Chapter 3 Exploring Proteins and Proteomes

A collective name for the genes existed in an organism C. elegance (roundworm) : 97 million bases, 19,000 genes Drosophila melanogaster (fruit fly) : 180 million bases, 14,000 genes Human : 3 billion bases, ~20,000 genes Static and absolute information Genome A collective name for the proteins expressed by the genome Dynamic and functional information It varies with cell type, developmental stage, and environmental condition such as the presence of hormones. Regulation of mRNA synthesis, alternative splicing, mRNA stability, rate of protein synthesis, post-translational modification, protein stability control, protein degradation Proteome

The purification of proteins is an essential first step in understanding their function. Purification should yield a sample of protein containing only one type of molecule of interest. Proteins can be separated from one another on the basis of solubility, size, charge, and binding ability. Assay : a test for some unique identifying property of the protein Specific Activity : the ratio of enzyme activity to the amount of protein in the enzyme assay Protein Purification NADH can absorb light at 340 nm. Nicotinamide adenine dinucleotide

Homogenation and Fractionation by Centrifugation

Salting Out : protein solubility decrease by very high concentration of salt Salting In : protein solubility increase by low concentration of salt Dialysis : separation of small molecules from proteins through membrane with pores such as cellulose membrane (cf. semi-permeable) Salting Out & Dialysis Ammonium sulfate for protein precipitation

Gel-Filtration Chromatography (Molecular Exclusion, Size Exclusion, Molecular Sieve) 1.Thyroglobulin (669 kd) 2.Catalase (232 kd) 3.BSA (67 kd) 4.Ovalbumin (43 kd) 5.Ribonuclease (13.4 kd)

Anion Exchanger Positively Charged Column Negatively Charged Proteins Cation Exchanger Negatively Charged Column Positively Charged Proteins Ion-Exchange Chromatography Depend on local charge on proteins

LC Liquid Chromatography FPLC Fast Pressure Liquid Chromatography HPLC High Pressure Liquid Chromatography Affinity Chromatography (concanavalin A) Highly specific - Histidine tag

Gel Electrophoresis v = Ez / f v : velocity of migration E : electric field strength z : net charge on the protein f : frictional coefficient f = 6  r  : viscosity of the medium r : radius of the protein Polyacrylamide gel electrophoresis

Polymer Formation of Acrylamide using Bis-Acrylamide for PAGE (Poly-Acrylamide Gel Electrophoresis) Ammonium Persulfate Sieving action!

SDS-PAGE : Denaturing Gel (Determination of the Molecular Weight of Protein) Coomassie Blue Staining; > 0.1  g) (cf. Silver Staining: > 0.02  g) Under BME, DTT One SDS anion for every two a.a. Except carbohydrate-rich proteins, membrane proteins Mobility ; Log of MW Resolution: 2% MW difference

Isoelectric Focusing & Two Dimensional Electrophoresis pI : Isoelectric Point (pH with net charge zero)

Evaluation of Protein Purification As purification continues, relative presence of contaminants should be decreased and the proportional amount of the protein of interest should be increased.

Centrifugation & Sedimentation Coefficient A more massive particle sediments more rapidly. A more compact shaped particle sediments faster. (i.e. elongated particles sediments more slowly than do spherical ones of the same mass. Frictional coefficient f) A denser particle sediments more rapidly. Buoyant force is smaller for the denser particle v  1 : float, v  = 1 : no movement s = m(1 - v  ) / f s : sedimentation coefficient m : mass of the particle v : partial specific volume; the reciprocal of the particle density  : density of the medium (1 - v  ) : buoyant force exerted by liquid medium f : frictional coefficient; a measure of the particle shape

a. Seidmentation process in the cell From Archimedes’ principle Buoyancy = weight of displaced fluid Or fluid density x submerged vol x g Principle of Analytical Ultracentrifugation

b.solute distribution in the cell

S Value for Various Proteins

Density and Sedimentation Coefficient for Various Cellular Components

Gradient (Zonal or Band) Centrifugation: Separation of Non-Denatured Proteins with different sedimentaion coefficients (Size, Density and Shape) Sedimentation equilibrium : centrifuged at low speed so that sedimentation is counter balanced by diffusion -Very accurate in mass determination without denaturing. - useful for large multimeric proteins. Sedimentation velocity

Determination of Amino Acid Composition of the Peptide 1.Peptide hydrolyzation by heating it in 6N HCl at 100 o C for 24 hrs Ala-Gly-Asp-Phe-Arg-Gly (Asp, Gly 2, Ala, Phe, Arg) 2.Separation of amino acid hydrolysates by ion-exchange chromatography (e.g. sulfonated polystyrene resin; Dowex-50)

3A. Quantitation of Each Fraction by Ninhydrin; Yield Visible Color (usually blue except Pro for yellow); Detection Sensitivity = Microgram (10 nmol) of an Amino Acid 3B. Quantitation of Each Fraction by Fluorescamine; Yield Fluorescence; Detection Sensitivity = Nanogram (10 pmol) of an Amino Acid

Identification of N-Terminal Amino Acid (FDNB) Yield Fluorescent Sulfonamide FDNB, Dabsyl Chloride, or Dansyl Chloride Can Specifically React with the N-terminal Amino Group, and Yield DNB-Amino Acid, Dabsyl Amino Acid, or Dansyl Amino Acid, and These Can Be Identified by Their Chromatographical Properties.

Determination of Amino Terminal Residue of a Peptide using Dabsyl Chloride

Edman Degradation Sequentially Removes One Residue at a Time from the Amino End of a Peptide up to 50 times Each round can be complete within 1 hr and the Edman degradation can be repeated up to 50 cycles in Practice.

Phenyl Isothiocyanate (PITC) Can Specifically React with the N-terminal Amino Group, and Yield Phenyl Thiocarbamoyl (PTH) Amino Acid, and This Can Be Identified by Its Chromatographical Property. Separation of PTH-Amino Acids Current Sensitivity of PTH-AA Detection Using Gas-Phase Sequenator: Picomole Mild acidic condition

For sequencing of an entire Protein…?? Divide and Conquer !!!

Sequence Specific Cleavage by Cyanogen Bromide Sequence Specific Cleavage by Trypsin

Deduction of Full Amino Acid Sequence of a Protein by Overlapping the Sequences Obtained from individual Peptides

Reduction of Disulfide Bonds :  -Mercaptoethanol, Dithiothreitol (DTT) Sequencing of Multimeric Proteins Denaturation : Urea, Guanidium Chloride, SDS Preservation of Reduced Sulfhydryl Groups : Alkylation using Iodoacetate Separation of Each Polypeptide Chain : SDS-PAGE

Diagonal Electrophoresis to Determine the Positions of the Disulfide Bonds Reduction and Oxidation of Disulfide Bonds by Performic Acid

The Amino Acid Sequence Provides Insights into the Protein’s Function, Structure, and History 1.The sequence of a protein of interest can be compared with all other known sequences to ascertain similarities. (Family, function prediction possible) 2.Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathway. 3.Amino acid sequences can be searched for the presence of internal repeats. 4.Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing. (N-terminal 20 hydrophobic residues, signal sequence, nuclear localization signal) 4 Repeating Motifs in Calmodulin : Each Unit Binds a Calcium Ion

Amino acid sequence data provide a basis for preparing antibodies specific for a protein of interest. Amino acid sequence are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins. The nucleotide sequence of DNA (gene) directly reveals the entire amino acid sequence of the protein encoded by the gene. However, DNA sequence can not disclose the information regarding post-translational modification. Practical Usage of Amino Acid and DNA Sequences

Antibody Antibody (immunoglobulin) is a protein synthesized by an animal in response to the presence of a foreign substance (antigen). Antibodies have specific and high affinity against antigens. Proteins, polysaccharides and nucleic acids can be effective antigens. Epitope : a specific group or cluster (portion) of antigen to stimulate the synthesis of an antibody and recognized by a specific antibody (antigenic determinant) Hapten : a small molecule containing epitope attached to a carrier

Antibody (continued) Each antibody producing cell synthesizes only one type of antibody recognizing a single kind of epitope. The proliferation of a given antibody producing cell is stimulated by the binding of its designated antigen to the cell surface receptor of the antibody producing cell. Periodic injections of an antigen into the host animal can raise the antibodies specifically recognizing the injected foreign substance. Blood withdrawn from the immunized host animal  centrifugation  separation of blood cells (pellet) and serum (supernatant)  anti-serum Anti-serum contains multiple kinds of antibodies each recognizing a different surface feature of the same antigen. This heterogenic antibodies are called as polyclonal antibodies. This heterogeneity can complicate the use of these antibodies.

Monoclonal Antibody Monoclonal hybridoma cell lines can generate large amount of homogeneous antibodies. Monoclonal antibodies can serve as precise analytical, preparative and therapeutic reagents.(HCV, HIV, herceptin) Immuno- Staining of Drosophila Embryo using Monoclonal Antibody against Engrailed Plasma cell by antigen-antibody interaction

Monoclonal antibody drugs?

Herceptin Binds to the C-terminus of Domain IV Herceptin Fab I III II IV N C HER2

Ribbon Diagram of Her-3 ECD N C “Tethered” I II III IV Right-handed  helix Laminin-like folds Fig pp397

Surface representations of EGFR and HER2 in Antibody-Bound Conformations Herceptin

ELISA (Enzyme-Linked Immuno-Sorbent Assay) Antibody detection, anti-HIV antibody Antigen detection

Western Blotting Radioactive secondary antibody For protein expression and purification

Immuno-Fluorescence Microscopy Actin Filament Staining using  -actin antibody Immuno-Electron Microscopy Detection of a channel protein from the synaptic vesicles using antibodies tagged with electron-dense markers such as gold or ferritin (Resolution better than 10 nm) Fluorescence-labeled antibodies (resolution 200nm) ex) Glucocorticoid receptor

Synthetic Peptides Synthetic Antigens for antibody formation Receptor or Interacting Protein Isolation Clinical Drugs (ex, vasopressin) 3D Structure Study Increase water absorption in the kidney; Aquaporin Caffeine decreases release of AVP

Protection of Amino Group by t-BOC Activation of Carboxyl Group by DCC

Solid Phase Peptide Synthesis (more than 100 amino acids) Resin : Insoluble Matrix (Polystyrene Beads), HF : Hydrofluoric Acid

MALDI-TOF Mass Spectrometry MALDI : Matrix-Assisted Laser Desorption-Ionization TOF : Time of Flight F=ma Mass Spectrometry Are Often Combined with 2D Electrophoresis for Proteome Analysis

h Laser +20 kV Variable Ground Grid Grid AH + Sample plate 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules are ionized by proton transfer from matrix: MH + + A  M + AH +.

Why MALDI? -Less sensitive to salts -Lower PRACTICAL detection limits -Easier to interpret spectra(less multiple charges) -Quick and easy -Higher mass detection -Higher Throughput(1000>samples per hour)

The Mass Analyzer: TOF Time Of Flight(TOF) Flight TubeIon Source Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass to charge ratios kV + +

Peptide can be sequenced by MS

Individual proteins can be identified by MS Protein cleavage, followed by chromatographic separation and MS

NMR (Nuclear Magnetic Resonance) Basis for Resolution : Electron (Proton) Chemical Shift in Magnetic Field Advantage - Solving Protein Structures in Solution State - Requires Smaller Quantity for Analysis than X-ray Crystallography Limitations - Technical Difficulties for Bigger Size Proteins (> 40 kD) - Technical Difficulties for Multi-Subunit Proteins X-Ray Crystallography Basis for Resolution : X-Ray Diffraction and Electron Scattering Advantage - No Size Limitation for Analysis - Solve the Structure of Multi-Subunit Complexes Limitations - Solving Protein Structures in Solid State - Difficulties in Making Protein Crystals with Good Diffraction Properties