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Purification, identification, and stability analysis of Escherichia coli translation initiation factor 3

This is a paper I wrote for a Biochemistry laboratory class while getting my undergraduate degree. This paper ties together almost a dozen experiments my lab partner and I did over the course of a quarter. It was intended to be written like a real scientific paper, so it is written with a scientific audience in mind. (Please excuse the lack of figures and formatting errors, I'm still working on fixing it up.)

ABSTRACT

Translation is the final step of biological protein synthesis, which plays a crucial role in the development and survival of all living organisms. It is the process in which cellular ribosomes decode messenger RNA (mRNA) produced in transcription to produce specific polypeptide chains that will later fold into active proteins. Alongside the ribosome, several helper proteins called translation factors ensure that the process of translation can work efficiently and accurately. One of these translation factors, initiation factor 3 (IF3), is especially important during the initiation of translation in prokaryotes, where it starts the process of initiation and ensures its fidelity. A pSV plasmid encoding a gene for an unknown translation factor fused to an N-terminal His tag was obtained. In order to identify and characterize the unknown protein, chemically competent E. coli NiCo21 cells were transformed with the plasmid. The desired unknown protein was then overexpressed by inducing the cell culture with Isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were lysed by sonication, and the crude lysate was purified by affinity chromatography in a Ni-NTA resin. A Bradford assay was used to quantify the protein and calculate its mass extinction coefficient. The apparent molecular weight of the unknown protein was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Dialysis was performed to transition the protein to a buffer appropriate for storage and denaturation experiments. The unknown protein was identified as initiation factor 3 (IF3) by comparing the apparent molecular weight and mass extinction coefficient to the literature values of each possible unknown. To gain insight into the stability of IF3 during unfolding, thermal and chemical denaturation experiments were performed. The stability of the secondary structure was analyzed using far-UV circular dichroism, and the stability of the tertiary structure was analyzed by observing intrinsic tryptophan resonance.

BACKGROUND

Initiation is the process where the two ribosomal subunits (30S and 50S) join together with the help of several specialized proteins called initiation factors. The initiation process consists of several steps: Formation of the 30S pre-initiation complex, adaptation of mRNA, and formation of the 70S initiation complex. IF3 participates in almost all of these steps, and is involved in recycling of the 30S subunit. When translation is not happening, IF3 binds to the 30S subunit preventing its association with the 50S subunit, ensuring a supply of free 30S subunits. IF3 improves the fidelity of protein synthesis by proofreading the selection of fMet-tRNA and the AUG codon during initiation. It does this by causing a conformational change in the 30S initiation complex (30SIC) when it binds, and by dissociating preferentially (and allowing the 50S subunit to bind) from 30SICs where the correct tRNA has bound, as well as mRNA with the correct start codon. Escherichia coli has many advantages that make it a preferred choice for recombinant protein expression in comparison to other organisms – both the genetics and molecular biology of protein expression are well studied, high cell growth and expression levels are easily obtained, and cells and other materials required for expression are economical (1,2). By using calcium chloride and heat shocks, E. coli can be made competent, allowing foreign DNA to be transfected through its cell wall. By using a plasmid engineered with a highly expressing promoter, a target protein can be expressed in large amounts (1). Histidine tags are commonly used in protein purification, and have a number of advantages, the most important being that they often allow for single step purifications, without further purification needing to be done to obtain protein in usable amounts (3). Polyacrylamide gel electrophoresis (PAGE) separates macromolecules on the basis of size, conformation, and net charge (4). PAGE is advantageous to other fractionation techniques because of its high resolution, versatility, sensitivity, and low cost. By varying the concentrations of acrylamide and the crosslinker bis-acrylamide, gels with various pore sizes can be constructed, which allows for high resolution separation within the desired range of molecular weights. When a solution of macromolecules is loaded onto the gel and an electric potential is establish across it, the macromolecules will flow through the pores in the gel at different speeds according to their size, conformation, and net charge. To determine the molecular weight of a charged macromolecule, a technique that separates macromolecules based only on their size is preferable. One well-known solution to this problem is to add the detergent sodium dodecyl sulfate (SDS), which masks the charges of each protein by creating a uniform negative charge, and a disulfide reducing agent (such as β-mercaptoethanol) to eliminate the effect that differing conformations have on migration (5). It has been shown that this technique is effective at separating proteins based on molecular weight alone, and that by plotting the relative mobility of a protein against a set of known standards, its molecular weight may be determined with high confidence (6). Circular dichroism (CD) is a commonly used technique for analyzing protein secondary structure. Although CD cannot give secondary structure information at single residue resolution like X-ray crystallography or NMR, it has several important advantages, namely that it can be used to analyze proteins non-destructively, in low concentrations, and at physiological conditions (7). CD takes advantage of the fact that circularly polarized light interacts differentially with chiral molecules, which rotate the plane of light at different angles depending on their structure. The addition of the vectors of clockwise polarized light and counterclockwise polarized light traces out an ellipse, and measurements are typically reported in degrees of ellipticity (7). Amide bonds in a polypeptide backbone absorb light in the far-UV region (190-240 nm), and different structure elements (eg. alpha helices, beta sheets, etc.) show characteristic CD spectra (7). By monitoring the far-UV region with CD, the overall secondary structure of a protein can be observed under native conditions, and conformational changes can be observed by performing denaturation experiments. Tryptophan and tyrosine strongly absorb light at 280nm, and fluoresce at 340nm. The fluorescence of these residues can be quenched by interactions with the solution, therefore, by observing changes in fluorescence during thermal or chemical denaturation, changes in tertiary structure can be observed (8).

EXPERIMENTAL PROCEDURES

Transformation of Chemically Competent E. coli Cells

Chemically competent E. coli NiCo21 cells and pSV plasmid containing a gene coding for an unknown transcription factor were obtained. To transform the E. coli, 5 μL of plasmid was added to 100 μL of cells and mixed briefly. As a negative control, 2.5 μL of deionized water was added to 50 μL of E. coli cells. Cells were incubated on ice for 30 minutes, heat shocked at 42°C for 1 minute, then cooled on ice for 2 minutes. Cells were then resuspended in 1mL of sterile lysogeny broth (LB), then incubated at 37°C with shaking for 45 minutes. Cells were then plated in 100, 200, and 400 μL portions onto LB/Kanamycin-50 plates, and 200 μL of the negative control was plated onto a fourth plate. Plates were incubated overnight at 37°C. The next day, kanamycin (Kan) was added to a flask containing 50mL LB to a final concentration of 50 μg/mL, with 6mL of this mixture set aside for use as an OD600 blank. A single colony was located and transferred using a sterile stick to the prepared LB/Kan broth. This flask was then incubated overnight at 37°C with shaking. This culture was then used to inoculate a 1L bacterial culture.

Protein Purification

The seed culture was then transferred to a 3L flask containing 1L of sterile LB-Kan-50 and incubated at 37°C until an OD600 of 0.5 was reached. Protein expression was then induced by adding IPTG to a final concentration of 0.75mM IPTG. The culture was immediately transferred to an incubator at 24°C, and cells were allowed to grow for 18-20hrs. Cells were then harvested by centrifuging for 10min at 8671 x g at 4°C with a F10S-6X500Y rotor. The cell pellet was stored at -20°C, then resuspended by adding 25mL of load/lysis buffer (50mM Tris-HCl (pH 7.5), 150mM NH4Cl, 10mM MgCl2, 10mM imidazole (pH 7.0), 15% glycerol (v/v)) immediately prior to lysis. The resuspended pellet was then sonicated for 90 seconds (at a 50% duty cycle, in three 30 second intervals, with sonicator – need to look up), and 50 mL of whole extract was set aside for quantification. The rest of the crude cell lysate was centrifuged at 17469 x g with a F21S-8X50Y rotor. A 50 μL aliquot of the supernatant was filtered through a 5 μm filter, then a 0.45 μm filter to prevent clogging of the column. The insoluble cell pellet was discarded.

Affinity Chromatography

A column was obtained already prepared using approximately 5 mL of a slurry of Qiagen Ni2+-NTA His-Bind Resin and was washed with 20 mL of imidazole (1M, pH 7.0). The high speed supernatant was added to the column and the flow through collected at 1mL/min. The column was then washed with 30 mL of wash buffer 1 (50mM Tris-HCl (pH 7.5), 300mM NH4Cl, 10mM MgCl2, 10mM imidazole (pH 7.0), 15% glycerol (v/v)), and the wash collected at 2mL/min. Then the column was washed with 30 mL of wash buffer 2 (20mM Tris-HCl (pH 7.5), 150mM NH4Cl, 10mM MgCl2, 10mM imidazole (pH 7.0), 15% glycerol (v/v)), and the wash collected at 2mL/min. The bound protein was then eluted with elution buffer (20mM Tris-HCl (pH 7.5), 150mM NH4Cl, 10mM MgCl2, 250mM imidazole (pH 7.0), 15% glycerol (v/v)) and 2 mL fractions were collected at 1mL/min. After quantification, 100 μL samples of the crude lysate, high speed supernatant, the collected flow-through from column loading, the washes, and the column fraction with the highest A280 reading were saved for analysis by SDS-PAGE.

Quantification using the UV/Vis and the Bradford Assay

The A280 of each column fraction was determined using a Thermo Scientific Evolution 220 UV-Visible Spectrophotometer to determine which fractions contained protein. Several samples had to be diluted for usable absorbance readings to be obtained. Then, 50 μL samples containing 0, 1, 2, 5, 10, 20, and 40 μg of bovine serum albumin (BSA) standard were prepared, and 950 μL of Bradford Reagent was added to each. The samples were incubated for 5 minute on ice, then A595 readings were taken and used to generate a standard curve. Similarly, samples were prepared using 2, 5, and 10 μL from the column fraction with the highest A280 reading, and A595 readings were taken. The standard curve was then used to determine the concentration in each sample, which were then used to calculate the mass extinction coefficient of the unknown translation factor protein. Determination of Molecular Weight by SDS-PAGE A 12.5% SDS-PAGE gel was prepared (Table 1), and poured immediately after APS and TEMED were added. After gel polymerization was complete, the gel was loaded into an electrophoresis chamber with Tris-glycine running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS, pH 8.3). Samples of the column fractions, crude cell lysate, high speed supernatant, the flow-through from the column loading, and washes from affinity chromatography were prepared by combining 30 μL of each sample with 10 μL of loading buffer (50 mM Tris, pH 6.8, 100 mM β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). The samples were heated for 5 minutes at 101°C to denature the proteins, and then loaded onto the gel. A Spectra Multicolor Broad Range Protein Ladder was also loaded. Electrophoresis was performed at 120V until the tracking dye reached the bottom of the resolving gel. The gel was stained by shaking with coomassie brilliant blue stain (0.25g of coomassie brilliant blue R250 in 90mL of methanol: H2O (1:1 v/v) and 10 mL of glacial acetic acid). The gel was rinsed with water, then destained overnight by soaking in a 30% methanol/10% acetic acid solution. The relative migrations of the molecular markers were fitted to create a standard curve. The standard curve was used to determine the apparent molecular weight of the unknown translation factor.

Table 1: Components used to prepare SDS-polyacrylamide gel Component Amount Added (mL) ddH2O 10.4 40% acrylamide mix 7.8 1.5 M Tris (pH 8.8) 6.3 10% sodium dodecyl sulfate 0.25 10% ammonium persulfate (APS) 0.3 Ν,Ν,Ν’,Ν'-tetramethylethylenediamine (TEMED) 0.04

Dialysis of Translation Factors

To prepare the protein sample for storage and further biochemical characterization, it was dialyzed into a storage buffer (20mM Tris-HCl (pH 7.5), 150mM NH4Cl, 10mM MgCl2, 20% glycerol (v/v)). Dialysis tubing (MWCO 12-14000 Da) was hydrated in ddH2O for 3 minutes and immersed in dialysis buffer. Protein solution was added into the tubing using a 5mL serological pipette, and the tubing was sealed using dialysis clips. Dialysis was carried out at 4°C with gentle mixing. The dialysis buffer was changed twice over the following several days. After dialysis, the protein was aliquoted and stored at -20°C. Analysis of Protein Stability during Thermal and Chemical Denaturation Samples of the purified protein were diluted to a concentration of 0.5 mg/mL. CD and fluorescence spectra were recorded for three samples: dialysis buffer (negative control), the completely folded protein, and a completely denatured protein (in 5M Gdn-HCl) to determine the optimal wavelengths to monitor in thermal and chemical denaturation experiments. Low signal intensity was observed in both the CD and fluorescence spectra, so further experiments used undiluted protein. A thermal denaturation experiment was performed by taking CD spectra at 12 temperature intervals from 30-70°C using an Olis DSM-10CD Spectrometer. An unfolding curve was generated by observing the wavelength determine from the first CD spectra. A chemical denaturation experiment was performed by preparing samples with 0-5M Gdn-HCl in 0.5M increments and recording their fluorescence spectra with a Thermo Scientific Evolution 220 UV-Visibile Spectrophotometer.

RESULTS

Transformation of Competent Cells and Overexpression of Recombinant Protein

Chemically competent E. coli NiCo21 cells were obtained and transformed using the plasmid labelled “Unknown D”. After plating the transformed cells and waiting overnight, only a single colony was observed on the plate inoculated with 400 μL of cells. The single colony was used to seed a 1L bacterial culture.

Table 2: Transformation efficiencies of cells transformed with unknown D Amount Plated Colonies Observed Transformation Efficiency (CFU/μg) 100 μL 0 0 200 μL 0 0 400 μL 1 5.5 x 103 Negative Control (200 μL) 0 0

The seed culture was induced with IPTG, and incubated at 37°C until an OD600 of 0.55 was reached. OD600 readings were taken approximately every 30 minutes during the first phase of growth, then every 15 minutes in the second phase of growth using UV/vis (Figure 1). Figure 1. Growth curve of transformed E. coli cells

Affinity Chromatography

Absorbance readings of each column fraction collected were taken at 280 nm, and dilutions performed if the absorbance was greater than 1.0 (Figure 2). After fraction 12, readings were taken for every other fraction for the sake of time. Figure 2. Elution of protein during affinity chromatography

Quantification with the Bradford Assay

A Bradford Assay was performed by adding Bradford reagent to known amounts of BSA standard (1mg/mL), taking A595 readings, and constructing a standard curve (Figure 3). Absorbance readings from the combined fractions 4-7 were taken at three different concentrations, and the standard curve used to calculate the concentration of protein present in each sample. The concentration of protein was used to determine the mass extinction coefficient of the protein (Table 3). Figure 3. BSA standard curve used to determine protein concentration.

Table 3: Absorbance readings of protein Sample and calculated mass extinction coefficients Sample (µL) Abs595 Mass (µg) Conc. (µg/µL) ε (L/g*cm) 2 0.157 4.53 2.26 0.210 5 0.384 11.07 2.21 0.214 10 0.675 19.46 1.95 0.244

Determination of Molecular Weight by SDS-PAGE

A photograph of the gel was taken (Figure 4) and relative mobilities were calculated with the software GelAnalyzer (http://www.gelanalyzer.com). Figure 4. Purification of IF3 by SDS-PAGE. a) molecular weight standard ladder b) crude cell lysate c) high speed supernatant d) column flow-through e) 1st wash f) 2nd wash g) protein fraction A standard curve was generated from the relative mobilities calculated for the molecular weight standard ladder (Figure 5), and was fitted using a logarithmic regression. The three standards with the highest molecular weights were excluded from the standard curve to achieve a better fit. The standard curve was used to calculate the apparent molecular weight of IF3 (Table 4).

Table 4: Calculated Molecular Weight of IF3 Relative Mobility of Protein Molecular Weight (Da) Expected MW (Da) % Error 0.511 25831 20564 20.4% Figure 5. Standard curve used to determine molecular weight from relative mobility

Dialysis of Translation Factors

The fractions from affinity chromatography containing significant amounts of protein were combined and dialyzed into a storage buffer. After dialysis, an A280 reading was taken to assess the concentration of the combined fractions (Table 5). Table 5: Absorbance at 280nm and Concentration of Combined Protein Fractions A280 Concentration (g/L) ε of IF3 (L/g*mol) Concentration (M) 0.59539 2.74 4470 0.000142

Chemical and Thermal Denaturation

The stability of the secondary structure of the translation factor IF3 was studied using circular dichroism (CD) and thermal denaturation. The purified protein was diluted to a concentration of 0.5 mg/mL and CD spectra were taken for both the native and completely denatured states (Figure 6). The difference in signal was plotted to determine the best wavelength to monitor in a thermal denaturation experiment (Figure 7). The wavelength with greatest difference in signal was determined to be 218.2nm. Figure 6. Circular dichroism spectra for native and denatured states of IF3 with baseline subtracted. Protein was denatured with 5M GdnHCl. Figure 7. Difference in ellipticity between native and denatured states of IF3. Highlighted point indicates optimal wavelength that was selected. Next, a thermal denaturation experiment was performed by taking CD spectra in 12 increments from 30-70°C (Figure 8). The region from 217.0-222.4nm was selected for further analysis based on the wavelength found from the initial CD experiment. The signals from this region were averaged, and the fraction unfolded calculated to generate a thermal unfolding curve (Figure 9). AU and AF values were selected just outside of the minimum and maximum signal observed in the region of interest. A van’t Hoff analysis was performed to calculate the thermodynamic parameters ΔH° and ΔS° and the melting temperature Tm (Figure 10, Table 6). The free energy of unfolding ΔG°H20 was calculated from ΔH° and ΔS° at 298K (Table 6).

Table 6: Thermodynamics of Thermal Unfolding for IF3 Tm (°C) ΔH° (kJ/mol) ΔS° (kJ/mol*K) ΔG°H20 (kJ/mol) 55 144.95 0.44 13.44 Figure 8. Thermal denaturation of IF3. CD spectra were smoothed by averaging each point with the two neighboring points on each side of it. Legend entries are in degrees Celsius. Figure 9. Thermal unfolding curve of IF3 (average of 217.0-222.4nm). Figure 10. Van’t Hoff plot for thermal denaturation of IF3. Figure 11. Protein stability curve of IF3 during thermal denaturation. Next, intrinsic tryptophan fluorescence was monitored to analyze the stability of IF3’s tertiary structure during chemical denaturation. Samples of the native and fully denatured states (0.2 mg/mL) were excited at 280nm and emission spectra were recorded (Figure 12). Figure 12. Fluoresence of IF3 in its native and chemically denatured states. Protein was denatured with 5M GdnHCl. A low signal intensity was observed, so the concentration of the samples was doubled and a chemical denaturation experiment was performed, with fluorescence spectra taken at several increments from 0-5M GdnHCl (Figure 13). The wavelength 331nm was determined from Figure 7 to be the area with the greatest difference in signal, so this wavelength was used to analyze the unfolding under chemical denaturation (Figure 14). A linear fit was applied to the points in the transition region to calculate the chemical melting point Cm and the ΔG°H20 of unfolding (Table 7.)

Table 7: Thermodynamics of Chemical Unfolding for IF3 Cm (M) ΔG°H20 (kJ/mol) 1.94 5.65 Figure 13. Chemical denaturation of IF3. Fluorescence spectra were smoothed by averaging each point with its two neighboring points on each side. Box indicates region of interest. Figure 14. Free energy of unfolding during chemical denaturation of IF3 at 331nm.

DISCUSSION

Transformation of Competent Cells and Overexpression of Recombinant Protein

Transformation efficiency is an effective way to gauge the success of a transformation. The low transformation observed here (Table 2), especially considering the high concentration of plasmid DNA, is an indication that some aspect of the transformation was not as efficient as it should have been. One possible explanation is that the E. coli cells were heat shocked too long and/or at too high a temperature, causing many of them to die – heat shocked was performed for 60 seconds but the cell data sheet recommended 10 seconds. A further indication of the low competency observed is the slow growth curve observed after induction (Figure 1). There was almost no growth observed for the first two hours, while groups working with the same cells induced with different genes had already reached their desired optical density in that time period. Overall, the efficiency was not as good as desired, but the cells were successfully transformed and induced, but in the future, greater efficiency may be obtained by altering the heat shock duration and temperature. Affinity Chromatography and Quantification with the Bradford Assay The plot of A280 readings for each protein fraction reveals that the affinity chromatography was performed successfully. The negative peak corresponding to the first two fractions represents wash buffer being eluted. The absorbance appears negative because the spectrophotometer was blanked with elution buffer, which contains a higher concentration of imidazole, a compound that absorbs at 280 nm. After this negative peak, a sharp positive peak is observed, corresponding to the protein being eluted. The maximum concentration of protein appeared in fraction 4, which was selected for quantification with the Bradford Assay. The plot of BSA standards (Figure 3) generated a second-order polynomial curve, rather than a linear plot as expected (9). Values calculated with the polynomial fit agreed closely (within 1µg) to the linear fit, so only mass values calculated with the linear fit were considered. The calculated concentrations of the sample were largely in agreement.

Table 8: Molar and Mass Extinction Coefficients of Translation Factors Factor Molar Mass (g/mol) ε (L/mol*cm) ε (L/g*cm) IF2 97349.9 27515 0.282640249 IF3 20563.9 4470 0.217371218 EF-Tu 43313.5 20525 0.473870733 EF-G 77581.3 61435 0.791878971 EF4/LepA 66570.2 39935 0.599893045 RRF 20638.5 2980 0.144390338 RF1 40517.3 21555 0.531994975 RF3 59574 41745 0.700725149 Molar mass and extinction coeffecients sourced from UniProtKB (http://www.uniprot.org/) and calculated with ProtParam (http://web.expasy.org/protparam/)

SDS-PAGE

The image of the gel (Figure 4) reveals that the protein purification scheme was carried out successfully, but the gel was overloaded. The columns containing the crude cell lysate, the high speed supernatant, and the flow-through show a smear over nearly the whole gel, as we would expect from a crude mix of proteins. There is a decrease in overall intensity from the high speed supernatant to the column flow-through, and from the flow-through to the first wash. A decrease in intensity from the crude lysate to the high speed supernatant would be expected, but some of the lysate did not sink into the gel during the loading stage because it was especially viscous, which lowered the concentration of the lysate relative to the other samples in the gel. A drastic decrease in intensity was observed from the first wash to the second wash, indicating that a majority of the undesired proteins were removed with the first wash. Along with the decrease in overall intensity as more purification steps were performed, some bands became more distinct, indicating that fewer contaminating proteins remained, with the remaining ones becoming more concentrated. Finally, the column containing the sample of purified protein shows a very distinct band around 25 kDa containing the protein, with only a small smear in the 10-25 kDa region containing contaminants with relatively low molecular weight. This is evidence that a decent quality of purification was achieved, but to be certain a new gel would have to be made that was not overloaded. The band representing the protein was fairly broad, and very intense, which suggests a highly concentrated sample. This was confirmed by absorbance spectroscopy, which showed that the protein had a concentration of 2.74 g/L (Table 5). The standard curve generated from the molecular weight ladder formed a smooth logarithmic curve after neglecting the top three bands from the high molecular weight region. The high molecular weight region was near the top edge of the gel, and the bands that were neglected were quite small and bunched up, so their center points were difficult to determine accurately. The protein band appeared within the well resolved region of the curve, and the apparent molecular weight was calculated as 25831.4 Da (Table 4). This represents a 20.39% error from the expected molecular weight of IF3, which does not fall in the optimal range for SDS-PAGE of ±10% (10). The apparent molecular weight was closest to the molecular weights of IF3 and RRF, but since those weights only differ by ~75 Da, more information was needed to determine the identity of the protein (Table 4, Table 8). The average apparent mass extinction coefficient of the 3 samples (0.223 L/g*cm) was compared to the possible mass extinction coefficients of IF3 (0.217 L/g*mol) and RRF (0.144 L/g*mol), and since the apparent value was much closer to that of IF3, the protein was determined to be IF3 and not RRF (Table 3, Table 8). One reason the apparent weight might be off is because the protein band was so broad, which may have obscured the true center of the band and led to an inaccurate relative mobility value. To correct this, a gel could be run with the protein sample diluted to the point where the band is clear but not spread out.

Chemical and Thermal Denaturation

The initial circular dichroism experiment (Figures 6 and 7) showed a clear difference in secondary structure between the native and unfolded states. The wavelength with the largest change was approximately 218nm, which corresponds to the minimum signal intensity of β-sheets, but is also very close to the minimum signal intensity of α-helices. That the difference in signal between the native and denatured states was greatest here makes sense, because IF3 contains both α-helices and β-strands (11). From the unfolding curve, thermal denaturation appears to be a two-state process, and thermal denaturation of IF3 in another species (the chloroplast of E. gracilis) has been reported to be two-state, so a two-state analysis was applied (Figures 10 and 11, ref. 12). The melting temperature that was calculated was higher than reported by Yu, et al. (48°C), which indicates that IF3 in E. coli is more thermo-stable than IF3 in the chloroplast of E. gracilis (Table 6, ref. 12). The quality of the fluorescence spectra suffered from a low signal intensity, but this was expected because IF3 has a low aromatic amino acid composition – containing no tryptophan residues and only 3 tyrosine residues (3). However there were enough points in the transition region of 1-3M GdnHCl to calculate the melting point (Cm) and ΔG°H20 of unfolding. Although the ΔG° plot (Figure 14) was too noisy to tell if the chemical denaturation process is two-state, it probably is not, because the ΔG°H20 of unfolding during thermal denaturation (which ¬¬is two-state) and chemical denaturation differ by approximately 8000 J/mol (Tables 7 and 8). To confirm that this is the case, fluorescence experiments would need to be repeated with a higher concentration of sample to account for the low aromatic residue content.  

REFERENCES

1. Makrides, S. C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological reviews, 60(3), 512-538.
2. Gold, L. (1990). Expression of heterologous proteins in Escherichia coli. Methods in enzymology, 185, 11-14.
3. Schmitt, J., Hess, H., & Stunnenberg, H. G. (1993). Affinity purification of histidine-tagged proteins. Molecular biology reports, 18(3), 223-230.
4. Chrambach, A., & Rodbard, D. (1971). Polyacrylamide gel electrophoresis. Science, 172(3982), 440-451.
5. Shapiro, A. L., & Viñuela, E. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and biophysical research communications, 28(5), 815-820.
6. Weber, K., & Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry, 244(16), 4406-4412.
7. Greenfield, N. J. (2007). Using circular dichroism spectra to estimate protein secondary structure. Nature protocols, 1(6), 2876-2890.
8. Teale, F. W. J. (1960). The ultraviolet fluorescence of proteins in neutral solution. Biochemical Journal, 76(2), 381.
9. Zor, Tsaffrir, and Zvi Selinger. “Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies.” Analytical biochemistry 236.2 (1996): 302-308.
10. Weber, K., & Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry, 244(16), 4406-4412.
11. Biou, V., Shu, F., & Ramakrishnan, V. (1995). X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix. The EMBO journal, 14(16), 4056.
12. Yu, N. J., & Spremulli, L. L. (1997). Structural and mechanistic studies on chloroplast translational initiation factor 3 from Euglena gracilis. Biochemistry,36(48), 14827-14835.


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