Welcome to 5610A: Advances in Analytical Biochemistry

1. Welcome to 5610A: Advances in Analytical Biochemistry A survey course: We will cover many of the shiny new analytical tools available to biochemists Focus on Proteins Core of the course: Analytical Techniques UV/visible Fluorescence Circular Dichroism Cellular Level Analysis Mass Spectrometry Nuclear Magnetic Resonance Separations Assignments (12.5% ea): Jan 24th, Feb 7th, Feb 21st and March 13th Big Project / Exam (40%): 15min presentation, 7 page review

2. What is Analytical Biochemistry? Not a well defined field, but the main questions are… Analytical Chemistry questions: i) What’s there? ii) How much? Analytical Biochemistry Specific Questions: iii) Where is it? iv) Where did it come from? v) How is it shaped? vi) How does it move? vii) What does it interact with? In theory, Analytical Biochemistry stops there, but usually these questions are asked to support hypotheses about biological function

3. Diving Right in: Biochemistry is… Different Life uses only a select few elements: Mol % (Dry) H = 46.7% C = 42.0% N = 6.4% O = 4.75% The distribution of elements in biological systems differs from the distibution of elements in non-biological systems

4. The First Analytical Biochemistry Question… First ever analytical biochemistry question: What kinds of molecules do these elements form? Sugars Fatty Acids Nucleic Acids Emil Fischer 1852 - 1919 Johann Miescher Michel Chevreul 1844 - 1895 1786 - 1889 Elemental Analysis by: solvent solubility, crystallization, saponification, melting point, distillation, and salt fractionation Invented alkaline extraction / acid precipitation of DNA (which he called Nuclein). Elemental analysis was likely by the Bunsen method. Bunsen and Kirchhoff Spectroscope

5. The First Analytical Biochemistry Question… Amino acids… Heinrich Hlasiwetz and Josef Habermann - 1867 Leucine and Tyronsine from Casein Henri Braconnot http://web.lemoyne.edu/~GIUNTA/hlasiwetz.html 1780 - 1855 Isolated Glycine Other Amino Acids Karl Ritthausen – 1866 Glutamic and Aspartic Acid Serine – 1865 Phenylalanine - 1881 Alanine – 1888 Arginine – 1895 Histidine – 1897 … … … Cysteine – 1935!! (still debated!)

6. First Analytical Biochemistry Techniques… These discoveries have one thing in common: Elemental Analysis Robert Bunsen (1811-1899) Gustov Kirchoff (1824-1887) Joseph Gay-Lussac (1778-1850) and Louis Thenard (1777-1857) He Spectrum Made chemistry-based elemental analysis much more practical and accurate http://web.lemoyne.edu/~GIUNTA/thenard.html http://www.scitechantiques.com/1959/

7. Macromolecules: Polysaccharides So the principal biological molecules are: Sugars, Fatty Acids, Nucelic Acids and Amino Acids Sugars: Disaccharides Polysaccharides (i) nature and molar ratios of the contained monosaccharide building blocks? (ii) positions of the glycosidic linkages? (iii) monomer sequences and identification of repeating units? GC-MS http://scholle.oc.uni-kiel.de/lind/iteach/kh_struct/kh_struct_eng_kap3.pdf

8. Macromolecules: Phospholipids Fatty Acids Fats Sterols Analytical Questions: Phospholipids i) What types of phospholipids? ii) Under what conditions do Liposomes, Micelles and Bilayers form? iii) Membrane physics (how do the monomers move around)? iv) Interactions with proteins Analytical Tools: i) HPLC/UV-visible (monomers) ii) Fluorescence (membranes) iii) NMR (micelles/proteins) iv) MS (monomers-bilayers)

9. Macromolecules: DNA/RNA Nucleotides RNA DNA

10. Macromolecules: DNA/RNA Con’t… DNA is the hereditary molecule: Analytical biochemistry at it’s best… Hershey and Chase Experiment - 1952

11. Macromolucules: DNA/RNA Con’t… Biological Roles: Analysis: X-ray crystallography DNA: Heredity p31 NMR Genome encoding Hybridization Gene Expression (methylation) MS/MS RNA: Heredity (virus) Intron excision (genome decoding) mRNA (intermediate DNA – protein) tRNA (genome decoding) Ribosomes (genome decoding) Metabolism (e.g. mRNA IRE)

12. L M V G S T F H D K Peptides Macromolecules: Proteins Amino Acids Proteins Proteins are definitely absolutely and totally the most important and coolest biological molecule!! Analytical Questions: i) Which amino acids? iv) Biological Function? v) Dynamics? ii) In what order? vi) Quantitation / Localization? iii) Structure?

13. What is Analytical Biochemistry? Biological Roles… Structural: hair/nails, cytoskeleton, muscle, cartilage Keratin, Actin/Tubulin, Actin/Myosin, Collagen/Elastin Signaling and Transport: Insulin, Transferrin, Ion Chanels, Receptors/Kinases Storage: Ferritin (Fe2+), Calsequesterin (Ca2+), Seed Storage Catalysis: Gazillions (I’m not even going to try)

14. Proteins: Primary Structure Analysis Primary Structure Gerardus Mulder 1802-1880 Elemental analysis of whole proteins, coined the term ‘protein’. S-Q-D-A-G-M-Q-Q-G-A-D-M-D-Q-V-S-A Frederick Sanger (1918-1997) Sequenced insulin using limited Proteolysis and paper chromatography! http://web.lemoyne.edu/~GIUNTA/mulder.html Fibrin: C400H620N100O120S1P1 Ratios: C1H1.55N.25O.33S.025P.025 Fibrin: C2103H3108N550O642S20 Enzymatic hydrolysis Ratios: C1H1.47N.26O.31S.01 Dipeptides

15. Proteins: Secondary Structure Secondary structure is how the amino acid chain arranges itself Beta Sheet Alpha Helix The primary structure contains all of the information needed to predict how a protein will fold

16. The discovery of Secondary Structure… http://www.nap.edu/readingroom/books/biomems/lpauling.html Linus Pauling 1901-1994 Trained in theoretical physics, at the center of early X-ray crystallograhy Recognized the importance of the H-bond in stabilizing protein structure “I think that enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyse, that is, to the molecular configuration that is intermediate between the reacting substances and the products of reaction for these catalysed processes. The attraction of the enzyme molecule for the activated complex would thus lead to a decrease in its energy, and hence to a decrease in the energy of activation of the reaction, and to an increase in the rate of the reaction” - 1948 Published proposed -helix and -sheet in 1951

17. Proteins: Primary to Secondary Structure… Secondary structure is caused by the tendency of amino acids to ‘prefer’ specific rotations around the N-C () and C-C ()bonds Gopalasamudram Narayana Iyer Ramachandran (1922-2001) Ramachandran Plot

18. Primary Structure to Secondary Structure Con’t… Certain amino acids prefer ‘helical’ / angles and some prefer ‘beta strand’ / angles.

19. Proteins: Tertiary Structure… Tertiary structure is the organization of secondary structures Greek Key Helix Bundle Beta Barrel

20. Primary and Tertiary Structure are Linked… Hydrophobicity and Tertiary Structure Chymotrypsinogen

21. Proteins: Quaternary Structure… Quaternary structure describes how protein subunits that are separate peptide chains interact with each other Heptamer Gigantic Complex Tetramer Cooperativity Size Multiprocess function

22. Quaternary Structure and Activity Control… Blood Clotting Prothrombin (II) Proaccelerin (V) Thromboplastin (III) Fibrin Aggregation / Amyloidosis

23. Diving Right In: What is Analytical Biochemistry Volume = 7 x 109 cm3 Volume = 2 x 10-8 cm3 These are about as different as you can get… But on the molecular level…