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Chapter 5I. Part A: The thermodynamic hypothesisa. The thermodynamic hypothesis states that the native structure of a protein is determined solely by the properties of the protein and is not the result of an external template. In other words, the polypeptide chains will be driven to adopt conformations that maximize their intrinsic molecular properties.b. A native structure is the structure the protein adopts under normal physiological conditions.c. It is in this way that this hypothesis states that the sequence of the protein is what determines its folding because it doesn’t require any external force to make it form its structure, but rather occurs spontaneously over time. d. This spontaneous folding is crucial to protein folding, because it is what allows the extremely large diversity in protein structure. If every protein was folded via a similar mechanism, then the diversity would be non-existent. e. Molecular chaperones are molecules that assist in protein folding. They make sure that proteins do not become nonspecific clumps, which is often the case when the hydrophobic regions become exposed during folding. The chaperones prevent this aggregation by binding to the chains and then releasing them in a coordinated fashion.II. The thermodynamic hypothesis was first established for an enzyme known as ribonuclease-A, which can be unfolded and folded reversiblya. This hypothesis was first created by Christian Anfinsen during his experiments using ribonuclease A, which catalyzes the breakdown of RNA into ribonucleotides. At the time of his experiments, the structure of this enzyme was unknown. b. How did he do it? He understood the idea that in order for ribonuclease A to cleave RNA effectively, all of its components had to come together in the correct configuration to perform its catalytic duties. He measured the catalytic activity of the enzyme, and based on the values was able to determine to what extent the native structure of the enzyme was present in the sample. c. One of his experiments was with the 8-cysteine residues found in the enzyme sequence. In the native form, all 8 are oxidized to form 4 disulfide bonds. Knowing that catalytic activity could be used to measure the correct native structure, he also began to look at the bonding of these cysteine residues. He knewin order to be in the native structure, only 1 structure would work. In other words, the binding had to be ordered. If allowed to oxidized and renatured in the absence of urea, the native structure is obtained. But in the presence of urea, there are 105 different possible binding configurations, in which only 1 is the native structure. d. What this proves? If you were to remove the urea, the protein would refold into its native structure. This proves that all the information necessary for proper folding is found within the proteins sequence. It can be reversibly folded and unfolded to prove this case. The urea acted as a denaturing agent that mess with the hydrophobic effect between the protein and water, causing the RANDOM bonding and incorrect structure. III. Part B: Protein structure is conserved during evolution while amino acid sequences varya. The first two structures to ever be determined were that of hemoglobin and myoglobin. Both are in the globin family, which consists of a set of molecules that can bind and transfer O2 and CO.b. Myoglobin is a monomer with a single heme group. Hemoglobin is a tetramer with four heme groups. If you compare their structures, the alpha and beta chains of hemoglobin look strikingly similar to that of myoglobin. Also, both molecules adopt the globin fold which is a 3-D arrangement of the alpha helices. Since these structures are so similar, one would assume their sequences would also be very similar. c. However, comparison of their sequences shows that of the 150 residues, only 28 are identical to the alpha and beta hemoglobin. The two chains of hemoglobin are very similar to each other, but the degree of similarity decreases when compared to myoglobin. d. This proves that, as the fold of the protein may remain partially constant throughout evolution, the sequences vary. e. It can be further proven through comparison with hemoglobins from differing species. If you compare multiple, it shows that the only two residues held in common by all hemoglobins is that of a histidine which is used to hold the heme in place, and a phenylalanine which is meant to stabilize the heme group. In other words, although they all share the same structure, the sequences all vary. IV. Similarities in protein sequences can be quantified by considering the frequencies with which amino acids are substituted for each other in related proteinsa. In the last section, the way in which the sequences were compared was using the method of sequence identity. Sequence identity solely looks for the exact same base in areas of corresponding sequences. It fails to take into account why substitutions happened where they did, and the fact that certain substitutions may have little to no effect on the overall thermodynamic protein folding. i. Conservative Substitution : A substitution of one protein residue by another one with SIMILAR CHEMICAL PROPERTIES. For example, the substitution of serine for threonine has little effect because both have very similar chemical properties.ii. Non-conservative Substitution : A substitution in which the residue being replaced and the residue doing the replacement differ in their chemical properties. For example, the substitution of glycine for glutamate differs due to glutamates negatively charged character. b. One way to quantitatively measure the degree of similarity and difference between sequences is to create a conservation score on how similar the chemical structure of the two residues are. However, this fails to take into account how these work with other residues, and hence how it alters the structure.c. BUT, what we do know is that substitutions that occur say in the globin protein family, do not have a great effect on the overall function or structure because they have proven to be evolutionarily similar. Therefore, the substitutions made have already been filtered by natural selection and evolution. V. The BLOSUM matrix is a commonly used set of amino acid substitution scores.a. This is found on slide 16. What is shows is the probability of an amino acid in row “I” being replaced by an amino acid in row “j”. A positive number corresponds to a

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FSU BCH 4054 - Chapter 5

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