Weekly Notes for Lectures 4-5
Weekly Notes for Lectures 4-5 BCM 475 - M001
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BCM 475 - M001
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This 6 page Class Notes was uploaded by Annie Notetaker on Sunday September 13, 2015. The Class Notes belongs to BCM 475 - M001 at Syracuse University taught by M. Braiman, R. Welch in Fall 2015. Since its upload, it has received 148 views. For similar materials see Biochemistry I in Biochemistry at Syracuse University.
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Date Created: 09/13/15
The following includes material covered in lectures 4 amp 5 Detailed notes of material from textbook not covered in class will be included in the study guides Quotes indicate text obtained directly from textbook Lecture 0909 Protein Structure and Function III p 4559 amp 355361 Tertiarv Protein Structure 0 The three dimensional arrangement of a polypeptide chain Myglobin 0 Serves as an example of a protein possessing a tertiary structure 0 Compact oxygen binding protein responsible for providing oxygen to muscle cells 0 Single polypeptide chain of 153 amino acids 0 Contains a heme group nonpolypeptide prosthetic group that buries into the protein structure in an aqueous environment 0 The interior of the protein consists almost entirely of nonpolar residues such as leucine valine methionine and phenylalanine while the exterior of the protein contains both polar and nonpolar residues F or thermodynamic stability watersoluble polypeptide chains fold in a manner such that the hydrophobic side chains are clustered in the interior of the protein to avoid contact with the polar hydrophilic aqueous environment whereas polar charged chains are exposed on the surface Membrane Proteins 0 Membrane proteins differ from soluble proteins in the distribution of hydrophobic and hydrophilic groups 0 Integral membrane proteins extend through the lipid bilayer end to end 0 Peripheral membrane proteins do not span the entire lipid bilayer Can be fastened to the lipid bilayer membrane via hydrophobic amino acids Bacteriorhodopsin vs Bacterial Porin Bacteriorhodonsin 0 Membrane protein 0 Consists of seven membrane spanning alpha helices Membrane spanning alpha helices are the most common structural motif in membrane proteins 0 Consists of mostly nonpolar uncharged amino acid residues that are in contact with the lipid bilayer s hydrocarbon core or with other alpha helices Bacterial Porin 0 Bacterial membrane protein k nl39atcrfjl39lcd n La rgc39ly 39IIydro ph obit hydrophilic c 13 exterior nel Porin has a hydrophobic exterior with charged polar hydrophilic chains in its interior R groups towards exterior facing lipid bilayer are hydrophobic while R groups towards interior facing water channel are hydrophilic Beta barrel structure consists entirely of B strands Transmembrane Helices Can Be Accurately Predicted From Amino Acid Sequences 0 Membrane spanning alpha helices mostly nonpolar uncharged amino acid residues are the most common structural motif in membrane proteins 0 The hydrocarbon core of a membrane is typically 30A wide a length that can be traversed by an alpha heliX consisting of 20 amino acid residues 0 The free energy changes that accompany the transfer of a alpha helical segment amino acid residues from a hydrophobic membrane environment to a hydrophilic aqueous environment can be used to determine potential transmembrane helices amino acid residues of a alpha heliX that are located within the membrane bilayer 0 A hydropathy plot indicates potential transmembrane helices E g a peak greater than 84 kJ mol391 in a hydropathy plot for glycophorin indicates the single alpha heliX indicative of a transmembrane heliX Proteins Consist of Structurallv Independent Domains 0 Many polypeptide chains fold into domains 0 Domains are compact globular units with sizes ranging from 30 to 400 amino acid residues 0 Domains are connected with exible linkers Ouaternarv Structure of Proteins Multisubunit Structure 0 The spatial arrangement of subunits and the nature of their interactions 0 Subunit l polypeptide chain in a protein that may be identical to or different from other subunits within a protein 0 A dimer is a quaternary structure with 2 identical subunits Hemoglobin 0 Quaternary structure 0 Serves as an example of a protein composed of different subunits 0 Contains 2 identical alpha subunits 2 identical beta subunits to form a 02B2 tetramer Rhinovirus Protein Coat 0 Quaternary structure 0 Contains 60 copies of four distinct subunits that collectively become a spherical shell The Relationship Between the Amino Acid Sequence of a Protein and Its Three Dimensional Structure 0 Ribonuclease enzyme composed of 124 amino acid residues cross linked by four disulfide bonds 0 The following eXperiment with ribonuclease revealed how the amino acid sequence of a protein determines the three dimensional structure of the protein 1 The three dimensional structure of ribonuclease was destroyed with 8 M urea Chemical agents such as urea or guanidinium chloride disrupt secondary tertiary and quaternary structures of proteins Most polypeptide chains devoid of crosslinks assume a randomcoil conformation in 8M urea or 6M guanidinium chloride 2 Disulfide bonds within the enzyme were reduced with B mercaptoethanol Disulfides cystines converted to free sulfhydryls cysteines gtkTreatment with B mercaptoethanol in 8 M urea resulted in a fully reduced randomly coiled peptide without its normal activity denatured 3 B mercaptoethanol and urea removed by dialysis Urea removed before Bmercaptoethanol Removal of the two chemical agents responsible for denaturing the enzyme led to the oxidation of sulfhydryl groups by air the reformation of the enzyme into its active conformation and the restoration of enzymatic activity gtkConclusion the information needed to Specify the catalytically active Structure of ribonuclease is contained in its amino acid sequence Protein Folding 0 Neighboring proteins play a role in stabilizing a particular peptide conformation Protein Folding and Unfolding is a Highly Cooperative Process 0 Protein folding and unfolding is an all or none process that results from a cooperative transition 0 Proteins cannot directly jump from a folded state to an unfolded state or vice versa Unstable short lived intermediate structures form between folded states and unfolded states Lecture 911 Experimental Techniques I p 6574 Proteins Fold Bv Progressive Stabilization of Intermediates Rather Than Bv Random Search 0 The free energy difference between the folded and the unfolded states of a typical 100 residue protein is 42 k mol39l and thus each residue contributes on average only 042 kJ mol391 of energy to maintain the folded state 0 Fig 26 Depicts how the thermodynamics of protein folding resembles a funnel Some Proteins are Inherentlv Unstructured and Can EXist in Multiple Conformations 0 Intrinsically unstructured proteins IUPs have no distinct structure and therefore vary structurally based on interactions with other proteins 0 Lymphotactin eXists in two conformation Covalent Modification of Primary Proteins 0 Proteins can be covalently modified through the attachment of groups other than amino acids to augment their functions to achieve tertiary structures to change their activity andor localization 0 Fig 264 displays some common covalent modification of R groups Self Catalyzed Protein Modification 0 Self catalyzed post translational modification in GFP is responsible for the protein s ability to uoresce 0 Fig 265 The Proteome the Functional Representation of the Genome directlv from lecture 0 As of 2015 there are about 50000 genomes sequenced 22500 of Eukaryotes and 475000 for prokaryotes 0 The human genome contains about 3 billion base pairs of DNA and about 23000 genes 0 A genome represents a list of all the genes that make up an organism but it does not tell you when those genes are made into proteins or how and when they interact with each other 0 The proteome of an organism signifies a more compleX level of information content encompassing the types functions and interactions of proteins within its biological environment Protein Purification 0 Key to understanding protein function 0 Enables the determination of amino acid sequences the protein s biochemical function and the protein s structure Protein Purification Requires an Assav 0 Assay a test that will enable one to determine the presence of the protein of interest by identifying one of its unique properties 0 Increased assay specificity increased purification effectiveness 0 E g Enzyme lactate dehydrogenase catalyzes the reaction of lactate NAD to pyruvate NADH H An assay can be used to measure the enzyme activity an enzyme s ability to effectively catalyze a reaction and lower the activation energy of lactate dehydrogenase NADH absorbs light at 340 nm and NAD does not absorb light at this wavelength Therefore an assay can be utilized to determine how much of a sample absorbs light after a certain amount of time to subsequently determine the activity of lactate dehydrogenase increased absorption of light means the enzyme is present and working because its job is to catalyze the reaction and lead to the production of lightabsorbing NADH Protein Purification Requires the Determination of the Concentration of Protein in the Sample Being Assaved 0 Specific activity Enzyme activity Protein concentration The overall goal of protein purification is to maximize the specific activity Proteins Must Be Released From the Cell to be Purified 0 To release the proteins from the cells the cell initially undergoes homogenization 0 The homogenate then undergoes dz erential centrifugation to yield several fractions of decreasing density each still containing hundreds of different proteins From Fig 31 we can see how during differential centrifugation pellets of nuclear fractions form faster than pellets for mitochondrial fractions while pellets for mitochondrial fractions form faster than pellets for microsomal fractions 0 Finally each fraction is assayed and subsequently purified Proteins Can Be Purified According to Solubility Size Charge and Binding Affinity Separation bv Solubilitv 0 Salting out 0 Utilizes the principle that most proteins are less soluble at high salt concentrations Separation bv Size Dialysis Performed by placing a protein mixture concentrated solution into a dialysis bag with a semipermeable membrane and subsequently submerging the bag into a buffer solution Smaller molecules and ions diffuse out of the dialysis bag while larger molecules and protein aggregates remain inside the bag Not an effective technique for purifying proteins Gel Filtration ChromatographV Aka molecular exclusion chromatography Utilizes a column containing porous beads While small molecules can insert themselves inside the beads and in the solution between the beads large molecules can only ow to the aqueous solution between the beads Large molecules ow more rapidly through this column and emerge first because a smaller volume is accessible to them This purification technique is good for separating samples with a 50 difference in size Longer columns will enable the separation of samples with increased differences Separation bv Charge Ion exchange chromatography Negatively charged beads placed in a column will attract and bind proteins with a net positive charge at pH 7 opposite charges attract while negatively charged molecules will simply ow through the column Proteins bound to the negatively charged beads are eluted by increasing the salt concentration of the eluting buffer Sodium ions compete with positively charged groups on the protein for binding to the column Positively charged proteins can be separated by chromatography on negatively charged carboxymethylcellulose CM celluose columns Negatively charged proteins can be separated by anion exchange on positively charged diethylaminoethylcellulose DEAE cellulose columns Affinity Chromatography Highly selective purification technique A substrate molecule with high affinity selectivity for a protein of interest is covalently attached to a column When the sample is added to the column the substrate will selectively bind the protein of interest A wash buffer will then be poured through the column to remove unbound proteins Desired protein can be eluted off the column by the addition of a high concentration of a soluble form of the substrate molecule with high specificity for the protein of interest Gel Electrophoresis Method for separating proteins based on their molecular mass Electrophoresis the phenomenon in which a molecule with a net charge will move in an electric field V Ezf V velocity of migration of a protein or any molecule in an electric field E electric field strength z net charge on the protein f frictional coefficient Before gel electrophoresis proteins are initially dissolved in sodium dodecyl sulfate SDS a detergent to denature the proteins and to give the proteins an overall negative charge The overall negative charge on the proteins will then enable them to migrate to the positive pole when an electric field is applied during gel electrophoresis Polyacrylamide gels are commonly used for gel electrophoresis Polyacrylamide gels can have varying pore sizes Gels with a higher concentration of acrylamide are used for larger molecules The electrophoretic mobility of many proteins in SDS polyacrylamide gels is inversely proportional to the logarithm of their mass An exception to this relationship occurs with carbohydrate rich proteins and membrane proteins and some phosphorylated proteins For SDS polyacrylamide gel electrophoresis proteins that differ in mass by about 2 e g 50 and 5 lkd arising from a difference of about 10 amino acids can usually be distinguished Isoelectric Focusing Isoelectric point pl pH at which the net charge is zero and the point where electrophoretic mobility is zero Purification technique used to separate proteins based on their isoelectric points in the absence of SDS A pH gradient is first established in the gel The gel is then run enabling proteins to migrate to the region on the gel where the pH of the gel is equal to the pI of the protein Two Dimensional Electrophoresis The combination of isoelectric focusing with SDS PAGE
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