3 - Lecture Guide

Review of Lecture 3 Questions to think about…. The sigmoidal curve for the binding of oxygen by haemoglobin illustrates the phenomenon of cooperative binding.ddsadsa The process by which a protein in a random coil conformation assumes its native shape is called renaturation. The actual site of oxygen binding in the heme of myoglobin is iron. RECALL THAT HEME IS A PROTOPORPHOYRIN RING. Proteins that possess quaternary structure are called__. The decreased affinity of haemoglobin for oxygen due to elevated levels of carbon dioxide and H+ is called _____. An organic molecule in erythrocytes that lowers the affinity of haemoglobin for oxygen is____. 2-3 BISPHOSPHOGLYERATE ____are proteins that assist in the cellular process of protein folding such that a large fraction of the protein molecules affected achieve their native conformation and are thus biologically active. CHAPERONINS The activity of some proteins, such as Hb, is modulated by specific small molecules called ______. Such proteins are called ______. Where does 2,3-bisphosphoglycerate interact with haemoglobin? What bonds or interactions are responsible for this interaction? NON-COVALENT Why is haemoglobin better suited for its job as the oxygen-carrying protein in the bloodstream than myoglobin? (Include a discussion of oxygen pressure in the lungs and peripheral tissues, and the O2-binding curves of the two proteins.) THE PARTIAL PRESSURE OF OXYGEN IN THE VIENS IS AROUND 20 PO2 WHILE THE PARTIAL PRESSURE OF OXYGEN IN THE ARTERIES IS 100. THE MYOGLOBIN MOLECULE IS HALF SATURATED AT A PARTIAL PRESSURE MUCH LOWER THAN THE VEINS, THUS IT ACCEPTS OXYGEN MUCH MORE READILY. ALSO RECALL THAT MYOGLOBIN IS A SINGLE PROTIEN UNLIKE HEMOGLOBIN WHICH IS MADE UP OF FOUR SUBUNITS. MOREOVER, MYOGLOBIN IS NOT AFFECTED BY OTHER BIOCHEMICALS LIKE CO2, H, AND BISPHOSPHOGLYCERTATE, HEMOGLOBIN IS. THESE CHEMICALS ARE IMPORTANT FOR THE REGULATION OF HEMOGLOBIN BECAUSE IT INDICATES THE OXYGEN LEVEL CONTENT AND THE AMOUNT THAT IS REQUIRED TO REFEUL IN OXYGEN. In the arteries, the partial pressure of oxygen is nearly at 100. At this pressure, both hemoglobin and myoglobin are fully saturated due to the high pressure. In the veins, the partial pressure is around 20. At this pressure, hemoglobin is only half fully saturated and tends to release half the oxygen it carried from the lungs. The advantage with myoglobin is that at this pressure of 20, it is fully saturated, so muscles need a heme protein that will fully take up all the oxygen it can get even at very low pressures. In fact, at 20 PO2, myoglobin is fully saturated. This could be due to the fact that myoglobin is a single peptide unlike hemoglobin, which is an olimeric polypeptide made up of four subunits that work cooperatively. Moreover, hemoglobin is affected by external molecules such as co2, H+, and 2,3-bisphosphoglycerate. So, in order to answer the question, myoglobin is not better suited for carrying the oxygen in the blood stream because even at the low pressures of the veins, it will not release the oxygen it has carried. The pressure in our body rarely decreases less that 20 PO2, therefore there is an unlikely chance that oxygen will ever get to any muscles requiring O2. As mentioned earlier, hemoglobin is regulated by many chemicals, including 2,3-bpg… this binds to hemoglobin and encourages the release of the remaining two o2 molecules that are not normally released at vein pressures; myoglobin is not affected by this molecules and will not encourage the release of the single oxygen molecule it carries. Do proteins have a characteristic melting temperature? NO Would you be able to renature a fibrous protein? How about a globular protein? FIBROUS PROTEINS ARE VERY RIGID DUE TO THEIR PHI AND PSI ANGLES. What is a molten globule? A PROTIEN THAT IS SEMI FOLDED OR NOT FOLDED TO ITS EXACT NATIVE SHAPE. What are prions? What is the difference between the normal conformer and the rogue conformer? Prions come in two types, PrpC which is the soluble naturally degradable type and the PrpSc which is the insoluble/non degradable type. The PrpC has a highly nature level of alpha helices and a low level of B-sheets. The opposite is true for the PrPsc which have a high level of Bsheets and low number of Alpha sheets. When the infectious version enters a persons body, it causes improper folding of the healthy version. Why was Tikvah Alper considered a heretic? He suggested that an infectious agent that lacked nucleic acids could cause diseases. Why did Stanley Prusiner win a Nobel Prize? HE DISCOVERED THE PROTENIOUS INFECTIOUS DISEASES (PRIONS) What is Kuru? Cruetzfeld-Jacob disease? Scrapie? All the same disease caused by prions. What are the two classes of molecular chaperones we discussed in class? The two different classes are HSP60’s and HSP70’s… The HSP70s binds to hydrophobic regions of unfolded polypeptides and assists their binding. It also prevents unfolded peptides from folding before they are transported outside the cell. What is meant by ‘assisted folding’? Explain why a vitamin C deficiency leads to scurvy. VITAMIN C ACTS AS A COENZYME THAT REGULATES AN ENZYME THAT ADSS HYDROXYL GROUPS TO PROLINE THAT MAKE UP COLLAGEN, A STRUCTURAL FIBROUS PROTEIN. WITHOUT HYDROXYLTION OF PROLINE, THE STRUCUTRE LOOSES ITS RIGIDITY. Outline the key differences between myoglobin and haemoglobin. MYOGLOBIN – SINGLE SUBUNIT… ONE PROTOPORPHORIN RING THAT ACCEPTS OXYGEN.HEMOGLOBIN – 4 SUBUNITS, EACH WITH A PROTOPORPHRYIN RING THAT ACCEPTS OXYGEN MYOGLOBIN IS FOUND ONLY IN MUSCLES, WHERE AS HEMOGLOBIN IS FOUND IN BLOOD. MYOGLOBIN IS NOT REGULATED BY CO2, H+, OR 2-3 BPG… It is not regulated by PH. Describe the Anfinsen experiment. Why was this experiment an important turning point for biochemical characterization of proteins? ANFINSEN DENATURED PROTEINS (RNASE FROM BOVINE PANCREATIC RIBONUCLEASES) BY ADDING 2-MERCPTAETHANOL TO A PROTIEN WHICH DENATURED IT BY BREAKING DOWN THE DISULFIDE BRIDGES THUS ACTING AS A REDUCING AGENT. HE THEN ADDED 8M UREA TO RENATURE THE PROTIEN INTO ITS ORGINAL NATIVE STATE. What are chaotropic agents? Give examples. These agents allow water to solvate hydrophobic regions of a globular protein in order to assist its denaturation, urea is a chaotropic agents. What are reducing agents? Give examples. Beta mercaptoethanol is a reducing agent that allows amino residues to become oxidized, or allows the oxidation of amino acids. For instance, mercaptoethanol breaks the disulfide bridges in the bovine pancreatic ribonuclease (RNase) into separate cysteine residues. DTT - DITHIOTHREITOL Know the different conditions under which proteins can be denatured. PH, TEMPERATURE, ACETONE, ALCOHOL, HEAVY METALS, DETERGENTS, MECHAICAL STRESS, TEMEPETATURE CHANGES, STRONG ACIDS, STRONG BASES, SALTS, REDUCING AGENTS, DTT, BETA MERACAPTOETHANOL, CHAOTROPIC AGENTS. Describe the energy landscape for protein folding. THE FURTHER DOWN YOU GO, THE MORE NATVE IT BECOMES, THE HIGHER YOU GO THE GREATER THE ENTROPY AND THE MORE UNFOLDED THE PROTEINS ARE. What role do molecular chaperones play in protein folding? Describe the two classes of molecular chaperones. HSP70, HSP60 --) GROES, GROEL… are mostly found in cells that are heat shocked. How can mutagenesis be used as a tool for the study of protein structure? - it knocks out a particular gene that is thought to encode a protein – WHICH RESULTS INTO A PARTICULAR PHENOTYPE, THIS CAN BE SEEN AFTER HOMOLOGOUS RECOMBINATION. Know the structures for: Urea Guanidinium chloride 2-mercaptoethanol (aka ?-mercaptoethanol) 4-hydroxyproline Fe(II)-protoporphyrin IX heme Carbamate adduct 2,3-BPG