Biochemistry I Notes for Test 1 - I 4.0ed the test!

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University of Central Florida
BCH 4053

Biochemistry Spring 2014 January 6, 2014 January 8, 2014 *Bulk of test will be ch3 and 4 Ch.1 *Memorize table 1-2 (common functional groups and linkages in biochem *Know ch 4 structures of amino acids. *Don’t worry about section 3 -Biochemistry is the study of life -Hydrolysis: you need water to break the bond and energy is released -Condensation makes water and needs energy to make bond -Only DNAcan replicate Compartmentation: 1. Biological stems evolved to store biological molecules within a lipid membrane (cell) 2. Within this cell other compartments exits which promotes efficiency by maintaining high local concentrations of reactants 3. Metabolic pathways are compartmentalized so that they can efficiently synthesize molecules or generate energy. Thermodynamics (assigned): a spontaneous process occurs with a decrease in free energy. Entropy (ΔS): the process in which matter leans towards disorder. Ch.2 Water: 1. Excellent solvent for biological molecules 2. Polar, can form hydrogen bond with other molecules and with other water molecules. 3. Electrostatic attractions between water dipoles are crucial to the properties of water. Structure of ice: 1. Highly structured, tetrahedral 2. Becomes more disordered as water melts, but still most of the hydrogen bonds remain. 3. Hydrogen bonds cause water to expand upon freezing 4. Liquid water consists of a rapidly fluctuation 3 network of H-bonds 5. Water solvated most biomolecules, via hydrogen bonds, making them water- soluble and playing a role in their 3D structure. Biochemistry Spring 2014 Solvation of ions: Water of Hydration, Hydration shell -An “envelop” of water molecules surrounding the ion or molecule in water. -Being rather strong, these water molecules must be shed in order to approach another molecule before reaction. January 10, 2014 Hydrophobic effect 1. non polar molecules do not dissolve in water, but rather they exclude water causing the non polar molecule to aggregate 2. DNAand proteins assume their shape partially due to the hydrophobic effect When a non polar molecule is dispersed in water, it cannot form hydrogen bonds with the water molecules but rather the water molecules form a cage-like type of structure around the non polar molecule to avoid contact. Less # of water molecules around the non polar molecule in an ordered state Higher entropy state----greater ΔS -----entropically favored. Amphiphilic compounds are partially hydrophobic and partially hydrophilic -these compounds self assemble in solution to form micelles (sphere, all tails touch to a center) and bilayers (tails touching tails.) -polar head, hydrophobic tail Water moves by osmosis and solutes moves by diffusion Osmosis: movement of solvent molecules from a region of high concentration to a region of low concentration Osmotic pressure: is the pressure that must be applied to the solution to prevent the inward flow of water. It is proportional to the concentration of solute. Dialysis: technique used to de-salt or purified a protein solution. Asemi-permeable membrane with a particular molecular weight (size) cut off is used Low pH has a high number of protons and will protonate High pH has a low number of protons and will deprotonate The equilibrium constant for an acid-base reaction is expressed as dissociation constant. HA+ H2O  H3O+ +A- K=[H3O+][A-]/[HA][H2O] pKa is where you have 50% of your molecules deprotonated. *Good to know figure 2-17 Biochemistry Spring 2014 Ch3. Nucleotides, nucleic acids, and genetic info Nucleotides 1. Key DNAmonomers; consist of a nitrogenous base, a ribose or deoxyribose and one or more phosphate 2. Nitrogenous base: 2 purines and 3 pyrimidines 3. Memorize the core and the numbers of the image 4. Nucleotides (sugar component) Nucleotides consists of a nitrogenous base, a ribose or deoxyribose and one or more phosphate *note: 5’deocynucleotide and 3’-ribonucleotide also exits nucleosides: when the nucleotide is missing the phosphate group. Memorize the structures and the letters associated with the names in table 3-1 Biochemistry Spring 2014 Uracil is specific to RNA ADP and ATP, best known nucleotides “energy carrier” Know the structure ofATP andADP Nucleotide derivatives participate in a wide variety of metabolic processes Biochemistry Spring 2014 ADP- glucose: “activated” monomer used by plant cells during starch synthesis. NucleicAcid Structure • DNAand RNA, polynucleotides Biochemistry Spring 2014 • Linked by phosphodiester bonds • The phosphate of these polynucleotides are acidic and at physiological pH they are deprotonated (polyanions) • These polynucleotides contain a 5’-end and a 3’–end that are unique DNA: double helix, 2 antiparallel strands RNA: single helix, may form intramolcular base pairs. January 13, 2014 DNAand RNA, polynucleotides -linked by phosphodiester bonds practice theAU(T) C G structure NucleicAcid Structure -1953 watson-crick x-ray data taken by rosalind Franklin -DNAis a right handed double helix (clockwise) Watson-crick DNAmodel 1. Two polynucleotide chains wind around a common axis to form a double helix 2. The two strands are antiparalled, forming a right handed double helix 3. The bases (purines and pyrimidines) occupy the core of the double helix. Biochemistry Spring 2014 4. The phosphate groups are in the periphery in contact with the aqueous environment 5. The surface of the double helix contains two groves. 6. Each base is hydrogen bonded to another base in the opposite strand to form a plan for base pair commentary base paring. * Memorize the structures of the poses DNA: base-to-base hybridization (hydrogen bonding) Hydrophobic molecule (benzyene) will interclulated the two bases of a DNAmolecule. RNAis a single stranded nucleic acids -But it can base-pair with complementary bases within the same RNA strand forming RNAstem loops -Same of these RNAstem loops have been found to have catalytic properties or to bind metals and small molecules. DNAcarries genetic information Biochemistry Spring 2014 - non-pathogenic - transformed bacteria pathogenic bacteria Know definitions DNA Replication: When a cell divides, each DNAstrand acts as a template for the assembly of its complementary strand DNA transcription: DNAtranscribes into RNA DNA translation: DNAinfo translated into proteins mRNA- messengers RNA tRNA- transfer RNA *standard genetic code (will have for test) NucleicAcid Sequencing 1. nucleic acids can be cut at specific sequence my restriction enzymes 2. Nucleic acid fragments can be separated by size using electrophoresis. 3. Nucleic acid sequence not only give you into about the nucleotide sequence in genes, but also give you indirect information about the sequence of amino acids in proteins. 4. NAsequences can be duplicated, modified and explussed making it possible to study proteins that could not be otherwise be obtained in useful quantities Biochemistry Spring 2014 Overall strategy for NAsequencing. 1. Cleave NAinto small fragments 2. Determine the sequence of residues in each fragment 3. Determine the order of the fragments in the original piece by aligning fragments that contain overlapping sequences. Central Dogma of molecular Bio (not really) Flow id information is directional Retroviruses retroviruses: RNAviruses that contains RNAand two proteins: reverse transcriptase (RT) and an integrase (IT) upon infection the RNA, the IT and RT are injected into the infected cell where the RNA is converted to DNA, by RT and integrated to the cells genome by the integrase. January 15, 2014 First attempt: clearing RNAnon-specifically for sequencing. Snake venom: Phosphodesterase- an enzyme that cleaves phosphodiester bonds Discovery of restriction endonucleases accelerated and simplified the process. Restriction site: two most common enzyms Electrophoresis: separates nucleic acids according to size Biochemistry Spring 2014 -Anucleic acid treated with a restriction endonuclease will generate fragments small enough to be able to be separated by size, shape and charge. DNAsequencing: chain termination method dNTP, deoxynucleotide ddNTP: dideoxinuclotide DNApolymerase I -key enzyme use in the chain termination DNAsequence method. This enzyme adds I of the 4 deoxynucleotide (dATP, dCTP, dGTP, dTTP) into a complementary polynucleotide chain being elongates from 5’to 3’. -Apre-existing template is required. -you need a free hydroxide (OH) on the 3’and the 3 phosphates are in the 5’ -if the OH is missing it will stop. ddNTP (dideoxynucleotide or dideoxynuleoside triploshate) Paraphosphate is very unstable but interacts and drives most biological reactions. Fluorescent dideoxynucleotide analogs -eachATCG analog nears a different ldye Sequence variation can be liked to human disease. dsDNAcan adopt either a lineal or circular conformation. *DNAin bacteria is circular and is called a plasmid. Biochemistry Spring 2014 January 17, 2014 dsDNAcan adopt either a lineal or circular conformation -DNAin bacteria is circular and is called a plasmid. Genetic engineering, manipulating DNA 1. Segments of DNAcan be cloned; reproduced in a host organism (bacteria, insects, plants, etc.) 2. Using these techniques, (recombinant DNA), genes can be introduced in a host organism (transgenic organism) for the expression of; a. Aparticular RNAand protein in large amounts, b. Amutated protein, Bacterial DNAis circular; Plasmid (1-2000 kb) These plasimids contain Restriction sites Genes for antibiotic resistance (amp ) These plasmids (neg. charged) are inserted into the host cells by various artificial methods; Transfection method Shot guns Etc. How do you know that introduction of the gene occurred? 1. Check for a particular phenotype introduced by the foreign gene 2. Sometimes that is not possible and the plasmid has to be engineered to contain a gene that produces a protein contain a gene that produces a protein marker 3. Common protein marker; 4. beta-galactosidase-which is generated by the lacZ gene Insertion of a foreign DNAinto a cloning vector Biochemistry Spring 2014 Where would you insert a foreign piece of DNA? Blue v.s white colonies -Both blue and white colonies contain bacteria that is amp resistance only the white colonies contain a plasmid (cloning vector) with the inserted foreign gene, only if it was inserted in the lacZ region Genetic Engineering, manipulating DNA Bacteriophage: virus that infects *didn’t get to finish copying Cloning with bacteriophage (λ) Infective phage is then used to infect bacteria and introduce the forging phages that have failed to acquire a foreign DNA are unable to propagate Caution: generated pages are recovered from plaques not colonies. Expanding or amplifying the gene or interest by polymerase chain reaction Biochemistry Spring 2014 *you HAVE to write DNAfrom 5’to 3’ Site Directed Mutagenesis: Away to introduce an altered gene into a suitable organism (cell, plant or animal) Biochemistry Spring 2014 Transgenic mice: Clones (growth hormone gene) Other examples: Host lacking a particular gene Host producing a particular human protein In 1961 Osamu Shimomura discovered a protein in this jellyfish that glowed bright green under ultraviolet light. The protein, green fluorescence protein, was isolated and the gene that code for this protein was identified and cloned in bacteria. GFP and other florescence proteins has been expressed in other organisms -cats and mice Biochemistry Spring 2014 January 22, 2014 DNA– anionic, highly negative Electrophoresis : separates by size, shape, and charge Cathode – negatively charged Anode – positively charged (electrode that attracts negative species; where DNAis attracted to) HW #1 being discussed on Friday 1/24 Ch. 4 AminoAcids Amino acids 1. Monomers within proteins 2. All proteins composed of 20 “standard” amino acids 3. All standard amino acids are alpha-amino acids and contain both an amine and carboxylic acid group 4. Protein can be broken down into amino acids 5. Believed to have appeared early in the earth history 6. The presence or particular amino acids, standard or non-standard, give proteins their particular properties Ex: adhesive proteins Ex of how certain amino acids give proteins different properties: o Presence of a hydroxylated form of the amino acid tyrosine called L-3,4- dihydrophenlalanine (L-DOPA) in adhesive protein gives these proteins their interesting properties o Adhesive proteins found in clams that help attach to surfaces (boats, peers) o Proteins in the meat make it difficult to be detached. o Proteins contain a high amount of L-DOPA(amino acid not typically present); two hydroxyl groups allow for adhesive *If you don’t have the right amino acids, you don’t have the properties Alpha amino acids = amine + carboxylic group Memorize structures on table 4.1 *pkAon the table corresponds to the free amino acids, and change when in different solutions Neutral = No charge because they don’t have any groups that can be ionized All carboxylic acids can be deprotonated
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