Protein Review PCB 4024
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This 3 page Class Notes was uploaded by Justin Bartell on Saturday September 10, 2016. The Class Notes belongs to PCB 4024 at Florida State University taught by Dr. Stroupe in Fall 2016. Since its upload, it has received 8 views. For similar materials see Molecular Biology in Biology at Florida State University.
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Date Created: 09/10/16
PCB 4024– Week 2 – 03 (Thursday Lecture 9/8/2016) Protein Review I. 50% of the dry mass of a cell is PROTEIN (both prokaryotic and eukaryotic) a. Functionality i. Structural Ex. Keratin in hair ii. Enzymatic Ex. Catalase (2H O 2H O + O ) 2 2 2 2 iii. Receptors Ex. Taste receptors on tongue (sweet receptor) iv. Transport Ex. Hemoglobin (carries oxygen in blood) v. Storage Ex. Albumin (egg white protein) vi. Signaling/hormonal Ex. Insulin (regulates sugar levels in blood) vii. Contractile/motor Ex. Gactin and Factin viii. Defensive Ex. lysosome and antibodies b. Composition i. Proteins are polypeptides: polymers of amino acids 1. Amino acids are joined together by a large complex called the ribosome 2. Amino acids are zwitter ions: both + and – charge a. Has an amino group H2N (H3N+) and carboxyl group COOH (COO) centered around the alpha carbon connected to a side chain (R). 3. 20 chemical side chains: 20 different amino acids 4. To make polymer dehydration reaction a. Loss of h20 b. Amino acids are linked by peptide bond c. Structure i. Polypeptides take on a defined structure that is linked to its function ii. Four layers of structure 1. Primary: amino acid sequence 2. Secondary: (motifs alpha helical and beta strand / beta pleated sheets) a. Alpha Helix: The sidechain (R) substituents of the amino acid extend to outside. Hydrogen bonds form between the oxygen of the C=O of each peptide bond in the strand and the hydrogen of the NH group of the peptide bond four amino acids below it in the helix. These hydrogen bonds make this structure very stable. b. Beta Sheet: The hydrogen bonding is between strands rather than within strands (interstrand vs. intrastrand). The sheet conformation consists of pairs of strands lying sidebyside. The carbonyl (C=O) oxygens in one strand hydrogen bond with the amino hydrogens (NH2) of the adjacent strand. The two strands can be either parallel or antiparallel depending on whether the strand directions (N terminus to Cterminus) are the same or opposite. The anti parallel Beta sheet is more stable. c. Hydrogen bonds! Van der Waals interactions! d. Disulfide bridge! Ionic Bonds! 3. Tertiary: how protein folds 4. Quaternary: assembly – multiple polypeptides interacting d. Sequence and function i. The primary structure of a protein determines its chemical characteristics 1. That the influences and creates the secondary, tertiary, and quaternary structure ii. Errors in amino acid sequence directly changes primary structure (because primary structure is ONLY the amino acid sequence) 1. This would then affect the secondary, tertiary, and quaternary structures! 2. As a result, the protein can lose function (structure function) a. Sickle Cell Hemoglobin: replace 6 amino acid (Glu Val) e. Xray crystallography, electron microscopy, and Nuclear Magnetic Resonance i. 3 methods for learning about protein structure II. One gene – One protein a. Garrod: Alcaptonuria patients cannot break down phenylalanine b. Beedle and Tatum: mold as model organism. i. Introduced mutations and then looked for different growth requirements in the variants. Found position of mutations through genetic analysis. Then identified the enzymes involved in metabolic pathways. c. Why is there RNA in the cell? Crick’s hypothesis: rRNA = message. i. Jacob, Brenner, and Meselson tested it. 1. rRNA had been identified as an integral part of the ribosome 2. Use phage (similar to Hershey/Chase) – once infected, the phage DNA is transcribed and then translated. 14 13 3. Grow uninfected bacteria in N and C. They will make heavy ribosomes bound to “cold” RNA. Then, infect with regular phage and transfer to regular media + P. New ribosomes created by phage will be light but all phage RNAs (and DNAs) will be “hot”. Isolate RNA and ribosomes and use a centrifuge to separate them out. Look to see if the Plabeled RNA is associated with heavy or light ribosomes. a. From top of centrifuge to bottom of centrifuge (from lightest to heaviest): i. Disassociated subunits of heavy ribosomes [rRNA] (ribosomes created by bacteria in 14N and 13C) ii. Whole units of heavy ribosomes [rRNA] (ribosomes created by bacteria in 14N and 13C) iii. Heavy ribosomes with light RNA complex [rRNA and mRNA] (ribosomes created by bacteria in 14N and 13C and mRNA created by phage in 32P) iv. Light RNA [mRNA] (mRNA created by phage in 32P) b. Therefore, phage did NOT create new ribosomes, just new mRNA. i. Demonstrated the existence of mRNA c. If mRNA contains phagederived product, will the ribosomes with which it is associated be heavy or light? i. heavy d. If rRNA contains phagederived product, will the ribosomes with which it is associated be heavy or light? i. light e. Were the new hot RNAs (ie the messages from the phage) associated with the heavy or light ribosomes? i. heavy III. Central Dogma a. Transcription: DNA to mRNA b. Translation: mRNA to protein i. rRNAribosome (2 parts) ii. tRNA – grab amino acids to bring to rRNA as mRNA is read iii. gene structure (DNA) 1. Transcription initiation site – start transcribing here (untranslated region) 2. Open Reading Frame (ORF) – only part that gets translated (only part that results in amino acids) a. Initiation codon b. Stop codon 3. Transcription termination site – stop transcribing here (untranslated region) IV. Techniques a. Gel b. Western Blots
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