipper resulted in a reconstituted, or re-formed, GFP protein that could be visualised. The successful fluorescent signal indicated that the separate GFP peptide fragments were able to correctly reassemble and achieve tertiary folding
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
. It was, therefore, postulated that using this technique, fragmented GFP could be used to study interaction of protein–protein pairs that have their N-C termini in close proximity.
After the demonstration of successful fluorescent protein fragment reconstitution in mammalian cells, Hu et al. described the use of fragmented yellow fluorescent protein
Yellow fluorescent protein
Yellow Fluorescent Protein is a genetic mutant of green fluorescent protein, derived from Aequorea victoria. Its excitation peak is 514nm and its emission peak is 527nm....
(YFP
Yellow fluorescent protein
Yellow Fluorescent Protein is a genetic mutant of green fluorescent protein, derived from Aequorea victoria. Its excitation peak is 514nm and its emission peak is 527nm....
) in the investigation of bZIP and Rel family transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
interactions. This was the first report bZIP protein interaction regulation by regions outside of the bZIP domain
BZIP domain
The Basic Leucine Zipper Domain is found in many DNA binding eukaryotic proteins. One part of the domain contains a region that mediates sequence specific DNA binding properties and the leucine zipper that is required for the dimerization of two DNA binding regions. The DNA binding region...
, regulation of subnuclear
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
localization of the bZIP domains Fos and Jun
C-jun
c-Jun is the name of a gene and protein that, in combination with c-Fos, forms the AP-1 early response transcription factor. It was first identified as the Fos-binding protein p39 and only later rediscovered as the product of the c-jun gene. It is activated through double phosphorylation by the...
by their different interacting partners, and modulation of transcriptional activation
Activator (genetics)
An activator is a DNA-binding protein that regulates one or more genes by increasing the rate of transcription. The activator may increase transcription by virtue of a connected domain which assists in the formation of the RNA polymerase holoenzyme, or may operate through a coactivator. A...
of bZIP and Rel proteins through mutual interactions. In addition, this study was the first report of an in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
technique, now known as the bimolecular fluorescence complementation (BiFC) assay, to provide insight into the structural basis of protein complex formation through detection of fluorescence caused by the assembly of fluorescent reporter protein fragments tethered to interacting proteins.
Fluorescent labeling
FluorophoreFluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
activation occurs through an autocatalytic cyclization reaction that occurs after the protein has been folded correctly. This was advanced with the successful reconstitution of the YFP fluorophore from protein fragments that had been fused to interacting proteins within 8 hours of transfection was reported in 2002.
Selection of fusion protein expression system
There are different expression systemsProtein expression
Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins...
that can be used for the fusion protein created. Transient protein expression is used to identify protein–protein interactions in vivo as well as in subcellular localisation of the BiFC complex. However, one must be cautious against protein over-expression, as this may skew both preferential localisation and the predominant protein complexes formed. Instead, weak promoters, the use of low levels of plasmid DNA in the transfection, and plasmid vectors that do not replicate in mammalian cells should be used to express proteins at or near their endogenous levels to mimic the physiological cellular environment. Also, careful selection of the fluorescent protein is important, as different fluorescent proteins require different cellular environments. For example, GFP can be used in E. coli cells, while YFP is used in mammalian cells.
Stable cell lines with the expression vector integrated into its genome allows more stable protein expression in the cell population, resulting in more consistent results.
Determination of fusion sites
When deciding the linker fusion site on the protein surface, there are three main considerations. First, the fluorescent protein fragments must be able to associate with one another when their tethered proteins interact. Structural information and the location of the interaction surface may be useful when determining the fusion site to the linker, although the information is not necessary, as multiple combinations and permutations can be screened. Secondly, the creation of the fusion protein must not significantly alter the localisation, stability, or expression of the proteins to which the fragments are linked as compared to the endogenousEndogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
wild-type
Wild type
Wild type refers to the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard, "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele...
proteins. Finally, the addition of the fluorescent fragment fusion must not affect the biological function of the protein, preferably verified using assays that evaluate all of the proteins' known functions.
Designing linkers
A linker is a short amino acid sequencePeptide sequence
Peptide sequence or amino acid sequence is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group...
that tethers the fluorescent reporter protein fragment to the protein of interest, forming the fusion protein. When designing a linker sequence, one must ensure that the linker is sufficiently soluble
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
and long to provide the fluorescent protein fragments with flexibility and freedom of movement so that the fragment and its partner fragment will collide frequently enough to reconstitute during the interaction of their respective fused proteins. Although it is not documented, it is possible that the length or the sequence of the linker may influence complementation of some proteins. Reported linker sequences RSIAT and RPACKIPNDLKQKVMNH (single amino acid code) and AAANSSIDLISVPVDSR (Sigma) have been successfully used in BiFC experiments.
Creating proper plasmid expression vectors
When designing plasmid vectors to express the proteins of interest, the constructPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
must be able to express proteins that are able to form fusion protein
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
s with fluorescent protein fragments without disrupting the protein's function. In addition, the expected protein complex must be able to accept stabilisation of the fluorescent protein fragment interaction without affecting the protein complex function or the cell being studied. Many fluorescent protein fragments that combine in several ways can be used in BiFC. Generally, YFP is recommended to serve as the reporter protein, cleaved at residue
Residue (chemistry)
In chemistry, residue is the material remaining after a distillation or an evaporation, or to a portion of a larger molecule, such as a methyl group. It may also refer to the undesired byproducts of a reaction....
155 (N-terminal consisting of residues 1–154 and C-terminal consisting of residues 155–238) or residue 173 in particular, as these sets of fragments are highly efficient in their complementation when fused to many interacting proteins and they produce low levels fluorescence when fused to non-interacting proteins. It is suggested that each target protein is fused to both the N- and C-terminal fragments of the fluorescent reporter protein in turn, and that the fragments are fused at each of the N- and C-terminal ends of the target proteins. This will allow a total of eight different permutations, with interactions being tested:
N-terminal fragment fused at the N-terminal protein 1 + C-terminal fragment fused at the N-terminal protein 2
N-terminal fragment fused at the N-terminal protein 1 + C-terminal fragment fused at the C-terminal protein 2
N-terminal fragment fused at the C-terminal protein 1 + C-terminal fragment fused at the N-terminal protein 2
N-terminal fragment fused at the C-terminal protein 1 + C-terminal fragment fused at the C-terminal protein 2
C-terminal fragment fused at the N-terminal protein 1 + N-terminal fragment fused at the N-terminal protein 2
C-terminal fragment fused at the N-terminal protein 1 + N-terminal fragment fused at the C-terminal protein 2
C-terminal fragment fused at the C-terminal protein 1 + N-terminal fragment fused at the N-terminal protein 2
C-terminal fragment fused at the C-terminal protein 1 + N-terminal fragment fused at the C-terminal protein 2
Selection of appropriate cell culture system
As previously stated, it is important to ensure that the fluorescent reporter protein being used in BiFC is appropriate and can be expressed in the cell cultureCell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
system of choice, as not all reporter proteins can fluoresce or be visualised in all model systems
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
.
Selection of appropriate controls
Fluorescent protein fragments can associate and fluoresce at low efficiency in the absence of a specific interaction. Therefore, it is important to include controlScientific control
Scientific control allows for comparisons of concepts. It is a part of the scientific method. Scientific control is often used in discussion of natural experiments. For instance, during drug testing, scientists will try to control two groups to keep them as identical and normal as possible, then...
s to ensure that the fluorescence from fluorescent reporter protein reconstitution is not due to unspecific contact.
Some controls include fluorophore fragments linked to non-interacting proteins, as the presence of these fusions tend to decrease non-specific complementation and false positive results.
Another control is created by linking the fluorescent protein fragment to proteins with mutated interaction faces. So long as the fluorescent fragment is fused to the mutated proteins in the same manner as the wild-type protein, and the protein expression levels and localisation are unaffected by the mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
, this serves as a strong negative control, as the mutant proteins, and therefore, the fluorescent fragments, should be unable to interact.
Internal controls are also necessary to normalise for differences in transfection efficiencies and protein expression in different cells. This is accomplished by co-transfecting cells with plasmids encoding the fusion proteins of interest as well as a whole (non-fragmented) protein that fluoresces at a different wavelength from the fluorescent reporter protein. During visualisation, one determines the fluorescence intensities of the BiFC complex and the internal control which, after subtracting background signal, becomes a ratio. This ratio represents the BiFC efficiency and can be compared with other ratios to determine the relative efficiencies of the formation of different complexes.
Cell transfection
Once the fusion proteins and controls have been designed and generated in their appropriate expression system, the plasmids must be transfectedTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
into the cells to be studied. After transfection, one must wait, typically about eight hours, to allow time for the fusion proteins to interact and their linked fluorescent reporter protein fragments to associate and fluoresce.
Visualisation and analysis
After sufficient time for the fusion proteins and their linked fluorescent fragments to interact and fluoresce, the cells can be observed under an inverted fluorescence microscope that can visualise fluorescence in cells. Although the fluorescence intensity of BiFC complexes is usually <10% of that produced by expression of intact fluorescent proteins, the extremely low autofluorescenceAutofluorescence
Autofluorescence is the natural emission of light by biological entities such as mitochondria and lysosomes, and is used to distinguish the light originating from artificially added fluorescent markers...
in the visible range extremely most cells often makes the BiFC signal orders of magnitude higher than background fluorescence.
If fluorescence is detected when the fusion proteins are expressed, but is lacking or significantly reduced after the expression of the mutated negative control, it is likely that a specific interaction occurs between the two target proteins of interest. However, if the fluorescence intensity is not significantly different between the mutated negative control fusion protein and its wild-type counterpart, then the fluorescence is likely caused by non-specific protein interactions, so a different combination of fusion protein conformations should be tested.
If no fluorescence is detected, an interaction may still exist between the proteins of interest, as the creation of the fusion protein may alter the structure or interaction face of the target protein or the fluorescence fragments may be physically unable to associate. To ensure that this result is not a false negative, that there is no interaction, the protein interaction must be tested in a situation where fluorescence complementation and activation requires an external signal. In this case, if the external signal fails to cause fluorescence fragment association, it is likely that the proteins do not interact or there is a physical impediment to fluorescence complementation.
Relevant biological context
Proteins interact with different protein partners and other macromolecules to achieve functions that support different functions in cells that support survival of the organism. Identifying these interactions may provide clues to their effects on cell processes. As these interactions can be affected by both the internal environment and external stimuli, studying these interactions in vivo and at endogenous levels, as is recommended in BiFC, provides a physiologically-relevant context from which to draw conclusions about protein interactions.Direct visualisation
BiFC enables direct visualisation of protein interactions in living cells with limited cell perturbationPerturbation (biology)
A perturbation of a biological system is an alteration of function, induced by external or internal mechanisms. Biological systems can be perturbed through a number of means...
, rather than relying on secondary effects or staining by exogenous molecules that can fail to distribute evenly. This, and the ability to observe the living cells for long periods of time, is made possible by the strong intrinsic fluorescence of the reconstituted reporter protein reduces the chances of an incorrect readout associated with the protein isolation process.
Sensitivity
Unlike many in vivo protein-interaction assays, BiFC does not require protein complexes to be formed by a large proportion of the proteins or at stoichiometricStoichiometry
Stoichiometry is a branch of chemistry that deals with the relative quantities of reactants and products in chemical reactions. In a balanced chemical reaction, the relations among quantities of reactants and products typically form a ratio of whole numbers...
proportions. Instead, BiFC can detect interactions among protein subpopulations
Statistical population
A statistical population is a set of entities concerning which statistical inferences are to be drawn, often based on a random sample taken from the population. For example, if we were interested in generalizations about crows, then we would describe the set of crows that is of interest...
, weak interactions, and low expression proteins due to the stable complementation of the fluorescent reporter protein. In addition, successful fluorescent protein reconstitution has been reported for protein partners over 7 nm apart, so long as the linkers binding the fluorophore fragment to the protein of interest has the flexibility needed to associate with its corresponding fragment.
Furthermore, the strength of the protein interaction can be quantitatively determined by changes in fluorescent signal strength.
Spatial resolution
BiFC allows measurement of spatial and temporal changes in protein complexes, even in response to activating and inhibiting drugs and subcellularly, providing the highest spatial resolutionAngular resolution
Angular resolution, or spatial resolution, describes the ability of any image-forming device such as an optical or radio telescope, a microscope, a camera, or an eye, to distinguish small details of an object...
of in vivo protein–protein interaction assays.
No specialised equipment
BiFC does not require specialised equipment, as visualisation is possible with an inverted fluorescence microscope that can detect fluorescence in cells. In addition, analysis does not require complex data processing or correction for other sources of fluorescence.No structural information needed
BiFC can be performed without structural information about the interaction partners, so long as the fluorescent reporter protein fragments can associate within the complex, as multiple combinations of fusion proteins can be screened. This is due to the assumption that, since the protein functions are recapitulated in the in vivo context, the complex structure will resemble that of the intact proteins seen physiologically.Multiple applications
The BiFC technology has been refined and expanded to include the abilities to simultaneously visualise multiple protein complexes in the same cell, RNA/protein interactions, to quickly detect changes in gene transduction pathways, demonstrate hidden phenotypes of drugs, where the predicted treatment outcome (i.e. cell death, differentiation, morphological change) is not seen in vivo, study complex formation in different cellular compartments, and to map protein interaction surfacesReal-time detection
The fluorescent signal only is produced after the proteins have interacted, which is generally in the order of hours. Hence BiFC is unable to provide real-time detection of protein interactions. The delay for chemical reactions to generate fluorophore may also have an effect on the dynamics of complex dissociation and partner exchange.Irreversible BiFC formation
BiFC complex formation is only reversible during the initial step of fluorescent reporter protein re-assembly, typically in the order of milliseconds. Once the fluorochrome has been reconstituted, it is essentially irreversible in vitro. This prevents proteins from interacting with others and may disrupt the association/disassociation of protein complexes in dynamic equilibriumDynamic equilibrium
A dynamic equilibrium exists once a reversible reaction ceases to change its ratio of reactants/products, but substances move between the chemicals at an equal rate, meaning there is no net change. It is a particular example of a system in a steady state...
.
Independent fluorescent protein fragment associations
Fluorescent protein fragments have a limited ability to associate independent of the proteins to which they are fused. Although protein-independent association will vary depending on identities of the fusion proteins and their expression levels, one must provide the necessary and numerous controls to distinguish between true and false-positive protein interactions. Generally, this limitation is mitigated by ensuring that the fusion proteins of interest are expressed at endogenous concentrations.Altering protein structure and steric hindrance
Fluorescent fragment linkage may alter the folding or structure of the protein of interest, leading to the elimination of an interacting protein's surface binding site. In addition, the arrangement of the fluorescent fragments may prevent fluorophore reconstitution through steric hindrance, although steric hindrance can be reduced or eliminated by using a linker sequence that allows sufficient flexibility for the fluorescent fragments to associate. Therefore, absence of fluorescence complementation may be a false negative and does not necessarily prove that the interaction in question does not occur.Obligate anaerobes
Due to the requirement of molecular oxygen for fluorophore formation, BiFC cannot be used in obligate anaerobeObligate anaerobe
Obligate anaerobes are microorganisms that live and grow in the absence of molecular oxygen; some of these are killed by oxygen. -Metabolism:...
s, which cannot survive in the presence of oxygen. This limits the use of BiFC to aerobic organism
Aerobic organism
An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.Faculitative anaerobes grow and survive in an oxygenated environment and so do aerotolerant anaerobes.-Glucose:...
s.
Use of fusion proteins
Because endogenous wild-type proteins cannot be visualised in vivo, fusion proteins must be created and their plasmids transfected into the cells studied. These fusion proteins may not recapitulate the functions, localisation, and interactions common to their wild-type counterparts, providing an inaccurate picture of the proteins in question. This problem can alleviated by using structural information and the location of interaction sites to rationally identify fusion sites on the proteins of interest, using appropriate controls, and comparing the expression levels and functions of the fusion and wild-type proteins through Western Blots and functional assays.Temperature dependence
Although low temperatures favours the reconstitution of fluorescence when fragments are within proximity, this may affect the behaviour of the target proteins leading to inaccurate conclusions regarding the nature of protein interactions and their interacting partners.Exact interaction relationship unknown
Because fluorophore reconstitution can occur at a distance of 7nm or more, fluorescence complementation may indicate either a direct or indirect (i.e. within the same complex) interaction between the fluorescent fragments' fused proteins.Application
In addition to the validation of protein–protein interactions described above, BiFC has been expanded and adapted to other applications:Multicolour fluorescence
The fluorescent protein fragments used in BiFC have been expanded to include the colours blue, cyan, green, yellow, red, cherry, and VenusYellow fluorescent protein
Yellow Fluorescent Protein is a genetic mutant of green fluorescent protein, derived from Aequorea victoria. Its excitation peak is 514nm and its emission peak is 527nm....
. This range in colours has made the development of multicolour fluorescence complementation analysis possible. This technique allows multiple protein complexes to be visualised simultaneously in the same cell. In addition, proteins typically have a large number of alternative interaction partners. Therefore, by fusing fragments of different fluorescent proteins to candidate proteins, one can study competition between alternative interaction partners for complex formation through the complementation of different fluorescent colour fragments.
RNA-binding protein interactions
BiFC has been expanded to include the study of RNA-binding protein interactions in a method Rackham and Brown described as trimolecular fluorescence complementation (TriFC). In this method, a fragment of the Venus fluorescent protein is fused to the mRNA of interest, and the complementary Venus portion fused to the RNA-binding proteinRNA-binding protein
RNA-binding proteins are proteins that bind to RNA. They bind to either double-strand or single-strand RNAs through RNA recognition motif . RNA-binding proteins may regulate the translation of RNA, and post-transcriptional events, such as RNA splicing, editing.They are cytoplasmic and nuclear...
of interest. Similar to BiFC, if the mRNA and protein interact, the Venus protein will be reconstituted and fluoresce. Also known as the RNA bridge method, as the fluorophore and other interacting proteins form a bridge between the protein and the RNA of interest, this allows a simple detection and localisation of RNA-protein interactions within a living cell and provides a simple method to detect direct or indirect RNA-protein association (i.e. within a complex) that can be verified through in vitro analysis of purified compounds or RNAi
RNA interference
RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...
knockdown
Gene knockdown
Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene...
of the bridging molecule(s).
Pathway organisation and signal transduction cascades
BiFC can be used to link genes to one other and their function through measurement of interactions among the proteins that the genes encode. This application is ideal for novel genes in which little is known about their up- and downstream effectors, as novel pathway linkages can be made. In addition, the effects of drugs, hormoneHormone
A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
s, or deletion or knockdown of the gene of interest, and the subsequent effects on both the strength of the protein–protein interactions and the location of the interaction can be observed within seconds.
Complex formation in different cellular compartments
BiFC has been used to study nuclear translocation, via complex localisation, as well as interactions involving integral membrane proteinIntegral membrane protein
An integral membrane protein is a protein molecule that is permanently attached to the biological membrane. Proteins that cross the membrane are surrounded by "annular" lipids, which are defined as lipids that are in direct contact with a membrane protein...
s.Thus, BiFC is an important tool in understanding transcription factor localisation in subcellular compartments.
Quantifying protein–protein interaction surfaces
BiFC has been coupled with flow cytometryFlow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
(BiFC-FC). This allows protein–protein interaction surfaces to be mapped through the introduction of site-directed
Site-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
or random mutations that affect complex formation.
Comparisons to other technologies
Most techniques used to study protein–protein interactions rely on in vitro methods. Unfortunately, studying proteins in an artificial system, outside of their cellular environment, poses a number of difficulties. For example, this may require the removal of proteins from their normal cellular environment. The processing required to isolate the protein may affect its interactions with other proteins. In addition, isolating the protein from the intracellular signalingCell signaling
Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue...
and mechanisms that occur in the normal cell may provide a misleading picture of intracellular and physiological occurrences. Furthermore, proteins studied in vitro may be studied at concentrations vastly different from their normal abundance levels, may not necessarily be transported efficiently into the cells, or may not be selective enough to function in the host genome. Finally, by studying proteins in vitro, one is unable to determine the influence of specific protein–protein interactions in the cell on the functional or physiological consequences.
Other in vivo assays most commonly used to study protein–protein interactions include fluorescence resonance energy transfer (FRET) and yeast two-hybrid
Two-hybrid screening
Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of...
(Y2H
Two-hybrid screening
Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of...
) assay. Each of these assays has their advantages and disadvantages in comparison to BiFC:
Fluorescence resonance energy transfer (FRET)
Fluorescence resonance energy transfer (FRET), also known as förster resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET), is based on the transfer of energy from an excited (donorElectron donor
An electron donor is a chemical entity that donates electrons to another compound. It is a reducing agent that, by virtue of its donating electrons, is itself oxidized in the process....
) chromophore
Chromophore
A chromophore is the part of a molecule responsible for its color. The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls...
or fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
(if the chromophores are fluorescent) to a nearby acceptor
Electron acceptor
An electron acceptor is a chemical entity that accepts electrons transferred to it from another compound. It is an oxidizing agent that, by virtue of its accepting electrons, is itself reduced in the process....
. In this method, fluorophores are chemically linked or genetically fused to two proteins hypothesised to interact. If the proteins interact, this will bring the fluorophores into close spatial proximity. If the fluorophores are oriented in a manner that exposes the fluorophores to one another, usually ensured when designing and constructing the fluorophore-protein linkage/fusion, then the energy transfer
Energy transfer
Energy transfer is the transfer of energy from one body to another.There are a few main ways that energy transfer occurs:*Radiant energy *Heat conduction*Convection*Electrical power transmission*Mechanical work...
from the excited donor fluorophore will result in a change in the fluorescent intensities or lifetimes of the fluorophores.
Yeast two-hybrid (Y2H)
The yeast two-hybridTwo-hybrid screening
Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of...
(Y2H
Two-hybrid screening
Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of...
) is a genetic screening technique that can be used to detect physical (binding) protein–protein or protein–DNA
DNA-binding protein
DNA-binding proteins are proteins that are composed of DNA-binding domains and thus have a specific or general affinity for either single or double stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that...
interactions. It tests a 'bait' protein of known function that is fused to the binding domain of the transcription factor GAL4
GAL4/UAS system
The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It was developed by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes...
against potential interacting proteins or a cDNA library that express the GAL4 activation domain (the 'prey').
Technology comparisons
| Comparison Technology | Similarity to BiFC | Advantages | Disadvantages | |
|---|---|---|---|---|
| FRET | Ability to detect and locate protein interaction sites within live cells | Instantaneous real-time monitoring of protein interactions
Reversible fluorophore interaction
|
Close spatial proximity
Decreased sensitivity
Irreversible photo-bleaching
|
|
| Y2H | In vivo technique used to screen for interactions | Genetic interaction screen
|
Tentative bait-prey linkage
Erroneous transcription activation
Genetic complementation
Overexpression of proteins
Nuclear localisation
|
External links
| Kerppola Lab Online – BiFC| Dr. C-D Hu Presentation – Bimolecular Fluorescence Complementation (BiFC): Principles & Applications