219160 lecture 11
- 1. HBC1011 Biochemistry I Trimester I, 2018/2019 Lecture 11 – Exploring Protein Part III Ng Chong Han, PhD MNAR1010, 06-2523751 chng@mmu.edu.my
- 2. Overview • Protein quantitation • Peptide Sequencing • Antibody • NMR • X-ray crystallography 2
- 3. Protein quantitation • Protein quantitation is often necessary before and after processing protein samples for isolation, separation and analysis by chromatographic, electrophoretic and immunochemical methods. • Most colorimetric protein assay methods can be divided into two groups. – Protein-copper chelation: BCA (Bicinchoninic Acid) and modified Lowry assay – Protein-dye binding: Coomassie (Bradford) assay. • Choice of assay: Buffer compatibility (the presence of reducing agent and detergent in the protein solution may interfere chemical reaction of protein quantitation) 3
- 4. Protein quantitation • Protein standard curve: bovine serum albumin (BSA), known concentration of protein standard to determine the concentration of purified proteins by interpolation. 4
- 5. Protein quantitation 5 Protein Assay Detection Absorbance Compatibilities BCA 562nm Yes: Detergents No: Reducing agents; Chelators Coomassie (Bradford) 595nm Yes: Most reducing agents; Chelators No: Detergents Modified Lowry 750nm Yes: SDS No: Most detergents; Reducing agents; Chelators Bradford assay
- 6. Peptide sequencing – N-terminal amino acid analysis • React the peptide with a reagent that will selectively label the terminal amino acid. • Hydrolyse the protein. • Determine the amino acid by chromatography and comparison with standards. 6 Sanger analysis: The protein is hydrolyzed by 6M HCl first, the N-terminal group of amino acids are labeled with 1-fluoro-2,4- dinitrobenzene (Sanger’s reagent, DNFB). The labeled amino acids are separated and identified.
- 7. Peptide sequencing – N-terminal amino acid analysis • Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. • In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting other peptide bonds between other amino acid residues. • Phenylisothiocyanate (PITC) reacts with the free N-terminus of the chain to form PTC derivative. • Then, under acidic (TFA, trifluroacetic acid) conditions, this PTC derivative is cleaved as a thiazolinone derivative. 7
- 8. Edman degradation • The thiazolinone derivative is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. • This procedure can then be repeated again to identify the next amino acid. • Development of automatic sequencer has speed up the process, 1 cycle can be carried out in less than 1 hour. 8
- 10. Edman degradation: Advantage and Disadvantage • A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 residues. • This is because the Edman degradation reaction is not 100% efficient, meaning that the cleavage step does not occur every time. • However, this problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. • It is able to accurately sequence up to 30 amino acids with 98% efficiency per amino acid. • Advantage: it only uses 10 - 100 picomoles of peptide for the sequencing process 10
- 11. Protein sequencing strategies • Polypeptides and proteins with greater than 50 amino acid residues must be cut into smaller pieces for sequencing. • Proteins can be cleaved at specific sites by chemical such as cyanogen bromide (CNBr) or enzymatically with enzymes like trypsin, or chymotrypsin. 11
- 13. Protein sequencing strategies 13 • Overlapping fragments are joined.
- 15. Protein sequencing strategies • Proteins with disulfide bonds: Must be broken with reducing agents such as mercaptoethanol and modified to prevent re- formation • Large proteins (>500 a.a.) : Often fragmented with two or more different methods. 15
- 16. Immunology provides important techniques with which to investigate proteins An antibody (immunoglobulin, Ig) is a protein synthesized by an animal (human) in response to the presence of a foreign substance (antigen). It normally functions to protect the animal from infection. • Antibodies have high specificities & affinities to antigens. • They recognize a specific group or clusters of amino acids on a large molecule called antigenic determinant or epitope. • From the large repertoire of antibody-producing cells, each produces an antibody of a single specificity (polyclonal). 16
- 17. Antibodies 17 2 light chains and 2 heavy chains Antigen-antibody interaction
- 18. Types of antibodies 18 • Polyclonal antibody: Heterogeneous mixtures of antibodies, each specific for one of the various epitopes on an antigen. Useful for detection of a protein of low abundance, because protein molecules can be bound by more than one antibody at multiple distinct antigenic sites
- 19. Types of antibodies 19 • Monoclonal antibody: Identical, produced by clones of a single antibody-producing cell, recognizing one specific epitope. Highly specific, useful when working with impure proteins.
- 20. Preparation of monoclonal antibody 20
- 21. Application of monoclonal antibody • Used in affinity chromatography to purify proteins • Used in clinical laboratories – Detection of blood isozymes in MI – Screening donated blood for HIV, hepatitis etc • Therapeutic agent – Herceptin a drug used in breast cancer treatment • Used as catalytic antibody due to their very specific binding properties • Detection of proteins by immunofluorescence, Western blot, immunosorbent assay 21
- 22. Immunofluoroscence • Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. • This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. 22
- 23. Immunosorbent assay - ELISA ELISA: Enzyme-Linked Immunosorbent Assay • Ab used as specific analytic reagents to quantify the amount of a protein or other antigen. • Enzyme reacts with a colorless substrate to produce a colored product. • Enzyme linked to specific Ab that recognizes a target antigen. • If antigen is present the Ab-enzyme complex will bind to it . 23 96-well microtiter plate used in a typical ELISA assay
- 24. Immunosorbent assay - ELISA ELISA: Enzyme-Linked Immunosorbent Assay • If antigen is present the Ab-enzyme complex will bind to it • Upon addition of substrate, the enzyme will catalyze the reaction and genetrate the color product • The presence of a color product indicate the presence of antigen • The amount of color product proportional to amount of antigen • Need low amount of protein/antigen 24
- 25. ELISA: Enzyme-Linked Immunosorbent Assay
- 26. Indirect ELISA To detect presence of antibody Example: • HIV test detects the presence of antibodies that recognizes viral core protein, which is the antigen. • In indirect ELISA, the antigen at the bottom of the well is the viral core protein. • Serum from patient is added to the well. • If patient has Aids/infected with HIV, he/she will have Ab to the viral core proteins. • Ab from his/her sample will bind to the antigen at the bottom of the well. 26
- 27. Sandwich ELISA To detect presence of antigen Example • Can use to detect cardiac markers after heart attack • Bind Ab to creatine kinase to the bottom of the well • During heart attack, creatine kinase (CK) is released within hours of heart attack by cardiac muscle cells • The patients will have CK in the serum, CK will bind to the AB at the bottom of the well • A second Ab-labeled with enzyme that recognize CK will bind to CK. Second AB has enzyme that can react with substrate, and upon reaction elicit color production 27
- 28. Western blotting/Immunoblotting 28 • Analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. • It uses gel electrophoresis to separate proteins and the proteins are then transferred to a membrane (typically PVDF or nitrocellulose), where they are detected with antibodies specific to the target protein. Detection: Indirect method, secondary antibody can amplify the signal, colorimetric or chemiluminescent.
- 31. The 3D protein structure can be determined by NMR spectroscopy, X-ray crystallography and CryoEM 31 • Protein function depends on 3D structure. Nuclear Magnetic Resonance (NMR) spectroscopy reveal the dynamics of structure of proteins in solution. X-ray crystallography provide the best resolution for the determination of molecular structures. Cryo-electron microscopy (cryo-EM), a technique that fires beams of electrons at proteins that have been frozen in solution, to deduce the biomolecules’ structure.
- 32. Nuclear Magnetic Resonance • NMR depends on the fact that certain atomic nuclei are intrinsically magnetic. • The chemical shift of nuclei depends on their local environment. The spins of neighboring nuclei interact with each other in ways that provide definitive structural information. 32
- 33. X-ray crystallography Preparation of a protein in crystal form • Challenging process: A concentrated solution of highly pure material is required. It is often difficult to predict which experimental conditions will yield the most effective crystals. • Protein frequently crystallize in their biologically active configuration. 33
- 34. X-ray crystallography • Provide the best resolution for the determination of molecular structures because their wavelength approximately corresponds to that of a covalent bond. • An x-ray source generates a beam, which is diffracted by a crystal. The resulting diffraction pattern is collected on a detector. 34
- 35. X-ray crystallography • A crystal is mounted and gradually rotated while being bombarded with X-rays, producing a diffraction pattern. • The two-dimensional images taken at different rotations are converted into a three-dimensional model, combined with chemical data known for the sample. • The scattering pattern provide abundant information about protein structure. 35
- 36. Cryo-electron microscopy • Electron microscopy (EM) technique where the sample is cooled to cryogenic temperatures. • Limited information about dynamics • Can be used for very large molecules and complexes 36
- 37. Visualizing Protein Structures • The protein structure data are stored in Protein Data Bank (http://www.pdb.org) • The complexity of proteins with thousands of atoms presents a challenge for the depiction of their structure. • Several different types of representations are used to portray proteins, each with its own strengths and weaknesses. – Space-filling model – Ball-and-stick model – Ribbon diagrams 37
- 38. Space-filling model • The most realistic type of representation • Each atom is shown as a sphere with a size corresponding to the van der Waals radius of the atom. Bonds are not shown explicitly. • Advantage: useful in showing conformational changes in a protein from one set of circumstances to another. • Disadvantage: secondary and tertiary structures are difficult to see. 38
- 39. Ball-and-stick models • Not as realistic as space-filling models, but the bonding arrangement is easier to see because the bonds are explicitly represented as sticks. • Reveals a complex structure more clearly than a space-filling model. However, the depiction is so complicated that structural features such a alpha helices are difficult to discern. • Because space-filling and ball-and-stick models depict protein structures at the atomic level, the large number of atoms in a complex structure makes it difficult to discern the relevant features. 39
- 40. Ribbon diagram • Use schematic representation instead, such as ribbon diagram. • Used to accent a few dramatic aspects of protein structures, such as alpha helix (coiled ribbon), loop (thin tube), beta strand (broad arrow). • Allow the course of a polypeptide chain to be traced and readily shows the 2nd structure. 40
- 41. Ribbon diagram with highlights • Protein structures are tightly packed and little open space. • The openness of ribbon diagrams makes them particularly useful to highlight additional aspects of protein structure. • Active sites, substrates, bonds can be included in ball-and- stick or space-filling form within a ribbon diagram. 41
- 42. Summary 1. The purification of a protein is an essential first step in understanding their function. 2. Amino acid sequences of proteins can be determined experimentally. 3. Immunology provides important techniques with which to investigate proteins. 4. Mass spectrometry is a powerful technique for the identification of peptides and proteins. 5. Three-dimensional protein structure can be determined by X-ray crystallography, NMR spectroscopy and Cryo-EM. 42
- 43. Study questions 1. How do you sequence protein with more than 50 amino acid residues long? 2. What is the application of monoclonal antibody? 3. Name protein detection methods using immunological approach. 4. What is a Western blotting? 5. What is the purpose of using indirect ELISA and sandwich ELISA? 6. Name the methods to determine protein 3D structure. 7. What are the commonly used models to visualize protein 3D structure? 43