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MCDB 310 1st Edition Lecture 4 Outline of Last Lecture I. Protonated vs deprotonated: jargon for proteins, acids and basesII. Physiological buffersIII. Amino acids and proteinsa. Structure of 20 amino acids to memorizeIV. Ionization of amino acidsa. Amino acids with non-ionizable R groupsb. Amino acids with ionizable R groupsV. Formation of peptidesa. Polypeptide structure and function in the bodyOutline of Current LectureI. Protein biochemistryII. Protein purificationa. Purification by chargeb. Purification by sizec. Purification by affinity for a ligandd. Electrophoresise. Protein analysis (how do we know that the protein we have collected is pure?)III. 4 Levels of protein structurea. Primary structureCurrent LectureThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.I. Protein Biochemistrya. Each protein has a function determined by its structure which is determined by its amino acid sequenceb. You need purified protein to determine the sequence of amino acidsc. A protein’s biochemical function is called its activityII. Protein Purificationa. Grow cells chosen from an animal that contains a gene that codes for the protein of interest in bacterial DNA (most commonlyE. Coli or insect cells)b. Induce protein expressionc. Break the membrane of the cell so that all the proteins (thousands of them) come out of the cell and into a purification bufferd. Use chromatography to purify your protein of interest (because proteins are usually colorless, odorless, hard to identify)i. Column Chromatography: Put the protein sample in the top of the column, as they pass through the buffer at different speeds, they can be collected at the bottom of the column separatelye. Separation relies on difference in physical and chemical properties such as:i. Charge (dependent on the amino acids that make up this protein)1. Proteins also have a Isoelectric Point (pI) (they retain all the properties of the amino acids that make them up)2. Usually the net charge, however, is 0 (the charges coming from the amino acids cancel each other out)3. Basic Proteins: Have more positively charged amino acids (positively charged at a pH of 7)4. Acidic Proteins: have more negatively charged amino acids (negatively charged at a pH of 7)a. Remember: you can change the charge of a protein by changing the pH, but at very high or very low pHs the protein will denature (unfold)5. Separation by Charge: a. Use a cation exchanger (a form of column chromatography filled with a charged resin), which will bind to cationic proteins (proteins rich in lysine and arginine)b. Anions are repelled by the resin (which is negatively charged), and flow through the column the fastest and are collected at the bottom of the columnc. Take the cationic proteins off the column by introducing another cation in high concentrations that will compete with the cationic proteins causing them to be released and flow down the columnd. Can also change the pH of the column to switchthe flow of the column ii. Size (dependent on how many amino acids make up the protein)1. Separation by size:a. Inside the column, there are beads that have small pores within themb. Small proteins will flow through the pores, slowing them down as they move down the columnc. Whereas, the larger proteins cannot fit in the pores, so they flow down the column the fastestd. Elution: process of getting the protein out of the bottom of the columne. The larger the molecular mass, the lower the Volume of Elution (Ve) (larger proteins come out of the column without using a lot of buffer, so the final volume is small)f. Vo: void volume of the column (the volume thatis constant in the column)g. Relative elution volume (Ve/Vo) has an inverse linear relationshipi. The graph of log(molecular weight) vs Ve/Vo will be a line with negative slopeii. Can be used to find out the molecular weight of an unknown proteiniii. Affinity for a ligand (dependent on interactions between ligands and amino acids)1. Separation by affinity for a ligand (a molecule that binds to a protein):a. Example in the book: ATP column to purify an ATP binding proteini. ATP in the column only binds to the protein with an affinity for ATP, while the rest of the proteins in the column will flowthrough the column much fasterii. Elution is possible by “outcompeting” the ligand present in the column (add in a free ligand that will bind to the protein, causing the protein/ligand to flow throughthe column together)b. Could also attach a tag (sequence of a few amino acids that do not affect the function of the protein) to the proteini. A Nickel column binds to a polyhistidine tagii. Glutathione column binds to Glutathione S Transferase (GST) tagiii. Amylose column binds to Maltose BindingProtein (MBP) tagiv. These three tag/column combinations canbe used instead when the protein of interest does not naturally bind to a ligand f. How can you tell that your protein is purified?i. Use Electrophoresis: load a sample of proteins into the sample well in the top of gel matrix with a positive electrode at the bottom and negative electrode at the top1. An electric field pulls proteins according to their charge (positive electrode at the bottom will pull negative proteins down)2. Also, the gel matrix causes larger, more bulky molecules to flow through much more slowly3. Therefore, the size and shape of the protein matters4. But we want to separate them solely based on size:a. Use SDS-PAGE: sodium dodecyl sulfate (an ionicdetergent)b. SDS micelles bind to and denature the proteinsc. SDS gives all proteins a uniformly negative charge, and all proteins are completely unfoldedd. Therefore, the protein will flow down the matrixat a speed determined only based on sizei. This is different than separating based onsize (as mentioned above) because itseparates the proteins based on their DENATURED sizeii. Whereas, regular size exclusion chromatography is performed on active, folded proteins5. Also use Coomassie blue staining: stains proteins based on molecular weight, causes blue bands to separate on the gel matrix a. running known standards through and analyzing them can be used to calculate molecular weight of unknown proteins6. Could also use Western Blot:a. Proteins separated by SDS-PAGEb. Transfer onto nitrocellulose membranec. Specific antibodies bind to the proteind. Will cause one clean band to show up on the gel matrix (so you know


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