Exploration of Protein Folding Question: What conditions affect the folding of a protein? Pre-lab reading Atkins & Jones (5 th ed.): Sections 5.1 5.5; 9.8 9.9; and Section 19.13 Safety and Waste Disposal Eye protection should be worn at all times. Aqueous solutions can be washed down the drain with copious amounts of water. Place waste solids in the designated container. Background Proteins are biological molecules that play vital roles in the body. Proteins are responsible for a variety of functions, from making muscles contract to replicating DNA to carrying oxygen to cells. Proteins are made in the body when many amino acids small organic molecules found either in food or produced by the body are bonded together to make a very large molecule in much the same way that beads are strung together to make a necklace. The identity of the protein depends on the number and arrangement of these amino acids. For example, hemoglobin, the protein used by red blood cells to carry oxygen, contains 574 amino acids in a unique order. Depending on the chemical structure of their side chains, amino acids can be classified roughly as polar or nonpolar. The intermolecular forces between these amino acids determine the threedimensional structure of the protein. The protein s three-dimensional structure, in turn, determines the biological function of the molecule. In the case of hemoglobin, the interaction of the amino acids causes the protein to arrange itself in such a way that a binding site is created for oxygen molecules. The environment in which the protein is found also is critical in determining its three-dimensional structure. In aqueous solutions, amino acids that have polar side chains can form relatively strong dipole-dipole attractions or hydrogen bonds with water. These hydrophilic amino acids position themselves on the outside of the three-dimensional protein structure where they can be partially surrounded by water molecules. Since water is not able to form hydrogen bonds or dipole interactions with nonpolar side chains, hydrophobic amino acids are generally found inside the protein structure. The three-dimensional arrangement of the biologically functional protein is called its native structure. Denaturing Proteins There are a variety of ways in which the three-dimensional structure of the protein can be disrupted, that is, ways in which the protein can be denatured. The denatured protein is no longer able to carry out its biological function. Some possible ways in which a protein can be denatured include: 1. Addition of a large quantity of a small polar molecule. The added polar molecule interferes with intermolecular forces between the hydrophilic amino acids and the water by preferentially interacting with water. As a result, these hydrophilic amino acids, normally found on the outside of the protein, interact with other amino acids. These new interactions are weaker than the interactions between water and hydrophilic amino acids and therefore do not normally occur. 2. Addition of a detergent. A detergent is a molecule that has a nonpolar hydrophobic end and a polar hydrophilic end. The nonpolar end of the detergent interacts with the hydrophobic amino acids that are normally buried inside the protein while the polar end of the detergent interacts with the water. Thus the hydrophobic amino acids are pulled to the outside of the protein structure. 3. Increase in temperature. The increase in molecular motion caused by the increase in temperature disrupts molecular interactions.
4. Change in ph. Increasing or decreasing the ph changes the charge distribution in the protein and therefore alters the intermolecular forces between the protein and the solvent. 5. Mechanical shock. One example of permanently changing a protein structure by mechanical shock is beating egg whites to form a fluffy white foam (the meringue in lemon meringue pie). The egg white is a solution of albumin, a protein. The albumin is mechanically shocked or beaten until its structure changes and the protein is no longer water soluble. Stabilizing Proteins The three-dimensional structure of the protein can be made more stable by adding ions such as sulfate or phosphate to an aqueous solution containing the protein. Although the reason these anions stabilize the three-dimensional structure is not well understood, it is believed that sulfate and phosphate ions increase the surface tension of the solvent. In order to dissolve a protein in a solvent, a tiny cavity must be created in the solvent for the protein. If it is difficult to create the cavity in the solvent, the cavity will be very small and the protein has to be folded very tightly to fit inside. If it is relatively easy to make the cavity in the solvent, the protein doesn t need to be as tightly folded. Both sulfate ion and phosphate ion increase the surface tension of the solution, thereby making it much more difficult to form a cavity in the water and allowing the protein to remain folded. Detecting Structural Changes Cyanobacteria (commonly known as blue-green algae) contain an elaborate light-harvesting antenna complex in addition to chlorophyll molecules. The complex is made up of approximately 300 chromoproteins that absorb sunlight from 500 to 650 nm, a region of the solar spectrum in which chlorophyll does not absorb. Many health food stores and drug stores now sell dried Spirulina, one type of cyanobacteria, in capsule form as a protein source. As part of its antenna complex, this cyanobacterium contains phycocyanin, a chromoprotein composed of a chromophore called phycocyanobilin and a protein that is attached to the chromophore through the sulfur atom of the amino acid cysteine. Figure 1. The chomoprotein phycocyanin, which is composed of the four-ring chromophore phycocyanobilin and a protein. Phycocyanobilin is shown here in a linear conformation. The color of phycocyanin is strongly dependent upon its environment. In the native (folded) protein, the chromophore is held in a linear conformation by hydrogen bonding to nearby side chains of the protein. The protein acts as a scaffold to hold the chromophore in the desired linear position. If the protein is unfolded (i.e., denatured), the scaffolding of nearby side chains is removed from the phycocyanobilin, which then folds into a cyclic lockwasher conformation. The absorption spectrum of the chromophore in the cyclic lockwasher conformation is very different from the spectrum of the
linear conformation. Instead of absorbing in the visible region, the unscaffolded phycocyanobilin absorbs in the ultraviolet at around 370 380 nm. Therefore, the chromophore indicates the threedimensional arrangement of the protein structure: when in its native form, the chromoprotein has a dark blue color; when the chromophore is unfolded, its blue color fades away. This color can be observed visually and more precisely with a spectrophotometer. In addition to absorption spectroscopy, fluorescence can be used to determine the integrity of the protein structure. When the phycocyanobilin is held in a linear conformation by the protein scaffolding, it displays a red fluorescence that is apparent even in room light. When the chromophore assumes the cyclic lockwasher position, this fluorescence disappears. Recall that this chromoprotein is used by the bacterium to collect photons for photosynthesis. Since in this procedure the chromoprotein has been isolated from the other photosynthetic proteins found in the living organism, these photons cannot be transferred to photosystem II and therefore escape as light in the form of fluorescence. Thus the red fluorescence indicates that the chromoprotein is functioning properly. The lack of red fluorescence means the protein has been denatured, i.e., unfolded. Another visual indication of the denaturing of the chromoprotein is the appearance of a cloudy mixture. When the chromoprotein is not properly folded, the molecules tend to aggregate together and precipitate out of solution. When a protein precipitate forms, the solution will look milky. In this experiment, you will investigate the intermolecular forces at work in aqueous solutions of phycocyanin by examining the specific conditions under which the chromoprotein exists in its native and denatured forms. Spectrophotometry What we see as "color" is the result of the absorption and/or reflection of light of specific wavelengths. White light consists of electromagnetic radiation having wavelengths ranging from 360 nm to 700 nm. When white light strikes a colored object, radiation of certain wavelengths is absorbed. The radiation that is reflected from or transmitted through the object will not contain the absorbed wavelengths, and the object will appear colored. Table I below lists the colors of various wavelengths of visible light: Red- Blue- Red Blue Red- Yellow Blue- Yellow Yellow- Table I. Wavelengths and Corresponding Colors Wavelength (nm) Color 750-610 red 610-590 orange 590-570 yellow 570-500 green 500-450 blue 450-370 violet Figure 2: Color Wheel The color wheel in Figure 2 shows the complementary relationship between colors absorbed by a solution and those transmitted. When a sample absorbs light of a particular color, we perceive the object as the complementary color, i.e., the color opposite the absorbed color on the color wheel. For example, if a sample absorbs red light, the sample will appear blue-green to our eyes.
According to theory, the amount of light of a specific wavelength absorbed by a sample depends on 1) the concentration of the absorbing substance in the sample, 2) the thickness of the sample, and 3) the chemical characteristics of the absorbing (colored) species. When the sample is in solution, the relationship among these factors is expressed by Beer's Law: - log T = lc (1) where and T is transmittance, the fraction of incident light that is transmitted is the molar absorptivity, which is a constant characteristic of the absorbing species l is the sample thickness c is the concentration of the absorbing species in solution. The absorbance, A, of a sample is defined as follows: A = - log T = - log (%T/100) (2) The percent transmittance, %T, is 100 times the transmittance. Most instruments report %T rather than T. Substituting the definition of absorbance into Beer's Law yields the useful form: A = lc (3) In this investigation, you will measure the absorbance of a series of protein solutions to investigate how protein structure is affected by adding different amounts of acid to the solution. Measure the absorbance of each solution at 620 nm (orange light, since the protein solution is blue). Terminology Procedure Hydrophilic: having relatively strong intermolecular attraction to water Hydrophobic: having relatively weak intermolecular attraction to water Native structure: the three-dimensional arrangement of a biologically functional protein Denature: the disruption of the three-dimensional arrangement of a protein resulting in loss of the protein s biological function Isolation of phycocyanin from blue-green algae (Spirulina) Each group should do this part of the experiment and obtain a sample of phycocyanin. Once you prepare your sample of phycocynin, add your sample to the beaker at the front of the lab. All lab groups will use this pooled phycocyanin sample for the rest of the lab to ensure that everyone starts with the same concentration of phycocynin. Pour the contents of a blue-green algae (Spirulina) capsule into a clean, dry mortar. Add an approximately equal volume of silica to the mortar and vigorously grind the mixture with a pestle until the mixture has a fine, smooth texture (about 4 minutes). Divide the solid roughly equally between four clean, dry test tubes and add 5 ml of 0.1 M sodium phosphate buffer (ph 7) to each test tube. Stir each solution well using a glass stirring rod and centrifuge the mixtures for about two minutes. Use a disposable pipet to transfer the clear supernatant liquid from the four test tubes to a small beaker (avoid transferring any of the solid material from the test tube). Use this supernatant liquid containing the phycocyanin for the experiments below. Observation of phycocyanin protein fluorescence Observe and record the color of the phycocyanobilin protein. Shine a flashlight up through the bottom of the small beaker containing the protein solution and record your observations.
Observation of phycocyanin protein under various conditions Preparation of the Control Sample Use a graduated cylinder to measure 5.0 ml of 0.1 M sodium phosphate buffer (ph 7) into a small test tube. Use a micropipette to deliver 1 ml (1000 L) of phycocyanin solution into the test tube. Mix gently. This tube contains a solution of the intact (native) protein. Record your observations of the color and appearance of the control sample, and retain this solution to compare to the test samples. Remove a small amount of the solution and place it in a cuvette. Measure the absorbance of the sample at 620nm. Preparation of Test Samples Effect of ph You will be assigned two volumes between 0.5 and 5.0 ml of 0.1 M HCl to test. Use a graduated cylinder to deliver your assigned volumes of 0.1 M HCl to two different clean dry test tubes. Use a conditioned glass pipet to deliver enough 0.1 M Phosphate buffer solution to each test tube to bring the total volume up to 5.0 ml. To each test tube, use a micropipette with a clean tip to deliver 1.0 ml of phycocyanin from the class s pooled sample. Record your observations of both color and fluorescence (with the flashlight) for each sample. Be sure to compare the color and the fluorescence with the control sample. When you have recorded the observations for your samples, place your test tubes in a centrifuge for 5 minutes. Compare the samples again with the control and record the observations. Remove a small amount of the solution in the centrifuged sample and place it in a cuvette. Measure the absorbance of the sample at 620nm. The data for all groups sample will be pooled. Be sure to give your results to your instructor. References Atkins, P.; Jones, L. Chemical Principles: The Quest for Insight, 5 th ed.; Freeman: New York. 2010. Bowen, R.; Hartung, R.; Gindt, Y. M. J. Chem. Educ. 2000, 77, 1456. Heller, B. A.; Gindt, Y. M. J. Chem. Educ. 2000, 77, 1458. Jones, C. M. J. Chem. Educ. 1997, 74, 1306.