Welcome to 5610A: Advances in Analytical Biochemistry

A survey course: We will cover many of the shiny new analytical tools available to biochemistsFocus on ProteinsCore of the course: Analytical TechniquesUV/visible Fluorescen

ceCircular Dichroism

Cellular Level AnalysisMass

SpectrometryNuclear Magnetic Resonance

Assignments (12.5% ea): Jan 24th, Feb 7th, Feb 21st and March 13th Big Project / Exam (40%): 15min presentation, 7 page review

Separations

What is Analytical Biochemistry?

Not a well defined field, but the main questions are…

In theory, Analytical Biochemistry stops there, but usually these questions are asked to support hypotheses about biological function

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?

Diving Right in: Biochemistry is… DifferentLife uses only a select few elements:

H = 46.7% C = 42.0%N = 6.4%O = 4.75%

Mol % (Dry)

The distribution of elements in biological systems differs from the distibution of elements in non-biological systems

The First Analytical Biochemistry Question…

First ever analytical biochemistry question: What kinds of molecules do these elements form? Suga

rsFatty Acids

Nucleic Acids

Emil Fischer 1852 -

1919

Bunsen and Kirchhoff

Spectroscope

Michel Chevreul1786 -

1889

Elemental Analysis by: solvent solubility,

crystallization, saponification, melting point, distillation, and

salt fractionation

Johann Miescher1844 -

1895

Invented alkaline extraction / acid

precipitation of DNA (which he called

Nuclein). Elemental analysis was likely by the Bunsen method.

The First Analytical Biochemistry Question…Amino acids…

Henri Braconnot1780 -

1855Isolated Glycine

Karl Ritthausen – 1866 Glutamic and Aspartic Acid

Heinrich Hlasiwetz and Josef Habermann - 1867Leucine and Tyronsine from Casein

Other Amino AcidsSerine – 1865 Phenylalanine - 1881 Alanine – 1888 Arginine – 1895 Histidine – 1897 … … …

Cysteine – 1935!! (still debated!)

http://web.lemoyne.edu/~GIUNTA/hlasiwetz.html

First Analytical Biochemistry Techniques…These discoveries have one thing in common: Elemental Analysis

http://web.lemoyne.edu/~GIUNTA/thenard.html

Joseph Gay-Lussac (1778-1850) and Louis Thenard (1777-1857)

http://www.scitechantiques.com/1959/

Robert Bunsen (1811-1899)

Gustov Kirchoff (1824-

1887)

Made chemistry-based elemental analysis much more practical and accurate

He Spectrum

Macromolecules: PolysaccharidesSo the principal biological molecules are: Sugars, Fatty Acids, Nucelic Acids and Amino AcidsSugars:

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?

http://scholle.oc.uni-kiel.de/lind/iteach/kh_struct/kh_struct_eng_kap3.pdf

GC-MS

Macromolecules: PhospholipidsFatty Acids

Fats

Sterols

Phospholipids

Analytical Questions:

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

i) HPLC/UV-visible (monomers)

ii) Fluorescence (membranes)

iii) NMR (micelles/proteins)

iv) MS (monomers-bilayers)

Analytical Tools:

Macromolecules: DNA/RNA

Nucleotides RNA

DNA

Macromolecules: DNA/RNA Con’t…DNA is the hereditary molecule: Analytical biochemistry at it’s best…

Hershey and Chase Experiment - 1952

Macromolucules: DNA/RNA Con’t…Biological Roles:

DNA: Heredity

RNA: Heredity (virus)

mRNA (intermediate DNA – protein)

Genome encoding

Metabolism (e.g. mRNA IRE)

tRNA (genome decoding)Ribosomes (genome decoding)

Intron excision (genome decoding)

Gene Expression (methylation)

Analysis:X-ray crystallography p31 NMR Hybridization MS/MS

Macromolecules: ProteinsAmino Acids

H

K

T

D

S

F

VG

LM

Peptides

Whale AcP.pdb

Proteins

Proteins are definitely absolutely and totally the most important and coolest biological molecule!!

Analytical Questions:i) Which amino acids?

ii) In what order?

iii) Structure?

iv) Biological Function?v) Dynamics?vi) Quantitation / Localization?

What is Analytical Biochemistry?

Biological Roles…

Catalysis:

Structural: hair/nails, cytoskeleton, muscle, cartilage

Signaling and Transport:

Storage:

Keratin, Actin/Tubulin, Actin/Myosin, Collagen/Elastin

Insulin, Transferrin, Ion Chanels, Receptors/Kinases

Ferritin (Fe2+), Calsequesterin (Ca2+), Seed Storage

Gazillions (I’m not even going to try)

Proteins: Primary Structure Analysis

Gerardus Mulder1802-

1880Elemental analysis of whole proteins, coined the term ‘protein’.

http://web.lemoyne.edu/~GIUNTA/mulder.html

Fibrin: C400H620N100O120S1P1

Fibrin: C2103H3108N550O642S20

Ratios: C1H1.55N.25O.33S.025P.025

Ratios: C1H1.47N.26O.31S.01

Primary Structure

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!

Enzymatic hydrolysis

Dipeptides

Proteins: Secondary Structure

The primary structure contains all of the information needed to predict how a protein will fold

Secondary structure is how the amino acid chain arranges itself

Alpha Helix

Beta Sheet

Helix.pdb Beta Sheet.pdb

The discovery of Secondary Structure…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” - 1948Published proposed -helix and -sheet in 1951

http://www.nap.edu/readingroom/books/biomems/lpauling.html

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

Ramachandran Plot

Gopalasamudram Narayana Iyer Ramachandran (1922-2001)

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

Proteins: Tertiary Structure…Tertiary structure is the organization of secondary structuresHelix Bundle

Beta Barrel

Greek Key

Leucine zipper.pdb Yfp.pdb Plant Antimicrobial.pdb

Primary and Tertiary Structure are Linked…Hydrophobicity and Tertiary Structure

Chymotrypsinogen

Proteins: Quaternary Structure…Quaternary structure describes how protein subunits that are separate peptide chains interact with each other

Tetramer

Heptamer

Gigantic Complex

Cooperativity

Size

Multiprocess function

Hemoglobin.pdb

GroEL Octamer.pdb

Giant Complex.pdb

Quaternary Structure and Activity Control…

Blood Clotting

Aggregation / Amyloidosis

Prothrombin (II)

Thromboplastin (III)

Proaccelerin (V)

Fibrin

Diving Right In: What is Analytical Biochemistry

These are about as different as you can get…But on the molecular

level…

E coli AcP.pdbWhale AcP.pdb

Volume = 7 x 109 cm3

Volume = 2 x 10-8 cm3