Biochemistry - week 2 (chapter 5)
Biochemistry - week 2 (chapter 5) CHEM 4712
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This 6 page Class Notes was uploaded by ShayD on Monday February 8, 2016. The Class Notes belongs to CHEM 4712 at University of Missouri - St. Louis taught by Xuemin Wang in Spring 2016. Since its upload, it has received 45 views. For similar materials see Biochemistry in Chemistry at University of Missouri - St. Louis.
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Date Created: 02/08/16
Dudaie 1 Biochemistry: CH’s 5 Chapter 5 Primary Structure One of the keys to understand the function of a given protein is to understand its structure 1. Polypeptide Diversity a. The primary structure of a protein is the amino acid sequence (also called the polypeptide chain) i. Each amino acid is covalently bonded to the other with bonds called peptide bonds b. Theoretical possibilities i. Since there are 20 different amino acid residues (individual a.a that make up the polypeptide chain) we can deride a method of calculating all the possible polypeptide chain combinations 1. 20 , where n is the number of residues in the chain Perspective: Let’s say we calculate a single polypeptide chain that 100 residues long. 100 130 20 = 1.27 x 10 possible combinations Dudaie 2 It is estimated there are 9x10 atoms in the universe! c. Actual possibilities i. The vast majority of polypeptide are contain 1001000 residues 1. These number are due to several constraints a. 40 residues is the near minimum for a polypeptide to be able to fold into a functional shape b. Whereas polypeptide over 1000 not only increase the risk of introducing errors in transcription & translation, but also the approach the limit of protein efficiency 2. Protein purification and Analysis a. Environmental conditions that may affect a protein during the purification process} all of which could cause a protein to denature i. pH ii. Temperature iii. Degradative enzymes iv. Absorption to surfaces b. Protein concentrations via spectroscopy i. A solution containing a solute that absorbs light follows Beer lambert’s Law A=log Io =εcl 1. ( I a. Where A absorbance(optical density); I is transmitted intensity at the given wave length; ε absorptivity; cconcentration; l length that light had to travel ii. Graph worth remembering: All these a.a peak at ~280 mm Dudaie 3 c. Purification as a stepwise process i. The idea is to eliminate other components selectively so that the desired substance remains 1. Chromatography a. A mixture is put in a liquid (mobile phase) and it’s percolated through a column containing a porous solid matrix. As solutes flow through the column it interact with the stationary phase. i. High performance liquid chromatography (HPLC) 1. Uses automated systems with precise applied samples with controlled flow rates ii. Hydrophobic interaction chromatography purifies non polar molecules 1. The matrix material is substituted with hydrophobic groupsat high salt concentration the hydrophobic groups on proteins interact with the matrix and get washed through the column iii. Gel filtration chromatography separate molecules by size 1. The matrix is made up of bead that fit smaller molecules this was larger molecules in an aqueous solution transverse the column faster than the smaller molecules Dudaie 4 2. Electrophoresis separates molecules by size & charge a. It’s the migration of ion in an electric field i. In Polyacrylamide gel electrophoresis the molecular separation is based on gel filtration (size and shape) as well as electrophoretic mobility (electric charge) ii. Another form of electrophoresis is SDS PAGE, which adds SSD (a detergent) to denature the protein so the proteins get separated by molecular mass only 1. Small polypeptide chains move faster and vice versa iii. Twodimensional electrophoresis, this runs SDS PAGE analysis as well as Isoelectric focusing which adds a pH gradient so each polypeptide chain will go to the according pH that corresponds to its pI Dudaie 5 3. Protein Sequencing a. To sequence a protein it first must be broken down into fragments small enough to be sequenced individually and the protein is reconstructed from the sequences of overlapping fragments i. Nterminus analysis can reveal the number of different subunits we can ID the end groups to establish the # of chemically distinct polypeptides (example is insulin... SEE page 1) 1. Disulfide bonds between polypeptide are cleaved via mercaptan b. Polypeptide cleavage i. Any polypeptide longer than 100 residues needs to cleaved prior to sequencing 1. Proteases enzyme the breakdown proteins a. Endopeptidases enzymes that catalyst via hydrolysis on internal peptide bonds in the chain b. Exopeptidases enzymes that catalyst via hydrolysis on the N or C ends of the chain Example worth remembering= Trypsin (highly specific) c. Edman Degradation i. This process removes the first amino acid in the chain 1. Edman’s reagent is called PITC ii. After each a.a is removed it is sequence, and eventually you will sequence the chain 4. Mass spectrometry a. Mass spectrometry accurately measures the masstocharge (m/z) ration of ions in a gas phase Dudaie 6 i. By comparing the molecular masses of successfully larger members of a family of fragments, the molecular mass (and the identity) can be determined ii. A method of mass spectrometry is called electrospray ionization , where a solution of macromolecules are sprayed through a narrow tube (a high voltage) that causes ionization of certain group these ion are analyzed in the spectrometer to determine the molecular mass 5. Protein evolution a. Evolution occasionally occurs of the proteome of an organism i. By analyzing and comparing proteomes we can see evolution unfoldrelated species have evolved from common ancestor so in turn the genes they inherited code for similar proteins 1. We can see this via phylogenetic trees b. Sequence comparison can provide information on protein structure and function i. Comparing homologous proteins (evolutionarily related) can indicate where a residue is essential or not 1. Homologous protein with the same function in different organism are known as orthologous
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