In this model, hemoglobin is shown in a backbone format with important residues displayed in ball and stick format. Heme groups are shown here as essentially planar structures with an orange iron atom in the center. Move your mouse over one of the orange iron atoms and then keep the cursor there. A small pop up window with the 'HEM' label should appear. Based on their primary amino acid sequence, proteins adopt various secondary structures. The grey shows regions that don't have secondary structure or regions that contain loops another type of secondary structure that we'll talk about next week.
The N amino terminus is blue and the C carboxy terminus is magenta. Remember to interact with the protein by rotating it and zooming in and out. Interactions between residues R groups determined by primary and secondary structure determine tertiary structure protein folding and allow for the formation of functional domains.
Click the button below to view the histidines thatcoordinate the heme group. In this image the heme group has an orange iron atom in the heme group center.
Hold your cursor over one of the histidines that point towards the heme group. A yellow pop-up box will appear. The first three letters should be HIS for histidine , followed by the residue , the chain, the atom type and the atom number.
Which two histidine residues coordinate hold in place the heme group? In order to function, some proteins must adopt quaternary structure, which requires the association of two or more proteins.
Quaternary structure is largely driven by the need to bury hydrophobic regions from contact with the cytoplasm. Forming the entire hemoglobin molecule is favorable because burying these large hydrophobic regions in the interior contact surfaces of each monomer creates a highly stable protein.
Click on the button below to see the quaternary structure of hemoglobin. This model is again displayed as a backbone structure, but now hydrophobic residues are displayed in ball and stick and colored yellow. Although each globin monomer can independently bind an oxygen molecule, the quaternary structure of hemoglobin allows for more efficient oxygen binding.
This is because the binding of an oxygen to one globin subunit causes conformational changes in the whole protein complex. These conformational changes make it easier for oxygen to bind the heme groups of the remaining subunits. Primary structure ultimately determines how a protein folds into a functional unit. Often, changing a single amino acid results in dramatic changes in protein folding and thus protein function.
The green residues represent the position of the two glutamate residue 6's shown as GLU6 in the pop up window when you pause your mouse over a green residue. These two glutamate residues are mutated to valines in people with sickle cell anemia. The nonpolar valine is hydrophobic, and makes the hemoglobin molecule stick to another hemoglobin molecule in order to shield this region from the watery environment.
This results in the formation of long fibers that cannot carry as much oxygen, and which also distort the shape of the red blood cells. Do you think that hemoglobin function would change if the glutamate was replaced with another polar amino acid?
Briefly explain. The jellyfish also contains a bioluminescent protein, aequorin, which emits blue light. GFP absorbs this blue light and emits green light, which is what we actually see when the jellyfish lights up. Solutions of purified GFP glow bright green when exposed to blue light. Click on the button below to see a model of GFP. In this model, GFP is shown in a backbone format with important residues displayed in ball and stick.
Click on the button below to view GFP's secondary structure. The amino N terminus is blue and the carboxyl C terminus is magenta. Locate the secondary structures of GFP on your model s. Click on the button below to see hydrogen bonds displayed in white within the secondary structures.
These three amino acids are positioned in such a way that they undergo a series of chemical reactions that result in a fluorescent chromophore region where light can be absorbed and emitted in a visible wavelength.
The fluorescence could not occur if these amino acids were exposed to the surrounding environment. Ratiometric GFP pH biosensors have been generated by modification of a few key amino acids in the vicinity of the chromophore. TheSH mutation was shown to be important for the ratiometric property; pHlourins lacking the SH were non ratiometric. The deGFPs are dual emission ratiometric GFPs emitting both blue and green light; blue light emission decreases with increase pH while green light emission increases with increased pH.
In addition to its pH sensing properties, fluorescence emission from E 2 GFP is affected by the concentration of certain ions, including Cl -. In addition to single molecule based pH biosensors, ratiometric pH biosensors using tandem fluorescent protein variants have been constructed in which a pH sensitive GFP variant is linked to a less sensitive or pH insensitive GFP. Genetically-encoded FRET-based biosensors can be applied in a variety of capacities to visualize intracellular spatiotemporal changes in real time.
The evolution of these applications has progressed from cell culture systems that transiently express FRET biosensors to transgenic mouse models that express them in a heritable manner [ 57 ].
Production of transgenic mice with FRET biosensors arose in an effort to enhance our understanding of the differences that exist between tissue culture and living systems. Transgenic FRET GFP biosensor systems are very efficient and their fluorescence signals are easily distinguished from autofluorescence, which is analyte-independent fluorescence.
The sensors themselves can be used to probe a variety of pathways for the activity of signaling enzymes as well as a number of post translational modifications. Kinase induced conformational changes are important because they are involved in the control a number of critical cellular processes that include glycogen synthesis, hormonal response, and ion transport [ 70 ]. A number of signaling cascades that involve kinases require a means of dynamic control and spatial compartmentalization of the kinase activity; a requirement highlights the need for a mechanism to continuously track kinase activity in different compartments and signaling microdomains in vivo.
Traditional methods of assaying kinase activity fail to capture its dynamicity; a void that is filled by genetically encoded FRET-based biosensors. These sensors are constructed so that the substrate protein of the kinase of interest is flanked with a fluorescent protein pair in such a way that the conformational change imparted by phosphorylation translates into a change in the FRET signal Figure7 [ 70 ].
These biosensors can be localized to particular sites of interest with the aid of appropriate targeting signal sequences, allowing the imaging of site-specific kinase activity. G-protein coupled receptors, when used in a biosensor, provide a mechanism for transducingdrug mediated effects on PKA activity into a light signal. Transgenic mice expressing FRET based biosensors provide an ideal system for studying the pharmacodynamics of these drugs. Representation of the mode of action of an intramolecular FRET biosensor containing a molecular switch.
This switch can perceive various molecular events, such as protein phosphorylation, through binding to the ligand domain. This in turn induces an interaction between the ligand and sensor domains that facilitates a global change in the conformation of the biosensor, which serves to increase the FRET efficiency from the donor to the acceptor CFP to YFP in this case [ 71 ].
When used to study the signaling events in wound healing, the strength and duration of the fluorescent signals that are generated by these biosensors are dependent on the location within the tissue tissue depth has a negative impact on the intensity of the fluorescent signal , its vicinity in relation to the site of injury, as well as the contributions made by chemical mediators drugs in sustaining kinase activity [ 57 ].
They also provide a tool for resolving the maze of upstream signaling pathways that contribute to chemotaxis in the animals. Ras GTPases play essential roles in regulating cell growth, cell differentiation, cell migration, and lipid vesicle trafficking. Such a design allows for the monitoring of Ras activation in living cells on the basis of fluctuations in the FRET signals generated. FRET-based GFP biosensors can also be employed in in vitro applications as an alternative tool for high throughput screening assays.
These assays are simple, inexpensive, reproducible and highly specific. A good example can be observed in the use of bacterial cell-based assays for screening antioxidant activity of various substances for biological activity [ 72 ]. To achieve this objective E. After paraquat treatment of E. Genes sodA and fumC are turned on by SoxR and OxyR, respectively, which are the two main regulatory proteins involved in oxidative stress sensing. GFP fluorescence is therefore diminished by successful antioxidants.
These constructs are important because they function as alternative screening tools that can be utilized to assess the activity of compounds with therapeutic potential against oxidative stress. Antioxidants have been shown to play a role in disease prevention. When used in vivo , the calmodulin-based biosensors suffered from endogenous interference by host proteins and did not always work [ 73 ].
GFP has great potential to work as an in vitro biosensor. Because of its remarkable stability, it can be used and manipulated in multiple ways to impart sensor functionality to the protein. The goal of GFP-antibody chimeric proteins GFPAbs is to convert a multi-step experimental process for locating molecules via antibodies and enzyme-linked secondary antibodies, into a one-step process using a GFPAbs.
This molecule could then work as a detection reagent in flow cytometry, for intracellular targeting, or fluorescence-based ELISAs [ 38 ]. However, in order to replace antibodies in these techniques, it is important to achieve the same nanomolar sensitivity that is found in the natural antibodies.
To do this, [ 38 ] inserted two antigen-binding loops into the GFP structure, counting on cooperativity in binding to enhance affinity. It became clear that adding loops impinges on the integrity of the native GFP structure. The binding loops must be placed such that their presence in the fluorescent protein does not jeopardize its structural fidelity, or that of the chromophore.
The latter two are too far apart in three-dimensional space to provide for cooperative binding see Figure 5. The yeast secretory pathway does not allow unfolded protein to reach the surface of the cell, thus only mutants that yield fully folded GFP were displayed by yeast cells.
The F64L mutation has been shown to increase fluorescence of GFP and also to shift the excitation maximum to nm. Y39H and NT have been shown to improve refolding kinetics and refolding stability, respectively. VA is linked to improved folding as a result of its increased expression in yeast surface display [ 38 ]. With dual loop insertion, dissociation constants as low as 3.
The success of this construct means that molecules such as GADPH can be located within cells without having to engineer a second round of antibodies, saving both time and resources.
A general method for developing a biosensor for a specific receptor-ligand interaction has been described [ 76 ] in which a receptor protein is inserted into the GFP sequence between strand 8 and strand 9. The insertion puts enough of a strain on GFP that its fluorescence is reduced.
This change may be found by plate screening for fluorescence. In [ 76 ], the receptor Bla1 was cloned into the loop, and random mutations were made to this construct. Using this method, a double mutant was found that was shown to detect BLIP in vitro with micromolar affinity. In principle, this method could be used to generate a sensor for any ligand that can be expressed in bacteria or added exogenously, as long as a receptor protein exists that can be inserted into the GFP loop.
FRET-based in vitro biosensors may be constructed by linking fluorescent proteins to quantum dots QDs. QDs are inorganic molecular nano-crystals whose absorption and emission spectra are dictated by the size of the QD. For example, a QD may be engineered to absorb ultraviolet light and emit light at nm, which overlaps well with the excitation spectrum of mCherry, a variant of GFP [ 77 ], and produces FRET when the two fluorophores are in close proximity. In order to make the FRET emission analyte-dependent, the QD was linked to the mCherry via an N-terminal linker peptide that contained a protease cleavage site and a 6 histidine tag.
The imidazole side chains of the histidines electronically coordinate with the zinc atoms of the CdSe—ZnS core-shell semiconductor of the QD [ 77 ].
Multiple mCherry molecules can be coordinated with each QD. By placing the caspase-3 cleavage sequenceinto the linker between GFP and the QD, the FRET complex becomes a biosensor for the presence of caspase-3, glowing red at nm in the absence of the protease, and reverting to the yellow fluorescence of the QD at nm when the protease is present Figure 8. When the two are split by caspase activity, FRET is lost. Figure used with permission from [ 78 ].
It has been shown that fluorescent proteins such as GFP and mOrange experience a shift in excitation and emission spectra with changes in pH [ 78 ]. At a slightly acidic pH, there is very little spectral overlap between the QD emission and the mOrange excitation, which means that the QD emission is seen, in this case around nm.
However, as the pH increases, the excitation spectrum of mOrange shifts such that there is more overlap with the QD emission, which subsequently causes an increase in FRET. The result is an upward shift in the emission wavelength with increasing pH. It is important to note that since there is a fluctuating hydrogen ion concentration, the histidine-QD coordination complex becomes unstable. In order to remedy this problem, a covalently linked quantum dot must be used.
Fluorescent proteins, especially E 2 GFP, have been shown to be sensitive not only to pH changes but also to the concentration of certain ions, particularly chloride ions.
E 2 GFP provides an avenue for single domain ratiometric analysis of pH because it contains two excitation and emission peaks. Only the longer wavelength emission peak is pH dependent [ 68 ]. Therefore analysis of pH based on the ratio of green fluorescence to cyan. By coupling E 2 GFP to another fluorescent protein in a fusion construct, it is also possible to measure other intracellular chloride ion concentration.
For example, DsRed is neither pH nor chloride ion sensitive, so it can be used to measure chloride ion concentration based on the ratio of its fluorescence to the cyan emission of E 2 GFP.
Making a few modifications can make GFP sensitive to the concentration of other ions. For example, superfolder GFP can be made sensitive to copper ions by mutating the arginine at position to a histidine, which, as previously mentioned, coordinates well with metal ions [ 79 ].
GFP can also become sensitive to ions by creating channels in the structure through which small molecules can pass through and access the chromophore Figure 9. This allows small molecules such as copper ions to enter the hydrophobic core of the protein and quench fluorescence [ 80 ].
GFP, thanks to its stability, has shown a remarkable ability to be modified, and thus shows great promise in visualizing a large variety of intracellular and extracellular substances. The analyte channel through which copper ions can pass through to the interior of the barrel structure and quench the fluorescence of the chromophore. Used with permission from [ 80 ]. Recent work in the Bystroff lab has focused on programming GFP to accept any desired protein as a binding partner, like an antibody, and to switch on fluorescence only when the targeted protein is bound.
The strategy combines Leave-One-Out split reconstitution with computational design and high throughput screening. A promising application of LOO-GFP, knowing that it binds to the left-out segment and fluoresces [ 39 , 40 ], is to engineer novel LOO-GFP molecules that recognize and sense desired peptides derived from other sources such as virus, bacteria and parasites.
LOO-GFP biosensors can be engineered by generating mutations that accommodate the shape and charge of a desired target peptide. The target peptide may be made available for binding by denaturing the target protein. Engineering LOO-GFP molecules to accommodate desired target peptides create specific sensing tools where fluorescence can be reconstituted upon adding back the left-out peptides and signals the detection.
Theoretically, this goal could be achieved by random mutation followed by high throughput screening to find mutants that glow in the presence of a peptide. However, random mutation would be extremely inefficient.
Computational protein design methods offer a much better alternative for rationally generating sequence diversity before the labor-intensive experimental screen. Computational protein design predicts protein sequences that fold into predefined protein structures. Instead of mutating residues experimentally, mutations are explored in silico and selected using a computed goodness of fit Figure Mutations predicted to cause collisions between atoms, leave unsatisfied hydrogen bonding partners, cause charge-charge repulsion, or employ rare amino acid side chain conformations are down-weighted by assigning them a higher energy value.
To facilitate the search for the best mutations, amino acid side chains are discretized into rotational isomers called rotamers [ 85 - 87 ]. Protein sequences that preserve the desired functionalities, such as the binding of a ligand, are obtained by searching the space of all side chain rotamers for the minimum free energy. There are few reviews of the methods used [ 88 ]. Computational protein design coupled with design library generation [ 89 ]. The entire designed sequence space of selected residues is computationally screened to determine the global minimum energy configuration GMEC for the given structure.
Starting from the GMEC, sequence space is explored to obtain sub-optimal sequences that are also potentially predicted to be functional.
A DNA library is constructed to cover all predicted sequences, and candidates are screened experimentally to select clones with desired functions. Information from analyzing obtained mutants is utilized to validate and improve the computational protein design strategy, and provides a better starting model for iterative optimization.
Biosensors for specific proteins and pathogens offer potential advantages over the current state of the art, notably speed and simplicity. The isolation method requires a culture system to inoculate a specimen, followed by the examination of specific characteristics produced by pathogens, such as the cytopathogenic effect of virus and the distinct metabolism of bacteria.
Although culture-based methods have higher detection sensitivity, they generally take three to ten days for diagnosis. Alternatively, immunoassays utilize pathogen specific antibodies and secondary anti-antibodies to detect and report a pathogen. Most of the rapid diagnostic tests only take 15 to 30 min for diagnosis, but raising specific antibodies against pathogens is time-consuming and expensive.
Thirdly, molecular diagnosis using PCR takes the advantage of the gene amplification and provides a highly sensitive detection in diagnosis from minute amounts of pathogen genome within a short time. However, the need for real-time PCR and gel electrophoresis apparati and reagents means it will not be possible in all settings, where a simple biosensor test would be possible.
PCR assumes that DNA is present, but some pathogens such as anthrax toxin, snake venom and bovine spongiform encephalopathy contain no genetic material. All these point to a need for developing a diagnostic tool for proteins that is fast and easy to use, and suitable for rural, point-of-care facilities in developing nations.
The following describes how the computer-aided design of LOO-GFP was done, and the encouraging but preliminary results. These patterns define the limits of mutation. Cysteines are also disallowed, for experimental reasons. Target peptides are selected by searching the sequences of the target organism for a match to the signature pattern. Other considerations including the location of protease recognition sites, cellular location, and protein expression levels.
DEEdesign uses a combination of physical properties and statistical knowledge to energetically evaluate the fitness of rotamers in protein structures, along with sampling algorithms to search the space of all possible mutations. The parameters used in the fitness scoring system are trained by a machine learning technique to reproduce the true sidechain conformations in high-resolution crystal structures [ 90 ].
An interesting mutation discovered by Ormo et al. This further supports the idea that Arg 96 is an important factor in the structural arrangement required for cyclization, perhaps by promoting the attack of Gly 67 on the carbonyl carbon of Thr In high protein concentrations, GFP has been found to dimerize under the influence of high ionic strength between the two monomers.
In Aequorea victoria , the aequorin is able to bind to the 1gfl , but not the monomer. As a result, dimers, and often even higher 1w7s , are predominant protein populations within the jellyfish. The first alpha carbon backbone model is colored by three-strand repeats, including red, blue, purple, and yellow. The second alpha carbon backbone model is colored by secondary structure, with alpha helices red and beta sheets yellow.
Both models show the fluorophore molecule at the center of the GFP structure. The MSOE Center for BioMolecular Modeling uses 3D printing technology to create physical models of protein and molecular structures, making the invisible molecular world more tangible and comprehensible. To view more protein structure models, visit our Model Gallery. Green Fluorescent Protein 3D structures. Crystal structure of the Aequorea victoria green fluorescent protein.
DOI Steinberger , David Canner. Category : Topic Page. Green Fluorescent Protein From Proteopedia. Jump to: navigation , search. Show: Asymmetric Unit Biological Assembly. Export Animated Image. European Bioinformatics EBI ; The molecular structure of green fluorescent protein. The Green Fluorescent Protein. Annual Review in Biochemistry. The green journey. J Fluoresc. The discovery of green fluorescent protein. Nobel Prize Lecture; ;; biographical background at Wikipedia. Aequorea victoria.
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