Thermo Stability Assay For Tnfr 1 Protein Stability Biology Essay

The mechanism by which tumour mortification factor receptor ( TNFR ) cause the disease TNFR associated periodic syndrome ( TRAPS ) is thought to be via protein misfolding, collection and ligand independent signaling. This survey aimed to develop a thermo stableness assay that will let the probe of the differences in structural stableness of wild-type TNFR 1 and mutant signifiers C33Y and R92Q. Protein flowering was measured utilizing SYPRO Orange protein dye which fluoresces when exposed to hydrophobic residues. Lysozyme, Carbonic Anhydrase and IgG1 were used to optimize the status prior to proving of TNFR 1. We observed unusual consequences for TNFR 1 and its mutations, the consequences showed an upside-down curve entering a lessening in fluorescence. We found that a big figure of variables affect the consequences of this type of check and as a effect reaction conditions are required to be tailored each person protein being tested.


Tumor mortification factor receptor ( TNFR ) associated periodic syndrome ( TRAPS ; OMIM 142680 ) is an autoinflammatory periodic febrility syndrome. It is associated with autosomal-dominant mutant in TNFR1, the cistron that encodes tumour mortification factor receptor ( TNF R ) [ 1 ] . There are more than 50 different mutants ( hypertext transfer protocol: // ) that have been identified in association with TRAPS within the TNFR superfamily 1A ( TNFRSF1A ) . The bulk of these mutants are likely to significantly impact the construction, conformation and stableness of the receptors. Studies back uping this hypothesis suggest that these mutants TNFR may ensue in a conformational alterations in the protein that leads to receptor misfolding, collection and ligand independent signalling, which explains the functional and clinical differences between the TRAPS-associated mutants [ 2, 3 ] . The leaning of the mutations to aggregate and independently signal suggest a verification that is less thermodynamically stable, this may consequences in drawn-out exposure of hydrophobic residues and unfolded provinces.

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TNFR1 is a transmembrane glycoprotein, with a conserved intracellular decease sphere ( DD ) that is responsible for receptor signalling, a transmembrane part and four tandem-repeat cysteine-rich spheres ( CRD 1-4 ) which contain three disulfide bonds. CRD1 is known as the preligand assembly sphere ( PLAD ) which mediates ligand independent trimetric receptor interactions. All the TNFR 1 mutants associated with TRAPS are located within the CRD ectodomian of the receptor.

This survey uses thermo stableness checks to look into differences in structural stableness between wild type TNFR 1 and two cardinal mutations, C33Y and R92Q. C33Y and R92Q are located in the CRD1 and CRD2 severally. C33Y is likely to be extremely riotous to the overall conformation of the receptor as it abolishes one of the disulfide bonds found in the extracellular part of the receptor which is hence expected to destabilise the protein. There is strong grounds to propose that every bit good as a alteration in conformation [ 4 ] and deficiency of TNF binding, this mutants lead to miss of TNF binding and unnatural receptor map [ 3, 5 ] . R92Q is a mutant with low penetrance, and is predicted to no major impact on the protein construction [ 3 ] , arginine is a basic amino acid which is replaced by a glutamine which has an uncharged polar side concatenation. It behaves really likewise to the WT receptor in footings of TNF binding and cell surface look [ 6-8 ] .

To day of the month there are no publications which show experimental differences in the stableness of TNF R1 and its mutants, although there are some computational surveies which foretelling the structural effects of theses mutants. All old work focal points on the functional and behavioural difference of the mutant receptors. There are a turning figure of diseases which are being recognized as conformational upsets such as Alzheimer ‘s disease, Huntington ‘s disease and Parkinson ‘s disease. The pathological consequence of these diseases is the consequence of misfolded proteins therefore understanding structural stableness and the consequence of mutants on a protein conformation is an of import tool.

The structural stableness of a protein can hold a cardinal consequence on its map at a physiological degree. The importance of amino acid interactions within the third construction of a protein means that a individual residue alteration can ensue in a important lessening in thermodynamic stableness of the protein construction which can take to collection, proteolysis or unwanted interaction. It would be possible, utilizing thermostability profiles, to look into difference in stableness and blossoming between wild type and protein mutations. If a mutant is involved in the structural stableness of the protein it can significantly impact the protein construction every bit good as the rate of folding and flowering, this in bend can impact how the protein maps at a biological degree ensuing in disease.

Previously thermo stableness checks have been applied as a high-throughput screen for protein technology [ 9 ] , construction finding [ 10 ] , structural optimisation of protein ringers [ 11 ] and monoclonal antibodies [ 12 ] . It has besides been used for crystalization testing [ 10 ] showing of stableness advancing ligands [ 13 ] and to analyze interaction between mark proteins and little molecules [ 13, 14 ] . Other techniques used to look into protein stableness and find the liquescent temperature of a protein included differential scanning calorimetry ( DSC ) , spectrometry and round dichroism but most of these techniques require big sums of protein and can be hard to modify for a high-throughput system.

The method being used in this survey has been adapted from [ 15 ] this survey used ANS ( 1-anilino-8-napthalenesulfonate ) but because the ANS has an emanation and excitement upper limit that is non accessible by standard PCR filters, this survey uses SYPRO orange, which has been successfully used in other thermo stableness assays [ 9, 16 ] . SYPRO orange is extremely fluorescent when exposed to hydrophobic parts in proteins. As the protein starts to blossom due to the addition in temperature, the hydrophobic parts of the protein become exposed adhering to the dye. This addition in fluorescence can be measured utilizing a Real-time PCR machine to bring forth a profile of protein flowering. This profile should be distinguishable for each protein and as a consequence this technique can be used to look into differences between mutated proteins.

Material & A ; Methods

Optimization of Thermal Stability Assays

SYPRO Orange dye was obtained from Sigma. It was stored in dimethyl sulfoxide ( DMSO ) solution at 5000 times concentration. The dye was stored at -20OC. Before use the dye stock is diluted 1:20 to 250 times concentration in 20mM Tris.Cl ( pH 7.1 ) ( Sigma-Aldrich, St. Louis, MO ) , 150mM NaCl buffer. The concluding concentration of SYPRO orange during the experiment was 5 times. Protein solution contained either ; Lysozyme ( Sigma ) , Carbonic Anhydrase ( Sigma ) or sneak IgG1 isotype control ( R & A ; D systems Minneapolis, MN ) at concentration between 1mg/ml – 0.005mg/ml, the proteins were diluted in one of three buffers ; 25mM MES pH 6.1 ( sigma ) 50mM NaCl 2 % DMSO, 100mM HEPES pH 7.0 ( Sigma ) 150mM NaCl 2 % DMSO or 20mM Tris pH 7.1 ( Sigma ) 150mM NaCl, as described in figure fables. The protein solutions ( 5 and 20Aµl ) were dispensed into 8x 0.2ml format strip tubings with optically clear caps ( Stratagene, La Jolla, CA ) . The checks were performed utilizing an MX4000 series multiplex quantitative Real-Time PCR system ( Stratagene ) the proteins were heated at a rate of 1oC/min and three informations point were collected at the terminal points of each rhythm. The fluorescence emanation was collected, ab initio utilizing standard Stratagene MX4000 filters ; FAM, HEX and ROX every bit good as SYPRO orange specific bandpass filters purchased from Chroma ( excitement 470/50 nanometer and emanation 590/55 nanometer ) . Assays were performed from a starting temperature of 30oC and stoping at 95oC. The consequences were analyzed utilizing the Mx4000 package.

Thermal stableness checks of TNF R1 wild type and mutants

TNFR 1 was supplied by Paul Radford from within the group. It was column purified FLAG tagged, full length TNFR 1, solubilised from the membrane utilizing NP40 detergent and stored in Tris ( Sigma ) buffer at -20oC until usage. The proteins were diluted to a concentration of 0.005mg/ml in 20mM Tris ( Sigma ) 150mM NaCl buffer, dye was added to a concluding concentration of 5 times. The MX4000 set up was as described above.

Flourescence excitement and emanation spectra of SYPRO orange


Assay optimisation

The thermostabilty profiles of Lysozyme, Carbonic Anhydrase and IgG 1

Low handiness of TNFR 1 meant initial experiments used Carbonic Anhydrase, Lysozyme and IgG1 to optimise the reaction conditions and measure the restrictions of the check. It besides allowed us to prove the compatibility of the dye with the standard real-time filters.

Figure 2 ( a-c ) shows the thermo stableness profiles of the Lysozyme, Carbonic Anhydrase and IgG1. Samples were heated at a steady rate and the fluorescence alteration was measured over clip. Rapid rise in fluorescence strength seen in all three profile suggest an addition in hydrophobicity in the environment of the dye this indicates protein blossoming. The dissociation points of each on the proteins can be seen in figure 2 ( d-f ) , which represent derivative secret plans of the thermic fluorescence profile ( alteration in fluorescence, DF, between temperature points ) . These are shown as negative extremums because the set up of the package is for real-time PCR reaction which uses lessening in fluorescence to bespeak DNA thaw and dye dissociation, in this survey this map has been used to look at rapid alterations in emanation associating to temperatures where the most protein unfolding occurs. This temperature is referred to in this survey as the TD ( dissociation temperature ) it relates to the steepest portion of the thaw curve. The thaw curve and dissociation profile of all three protein are distinguishable, with different TD and swerve forms. It was observed that in all three profiles the fluorescence drops off somewhat after the maximal point.

Lysozyme is a simple ball-shaped protein with merely a individual sphere ; Carbonic Anhydrase and IgG1 are more complex with defined sphere construction. This can be seen in the thaw curves, Lysozyme produces a simple smooth curve, whereas the consequences for the other proteins look more complex with a less rapid diminution in fluorescence after the first rise in fluorescence.

The consequence of check buffer on thermo stableness assay

Originally the proteins were tested in Tris buffer but we were unable to detect any consequences utilizing Carbonic Anhydrase, because we were cognizant that there had been old success with this protein we chose to execute farther experiment utilizing MES pH 6.1 and HEPES buffers pH 7.0. Using these reaction buffers we were able to derive consequences for Carbonic Anhydrase. All three proteins were repeatedly tested utilizing all three buffers but no consequences were obtained for Lysozyme in HEPES. We assessed the consequence of the buffer by looking at the TD of the protein under the different status. Little fluctuations can be seen in the consequences ( table 1 ) .









Carbonaceous Anhydrase




IgG 1 isotype




Table 1. The TD ( o C ) of Lysozyme, Carbonic Anhydrase and IgG1 under assorted buffer conditions. Buffers are as stated in the stuff and methods. n=3

N/A indicates non clear TD was obtained during this survey.

The consequence of filter sets on thermo stableness assay

Fluorescence strength varied greatly depending on the filter set used to roll up the thaw curve informations. The scope of the filters can be seen in figure 1, the maximal fluorescence which was seen by each of the filters set is summarized in table 2. Initial experiments used bing ROX and HEX filters for informations aggregation, the set base on balls for these filters fell within the excitement and emanation spectrum of SYPRO orange ( see figure 1 ) and required no internal accommodation to the Mx4000. Unfortunately the maximal fluorescence collected was 376 comparative fluorescence units ( rfu ) . Because of this low end product alternate filters were tested. Previous surveies [ 9 ] have used a combination, FAM ( ex ) /HEX ( mutton quad ) , of the standard filters to increase the fluorescence strength collect during their experiments. Although the FAM/HEX filter combination did better the degree of fluorescence collected to some extent, it was decide that to better the sensitiveness of the check further we would buy SYPRO orange specific filters from Chroma. This saw the most drastic betterment in maximum fluorescence. There was an about 17 clip addition in fluorescence when comparing the FAM/HEX filter set and specific filters in experiments utilizing Lysozyme, and a 5 times increase when looking at the same status utilizing Carbonic Anhydrase. IgG1 showed a 14 times increase in maximum fluorescence utilizing specific filters.

Filter sets




Specific SYPRO orange






Carbonaceous Anhydrase





IgG 1 isotype





Table 2. The maximal fluorescence ( rfu ) for Lysozyme, Carbonic Anhydrase and IgG1 utilizing criterion ( ROX, FAM/Hex and HEX ) filter set. In comparing with SYPRO orange specific filters. All reaction volumes 20Aµl. Lysozyme ( 0.6mg/ml ) and IgG1 ( 0.5mg/ml ) in 20mM Tris, 150mM NaCl, Carbonic Anhydrase ( 0.6mg/ml ) tested in 25mM MES, 50mM NaCl, 2 % DMSO

*No experiments were performed looking at the maximal fluorescence of IgG1using HEX filters

The consequence of concentration on thermo stableness assay

The concentration of the TNF R1 available to us was rather low and for this ground we chose to prove the bounds of this method by diminishing the concentration of protein used. Decease in protein concentration resulted in lower degrees of fluorescence. Below a concentration of 0.01mg/ml signal became deformed and we were unable to obtain clear consequences utilizing proteins at lower concentrations. Table 3 shows the maximal degrees of fluorescence diminishing with diminishing concentration of protein.

Protein concentration mg/ml







Carbonaceous Anhydrase







Table 3. The maximal fluorescence for Carbonic Anhydrase utilizing varing concentrations of protein. Reaction volume 5Aµl, dilution made utilizing 25mM MES, 50mM NaCl, 2 % DMSO.

* 20Aµl volumes

The consequence of volume on the result of the thermo stableness profile

It was observed that reaction volume played a big portion in optimisation of the check. Increasing the reaction volume resulted in an increased the maximal fluorescence, Figure 3 show the consequence of increasing the reaction volume on the form and tallness of the curve. The consequences were obtained at the same time and all three reactions were performed utilizing the same reaction conditions. For Lysozyme we observed an about 14 fold lessening in fluorescence which resulted from halving the reaction volume. For Carbonaceous Anhydrase the decrease in fluorescence was less drastic but we still observed that the fluorescence was decrease by a one-fourth as a consequence of the reaction volume being halved.

Thermo stableness check for TNF R1 WT, R92Q and C33Y

From the optimisation experiment we selected conditions which would supply the best opportunity of bring forthing a successful thaw curve of TNF R. Unfortunately the concentrations of the wild type and mutation TNFR1 which were available were much lower than the concentration used in the initial experiments with trial proteins. To get the better of this we used larger volumes and the specific filters to maximise fluorescent end product. Tris buffer was used as this was the buffer the protein was stored in and hence seemed most suited.

Despite this the degrees of fluorescence are significantly lower when compared with the other proteins used in this survey. We unable to obtain a clear TD from these consequences, and although the three curves were consistent some fluctuation can be seen between the three repetitions. We were unable to place any clear difference between the thermo stableness profiles of wild type and mutant TNF R ( figure 4 ) .

The form of the thermo stableness profiles of TNF R mutants we collected is unlike any of the others seen in earlier experiments ( calculate 2 ) the curve appears to be inverted with fluorescence diminishing over clip and as the temperature increases. This is farther discussed in the following subdivision.


The ability to analyze protein stableness is an of import tool when look intoing the consequence of protein mutants. The purpose of this survey was to develop a method which would let the survey the thermo stableness of wild type and mutant TNF R 1. The concentration and volumes of TNFR 1 available to us were low therefore we decided to optimise the method utilizing Lysozyme, Carbonic Anhydrase and IgG1. Previous surveies have used thermic stableness checks to analyze the effects of ligands on the protein stableness of Carbonic Anhydrase [ 15 ] . This survey showed Carbonic Anhydrase to be an ideal protein for this type of check. Lysozyme and IgG1 are both proteins which were freely available and have good known defined constructions.

The thermostability secret plans for all three proteins appeared distinguishable, giving different profiles ; the Lysozyme secret plan was, as expected, the simplest of the three with a smooth addition to a peak fluorescence. The profiles for the other two proteins appear to be more complex with a rapid initial addition in fluorescence with some fluctuations, emphasized in the derivative secret plan which may be the consequences of specific sphere flowering. All three profiles showed a lessening in fluorescence towards the terminal of the tally ( ie higher temperature ) , this is largely likely due to protein collection ; an alternate account is that at the higher temperatures the dye becomes less stable, hence cut downing the degree of fluorescence.

The consequence of reaction buffer is an of import factor to see when construing these consequences. The difference between consequences which we observed when altering buffer means that in order for comparing to be draw between the two proteins both need to be assayed utilizing the same buffer to forestall unwanted fluctuation which resolved from the buffer. It may be that salt concentration and pH of the buffer has a big consequence on the solubility of the protein, buffers with a pH near the pi of the protein may ensue in the protein coming out of solution. To guarantee that this method will work for any given protein it is of import to happen a suited buffer that will stabilise the protein and that is compatible with this technique.

There were similarities in the TD of Lysozyme and IgG1, both proteins gave a TD of 70.5-72.5 ( buffer dependant ) , therefore it is of import to see whether the consequence obtained in the survey were a true contemplation on single protein blossoming. Despite these similarities in dissociation of these two proteins the fluorescence profile obtained during these experiments were distinguishable ( figure 2 ) the profile for IgG1 seems to plateau after the crisp rise in fluorescence whereas the profile for Lysozyme shows a rapid lessening in fluorescence after the extremum in strength. The forms of these profiles were consistent thought out the survey proposing that they are in fact a merchandise of the protein instead than the dye or an external contamination.

It is deserving observing that although Lysozyme, Carbonic Anhydrase and IgG1 were compatible with this method and gave clear profiles, we tested a broad scope of proteins. Ribonuclease A and BSA were both used in initial experiments but neither gave clear or consistent consequences. We were able to obtain some consequences utilizing Nuclear Histone protein and anti TNFR2 antibody but they were inconsistent and required farther optimisation to better the definition of the curves. Anti TNFI± antibody, Anti TNF1 antibody and TNFI± were besides tested but the profiles were ill-defined which is most likely the consequence of low protein concentrations. This method was foremost designed for high throughput testing but the faithlessness of the check in respect to both protein and buffers used, means that in order for this check to be successful the right experimental environment demand to be tailored to each person protein being used.

We found that with more specific filters the concentration of protein required could be lower. The observation was besides made that higher volumes of protein solution resulted in less intervention and allowed the concentrations protein to be lowered even further. Due to the consequence of the filter set on the reaction sensitiveness we believe it is deserving utilizing optimum filters for this type of survey.

It may be that this technique is non sensitive plenty to distinguish between different spheres blossoming. Higher declaration machines to supply a more elaborate thermic profile and higher concentration of proteins may be adequate to specify the thaw curve at a high adequate declaration which will let us to place single sphere unfolding events.

The TNFR wild type and mutations gave surprising consequences with the fluorescence decreased as the temperature increased ensuing in an upside-down profile, which is unlike the consequences obtained when utilizing the other proteins. Because these proteins are membrane associated, they must be solubilised utilizing a detergent to let purification before this type of experimental testing. It is possible that it is the interaction between SYPRO orange and the detergent at the start of the check that resulted in the unexpected lessening in fluorescence. For this ground it may be that SYPRO orange is an unsuitable dye for this type of check when utilizing membrane proteins, hence future experiment may necessitate a alteration of dye to an alternate such as 1-anilino-8-naphthalenesulfonate ( ANS ) [ 15 ] which fluoresces in response aqueous environment, or N- [ 4- ( 7-diethylamino-4-methyl-3-coumarinyl ) phenyl ] maleimide ( CPM ) [ 17 ] which is a thio-specific dye which can uncover thermic stableness of disulphide bonds within a protein.

Another account for the unusual consequences seen by TNFR1 is the protein itself. If the hydrophobic resides usually buried in the plasma membrane were exposed because of the solubilisation this would ensue in a high initial blossoming. The conformation the receptor takes up after it ‘s been removed from the membrane is unknown. Carbonaceous Anhydrase acts as a monomer whereas TNFR 1 is found to organize trimers via the PLAD sphere in the absence of TNF. It may be that the lessening in fluorescence histories for the initial breakage of these interactions as the temperature increases. Further surveies utilizing site directed mutagenesis could look at the consequence of mutant in the PLAD sphere on the thermo stableness profile of TNFR 1.

It may be that the concentration of TNFR 1 were excessively little for it to be assessed utilizing this method, as a consequence farther purification may be required. Although this may non be easy possible as other members of the research group have found that TNFR 1, and in peculiar C33Y, has a inclination to come out of solution when extended purification is performed. This may be because WT TNFR 1 is itself is an unstable protein. Surveies have shown that if extremely expressed within cells, WT receptor besides has a leaning to organize sums [ 18 ] . For this ground it may be that this check system is unable to get by with the instability of the protein every bit good as the size of the receptor.

Further surveies could look at simplifing TNFR 1 for the intents of the check, executing experiments utilizing the single domains instead than the whole protein ( figure 5 ) . As the mutant for TRAPS are all located in the ectodomain it would be sensible to analyze merely this subdivision of the protein, this would necessitate no solublisation from the plasma membrane and therefore may be easier to sublimate. We could besides utilize this technique to look into the structural stableness of the intracellular decease sphere ( DD ) and TNFR 1- TNF association. Alternatively we could simplify the theoretical account farther by analyzing the local effects of the mutant on single CRD.

Current interventions for TRAPS have had varying success, some patients respond to anti-TNF interventions but for others this intervention is uneffective and can take to increased disease flares [ 19 ] . This is due to the differences in behaviour between different TRAPS mutants. If we were able to successfully modify this technique so that it would let us to analyze TNF R1 it would be possible to this check to test for ligands which are able to stabilise the mutant signifiers of the protein, this may let us to look into fresh intervention for TRAPS. This would let us to analyze mutants as single entities and from this interventions could be tailored to the patient depending on the specific mutant they have.

There are many variable to see with this technique. The set up for this method required extended testing to specify its optimum conditions and restrictions. We had limited success utilizing TNFR 1 and in order for this testing to come on any farther simplification of the protein may be required.


I would wish to thank Patrick Tighe for all his support and counsel throughout this undertaking. I would besides wish to thank Paul Radford for providing the TNFR 1 and Sue Bainbridge for helping me with protocols and other research lab work.


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