* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Equilibrium and Free Energy of Protein Denaturation
Amino acid synthesis wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Genetic code wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Paracrine signalling wikipedia , lookup
Gene expression wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Biochemistry wikipedia , lookup
Point mutation wikipedia , lookup
Expression vector wikipedia , lookup
Magnesium transporter wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Metalloprotein wikipedia , lookup
Interactome wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein structure prediction wikipedia , lookup
Western blot wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Estimation of the Free Energy of Stabilization of a Folded Small Globular Protein, Myoglobin: Determination of the Denaturation Equilibrium Introduction: The denaturation of some proteins can be described by a two-state transition model in which the protein exists in either the native (N) or completely unfolded, denatured (D) conformation. In large and more complex proteins, there may be multiple unfolding intermediates where only part of the protein is unfolded or the protein adopts a different conformation. With these more complex protein structures the two-state transition may not apply and it may be necessary to account for multiple intermediates. The two-state transition model however, can often be applied to small globular proteins. Acid (pH), heat, and denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate are can be used to unfold and denature the protein. The denaturation process can be monitored by changes in physical properties of the protein that can be measured. Spectroscopic changes such as UV absorbance of amino acid side chains or other chromophores attached to the protein, changes in the fluorescence spectrum, or changes in circular dichroism (the way the protein interacts with circularly polarized light) have been used to monitor the native and denatured states of the protein. The aromatic amino acids that are responsible for the absorption spectrum in the UV range are usually buried when the protein is in the native conformation. Upon unfolding, these amino acids are exposed to solvent and may show a change in both the wavelength of maximum absorbance and the molar absorbtivity due to the change in chromophore environment. If the protein has tryptophan residues there is often a change in the fluorescence of the protein. The denaturation of ribonuclease A, lysozyme, α-lactalbumin and myoglobin by urea and guanidinium chloride fit the two-state model and show maximum differences in molar absorbtivity at 287, 292, 292 and 409 nm respectively. Chromophores for ribonuclease A, lysozyme, and α-lactalbumin are aromatic amino acids while the chromophore for myoglobin is heme. native configuration denatured A protein exists in its most stable form. Under biological conditions, this is the folded native state that retains biological activity. In this experiment, you will determine the free energy that is required to convert the protein from its native structure to the denatured state. This requires determination of the equilibrium distribution of the protein between the two states. This will be done in the presence of increasing amounts of denaturant. Solutions: 1) 2) 3) 0.1M NaH2PO4•H2O with 0.1 M KCl, pH 6.6 (NaOH) 9 M urea in buffer (This solution is nearly saturated. Warm to dissolve and then cool to room temperature before use.) protein stock solution - 0.5 mg myoglobin/mL buffer (pH 6.6). Do not shake the protein solution or cause foaming of the solution. Filter through a small plug of cotton to remove insoluble material. Abs409nm of 1:10 dilution should be 0.4 to 0.8. Procedure: Part 1: A) Prepare solutions 1-25 as shown in the table below. Calculate the urea concentration for each solution. Mix buffer and denaturant first, then add the myoglobin. ACCURATE PIPETING IS ESSENTIAL FOR GOOD RESULTS. Allow solutions to stand for at least 60 min. before reading absorbances. n. Read absorbance at 409 nm. Solution 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PBS, pH 6.6 mL 2.70 2.50 2.30 2.10 1.90 1.70 1.60 1.50 1.40 1.30 1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 9 M urea mL 0.00 0.20 0.40 0.60 0.80 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 Mb, 0.5 mg/mL 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.80 4.00 1.20 calculated urea concentration, M Abs at 409 nm 2.00 mL solution #24 + 2.00 mL buffer B) Analyses like the one performed in this lab should include a demonstration that the transition between native and denatured state is reversible. Solution 24 represents a protein that was denatured at a high urea concentration and then presented with a diluted urea solution which should allow rapid reversal of denaturation upon decrease in denaturant concentration. Prepare solution 24 and allow to sit for 45 minutes, then dilute to prepare solution 25. Allow this to sit for 45 minutes before reading absorbance. Solution 5 corresponds to a partially denatured protein. The absorbance of tube 24 should be divided by two for comparision with tubes 5 and 25. Does your protein renature? Part 2: Obtain a uv-visible spectra of the native and denatured protein to show the spectral differences in the two conformations. Use samples 1 and 18 and the visible to near uv region. Calculations: A. Determine Kd: Plot absorbance as a function of denaturant (urea) concentration. The measured absorbance at each urea concentration is Y. Plotted data should generate a graph like the one below. To calculate Kd you also need the values for YD (absorbance due to denatured) and YN (absorbance due to native) at each urea concentration. This is done through extrapolation. At very low urea concentration, the majority of the protein is still in the native configuration and the measured absorbance (Y) is effectively YN. A line through these points and extrapolated to higher urea concentrations can be used to determine YN at increasing urea concentrations. In a similar fashion, at high urea concentration, most of the protein is denatured the measured absorbance (Y) is effectively YD. A line through these points and extrapolated to lower urea concentration can be used to determine YD at lower urea concentrations. Because at low urea, the amount of denatured protein is low and hard to determine and because at high urea, the amount of native protein is low and difficult to determine, those points are only useful for the extrapolation and should not be used to find Kd. The range of interest for determination of Kd is the linear range where there is a rapid change in absorbance. B. Determine ΔGd: Calculate the fraction of the protein that is denatured at each urea concentration as measured by absorbance using the formula Yobs - Yn fd = Y - Y d n where fd is fraction denatured, Yobs is the observed quantity (absorbance), and Yn and Yd are the values for the native and fully denatured proteins at each of the urea concentrations, respectively. Plot fd vs urea concentration. Calculate the equilibrium ratio for each concentration of urea using the formula [D] fd Yobs - Yn Keq = [N] = f = Y - Y n d n Calculate the free energy change for denaturation at each denaturant concentration. ∆Gdenaturation = -RT(ln Keq) If we assume that ln Keq (and ΔGdenaturation) is a linear function of the denaturant concentration, a plot of ΔGdenaturation versus urea concentration will be a straight line and the y-intercept will be an estimate of the free energy of denaturation of myoglobin in the absence of urea, i.e., in buffer alone.