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Biochemistry - week 2 (chapter 5)

by: ShayD

Biochemistry - week 2 (chapter 5) CHEM 4712

Marketplace > University of Missouri - St. Louis > Chemistry > CHEM 4712 > Biochemistry week 2 chapter 5
GPA 3.74

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About this Document

These notes combine both the notes from class and a detailed summary of chapter 5. It covers material such as: polypeptide diversity, different purification analysis (mass spectrometry and electrop...
Xuemin Wang
Class Notes
polypeptide diversity, different purification analysis, mass spectrometry, electrophoresis, protein sequencing techniques, protein evolutio
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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 groupsat 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  unfoldrelated 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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