Biochemistry Sixth Edition Chapter 3: Exploring Proteins and Proteomes Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer

The proteome is the functional representation of the genome Proteome: proteins expressed by the genome Represents functional expression of information Larger than the genome

3.1 The purification of proteins is an essential first step In understanding their function Never waste pure thoughts on an impure protein

The assay: how do we recognize the protein that we are looking for? Assay: a test for some unique identifying property of the protein Specific activity: the ratio of enzyme activity to the amount of protein Purification: to maximize the specific activity

An assay for LDH

Proteins must be released from the cell to be purified Fractionation Homogenate Differential centrifugation

Proteins can be purified according to solubility, size, charge, and binding affinity Salting out with ammonium sulfate Dialysis Gel-filtration chromatography Ion-exchange chromatography Affinity chromatography High-pressure liquid chromatography: high resolution, rapid separation

Dialysis

Gel-filtration chromatography

Ion-exchange chromatography

Affinity chromatography

HPLC

Proteins can be separated by gel electrophoresis and displayed Gel electrophoresis v = Ez /f, f = 6  r The gel serves as a molecular sieve SDS-PAGE: separation on the basis of mass under denaturing condition One SDS for every two amino acid residues Mercaptoethanol: reducing agent Staining

PAGE

Formation of a polyacryamide gel

SDS

staining

Mass vs. mobility

Isoelectric focusing Isoelectric point Generation of pH gradient, polyampholytes Two-dimensional gel electrophoresis Combination of isoelecrtic focusing and SDS-PAGE

IEF

2D gel elctrophoresis

E. coli proteins

A protein purification scheme can be quantitatively evaluated Total protein Total activity Specific activity Yield Purification level

Electrophoretic analysis of purification

Ultracentrifugation is valuable for separating biomolecules and determining their masses s = m(1-  f  partial specific volume  density of the medium Sedimentation coefficient x   r = velocity Svedberg unit (S): s Sedimentation velocity depends on mass, shape, density, and density of the medium Gradient centrifugation

Density and sedimentation coefficients

Gradient centrifugation

3.2 Amino acid sequence can be determined by automated Edman degradation Sequencing a peptide Amino acid composition – heating in 6N HCl at 110 C for 24 hrs Identification using HPLC Color reaction with ninhydrin Fluorescamine

Determination of amino acid composition

Quantification of amino acids

fluorescent derivative of amino acid

Amino acid sequence can be determined by automated Edman degradation Automated sequencer

Edman degradation sequentially removes one residue at a time

phenythiocarbamoyl derivative Under a mildly acidic condition Phenylthiohydantoin (PTH)-amino acid

Separation of PTH-amino acids

Proteins can be specifically cleaved into small peptides to facilitate analysis Divide and conquer CNBr, trysin Overlap peptide Reducing agent: DTT Alkylating agent: iodoacetate Diagonal electrophoresis

Cleavage by CNBr

Cleavage by Trysin

Overlap peptides

Disulfide bond reduction

Oxidation of cystine

Diagonal electrophoresis

Amino acid sequences are sources of many kinds of insight Homology - function Evolutionary pathway Internal repeats Localization signal Generation of antibodies Generation of DNA probes

Repeating motifs

Recombinant DNA technology has revolutionized protein sequencing Still need to work with isolated proteins Genomic and proteomic analyses are complementary

DNA sequence yields the amino acid sequence

3.3 Immunology provides important techniques with which to investigate proteins Antigen and antibody Antigenic determinant or epitope Hapten plus macromolecular carrier antiserum Monoclonal and polyclonal antibodies Antibodies to specific proteins can be generated

Antibody structure fragment crystallizable

Antigen antibody interaction

Multiple myeloma generates a large number of cells of a single kind A clone producing immunoglobulin of a single kind Fusion of a short-lived antibody-producing cell with an immortal myeloma cell Hybridoma cells Monoclonal antibodies in clinical assays and affinity purification Monoclonal antibodies with virtually any desired specificity can be readily prepared

Staining with a fluorescent-labeled monoclonal antibody

enzyme-linked immunosorbent assay (ELISA) Indirect ELISA: detection of antibody Sandwich ELISA: detection and quantitation of antigen Proteins can be detected and quantitated by using an enzyme-linked immunosorbent assay

ELISA

Western blotting permits the detection of proteins separated by gel electrophoresis

Western blotting

Fluorescence-labeled antibodies – fluorescence microscopy GFP (green fluorescent protein) fusion Immunoelectron microscopy – gold conjugated antibodies Fluorescent markers make possible the visualization of proteins in the cell

Fluorescence microscopy

Steroid receptor-GFP fusion protein

Immunoelectron microscopy

Synthetic peptide: antigen, affinity tag or bait, drug, study of 3D structure Blocking of amino group: t-Boc Activation of carboxyl group: dicyclohexylcarbodiimide (DCC) 3.4 Peptides can be synthesized by automated solid-phase methods Solid-phase method

Released in the breakdown of bacterial proteins Useful in identifying the cell surface receptor

Synthetic peptides as drugs

More stable analog

Blocking of amino group: t-Boc Activation of carboxyl group: dicyclohexylcarbodiimide (DCC)

Amino acid activation

Solid-phase peptide synthesis

The mass of a protein can be precisely determined by mass spectrometer Matrix-assisted laser desortion/ionization (MALDI) Electrospray ionization (ESI) Time of flight MALDI-TOF 3.5 Mass spectrometry provides powerful tools for protein characterization and identification

Individual components of large complexes can be identified by MALDI-TOF mass spectrometry Peptide mass fingerprinting (PMF) – extensively used in proteomics

NMR spectroscopy: structure in solution X-ray crystalogrphy: solid 3.6 Three dimensional protein structure can be determined by NMR spectroscopy and X-ray crystalogrphy

Protein crystal, source of X-ray, detector Electron scatters x-rays The scattered waves recombine X-ray crystalography reveals three dimensional structure in atomic detail The atomic arrangement affects the scattering pattern Electron density map

Spin state of hydrogen nucleus Resonance Shielding by flow of electrons – chemical shift Nuclear magnetic resonance spectroscopy can reveal The structures of proteins in solution 1D NMR Transfer of magnetization: Nuclear Overhauser effect (NOE), an interaction between nuclei is proportional to the inverse sixth power of the distance Nuclear Overhauser enhancement spectroscopy (NOESY) Off-diagonal peaks identify pairs of protons that are less than 5A apart