Ch5-120201 - CHEM 350 Introduction to Biological Chemistry...

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Unformatted text preview: CHEM 350: Introduction to Biological Chemistry Brian Lee, Ph.D. [email protected] Office: Neckers 146G or 324 Phone: 453-7186 Hours: 9:30am to 10:30am or by appointment Website: https:/ / Textbook (required, U.S. edition only) Fundamentals of Biochemistry, 3rd Ed., Voet, Voet & Pratt. Study Guide (recommended) Student Companion to Fundamentals of Biochemistry, 3rd Ed. Help Desk Tuesday 6:30 to 7:30 pm in Neckers 218 Thursday 5:00 to 6:00 pm in Neckers 410 Announcements Undergraduate Research Opportunities Research for credit (such as CHEM 396 or CHEM 496) Student worker ($8.00 per hour) ( Undergraduate Assistantships ( McNair Scholars Program ( REACH Awards Competition ( Summer Research Experiences for Undergraduates (REU) For other REU programs, search the National Science Foundation site: Students must contact the individual sites for information and application materials. NSF does not have application materials and does not select student participants. A contact person and contact information is listed for each site. Assignments Read Chapter 5 Proteins Chapter 5 Problems • First Midterm Exam, Monday February 6th – Chapters 1 through 5 • Help Desk Tuesday 6:30-7:30pm in Neckers 218 Thursday 5:00-6:00pm in Neckers 410 Column chromatography: takes advantage of the size, net charge and binding properties of proteins. Proteins migrate depending on their properties. HPLC – high performance liquid chromatography uses increase pressure and small bead size to improve resolution FPLC – fast protein liquid chromatography controls flow rate with low pressure pumps. Ion-exchange chromatography (cation or anion exchange) Cation exchange: Positive charged solutes bind to the column. Negative charged solutes pas through without interacting with the column. Elution of positive charged solute with salt gradient. Figure 5-6 Size-exclusion chromatography (gel filtration) separates proteins according to size Small molecules “explore” the porous beads, slowing elution. Figure 5-7 Affinity chromatography separates proteins based on their binding specificity Electrophoresis: separation by charge and size PAGE – polyacrylamide gel electrophoresis SDS – a negatively charged detergent VZ μ= = Ef μ = electrophoretic mobility V = molecule velocity E = electric potential Z = net charge f = frictional coefficient Electrophoresis - Stain (Coomassie brilliant blue, silver) - autoradiograph (35S radioisotope) - immuno-blotting (Western blot) Denaturing conditions to separate by size only SDS-PAGE (sodium dodecyl sulfate) Reducing agent (2-mercaptoethanol) Isoelectric Focusing using a pH gradient in the gel Native conditions – proteins are folded Protein migration stops when pH = pI 2D gel separation by - isoelectric point - molecular weight Amino acid sequence of bovine insulin. The sequence was determined by Frederick Sanger who developed the first methods to sequence proteins. Sequencing insulin took ten years. Protein sequencing is faster, but still limited to short peptides (<100 residues) Protein sequencing strategy -separate chains -digest polypeptide -sequence fragments -reconstruct from overlap -repeat without reducing agent to find cross-links Figure 5-12 Sanger’s Reagent – (1-Fluoro-2,4-dinitrobenzene) DNFB – reacts with free amino groups of intact peptide - after hydrolysis DNP-amino acid identified Box 5-1 Pehr Edman’s improved method. Edman degradation Phenylisothiocyanate (PITC) PITC cleaves the peptide bond. N-terminal amino acid is labeled and can be separated by washing. After removal, the procedure is repeated to identify the next residue. Figure 5-15 DNFB 2,4-dinitrofluorobenzene Sanger method relied on incomplete hydrolysis of protein Edman Degradation can use a sequenator PITC = phenylisothiocyanate N-terminal identification Figure 5-13 Reduction of disulfide bonds with 2-mercaptoethanol Page 106 Free thiol groups can be protected by acetylation with iodoacetate to prevent oxidation and cross-linking Page 107 Disulfide bonds interfere with the sequencing procedure Peptide digestion with sequence specific proteases Trypsin cleaves after a positively charged residue: Arginine or Lysine The next residue can be anything but Proline Page 107 Table 5-3 Chemical cleavge by cyanogen bromide after Methionine Protein sequencing strategy – overlapping fragments Figure 5-18 Other methods for obtaining an amino acid sequence: 1) Indirectly from the DNA sequence: 2) Directly using mass spectrometry: tandem mass spectrometry Peptide fragments can be separated by their mass to charge ratio Figure 5-16b Tandem mass spectrometry: 1) separation of peptide fragments 2) collision with He produces smaller fragments 3) i dentify smaller fragments with 2nd mass spectrometer 4) fragments size matched to list of possible sequences Table 5-4 Protein sequences and evolutionary relationships: Sequence alignments show amino acid residues that are conserved by evolution. Why are some residues conserved? Sequences show evolutionary relationships between organisms The above sequences suggest that archae and eukaryotes are closer relatives than eubacteria such as E. coli and B. subtilis Cytochrome c sequence comparison Phylogenetic tree based on cytochrome c comparison Eukaryotes Myoglobin 1 chain Hemoglobin 4 subunits Figure 5-22 Hemoglobin evolved from gene duplication of early globin gene to produce subunits. Variable rates of evolution depend on protein function blood plasma and extracellular matrix oxygen transport electron transport and apoptosis DNA packaging into chromosomes Figure 5-23 Shared protein domains Most are derived from gene duplication Figure 5-24 ...
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