METHODS TO DETERMINE PROTEIN STRUCTURE
- 1. Topic: protein structure determination methods PRESENTED BY KHIZAR ABBAS ULFAT RANA SABAHAT ALI
- 2. CONTENTS: INTRODUCTION EDMAN DEGRADATION X-RAY CRYSTALLOGRAPHY WESTERN BLOTTING SDS-PAGE 2D-GEL ELECTROPHORESIS ISOELECTRIC FOCUSING
- 3. Introduction Proteins, also known as polypeptides, are organic compounds made up of amino acids. They are arranged in a linear chain and folded into a globular form. Proteins are essential parts of organisms and they participate in virtually every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism.
- 4. The Structure of Proteins Most proteins fold into unique three-dimensional structures. The shape that a protein folds into naturally is known as its native conformation. There are four distinct aspects of a protein’s structure:
- 5. Continued.. Primary structure: the amino acid sequence Secondary structure: regularly repeating local structures stabilized by hydrogen bonds Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another Quaternary structure: the structure formed by several protein molecules which function as a single protein
- 6. Protein Analysis Techniques 1)Edman Degradation Introduction The strategy of Sanger for the sequencing of insulin was to characterize series of small overlapping peptides produced by cleavage of the parent molecule. Determination of the overall amino acid content and the identity of the amino- (N- )terminal residue for each peptide allowed deduction of the sequence of the whole molecule.
- 7. Contd.. An alternative approach was that described by Edman (1950). This allowed determination of extended sequences of peptides or whole protein.
- 8. Continued: The method employs chemical reactions to remove and identify the amino acid residue that is at the N- terminus of the polypeptide chain, i.e. the residue with a Free a amino group. Repeate this process,we can get the sequence of the polypeptide.
- 9. The Basis of the Method Peptide sequencing by Edman chemistry may be divided into steps as given
- 10. Coupling: •Phenyl isothiocyanate (PITC) reacts with an a- amino group (or in the case of prolyl residue with an imino group) at the N-terminal end of the polypeptide chain, to form a phenylthiocarbamyl derivative of the terminal residue.
- 11. Contd.. Basic conditions are required for this reaction. Edman originally used pyridine to generate a pH of 8.6. Clearly, a free a-amino group is required for this reaction to occur. If the amino group has become modified and will no longer react with PITC, the polypeptide is said to be ‘blocked’
- 12. Cleavage: In the presence of strong acid, cleavage occurs at the first peptide bond, giving the peptide (minus the first residue) and the liberated first residue as the anilinothiazolinone (ATZ) form.
- 13. Cleavage conti… Once other reactants and products have been washed away, the shortened polypeptide can be taken through another round of coupling and cleavage to release the second residue, and so on in a cyclical fashion
- 14. 3-Conversion: The ATZ residue is separated from the peptide by extraction in organic solvent (ethyl acetate or chlorobu- tane), and is then converted to a more stable form to allow better analysis. Conversion to the more stable phenylthio- hydantoin (PTH) form is done in aqueous acid (25% TFA, v/v in water). .
- 15. Analysis of PTH residues: The PTH residue generated by each cycle of Edman chemistry is typically identified by chromatography, originally thin-layer chromatography and latterly re- versed-phase high-performance liquid chromatography
- 23. 3)Western blotting Blotting is a technique used to transfer DNA , RNA, Protein into a carrier so they can be separated, often follows the use of gel electrophoresis. Three blotting techniques are used: Southern Blot Northern Blot Western Blot
- 24. Western blotting So we can define western blotting as “An analytical method that involves the protein immobilization on membranes before detection using monoclonal or polyclonal antibodies”
- 25. Principle • Immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of molecule of interest In a mixture of molecule.
- 27. Principle… • The molecule of interest is a protein and the probe is an antibody raised against that particular protein. • The SDS PAGE is prerequisite for western blotting.
- 28. Procedure Tissue preparation (Sample can be taken from whole tissue or cell culture Detergents, salt and buffers are employed to encourage lysis of cell) Gel electrophoresis( Protein of sample separated by using gel electrophoresis on the basis of molecular weight, electric charge )
- 29. Procedure… Transfer( Proteins are moved from the gel onto membrane made of nitrocellulose .Electro blotting can also be used.) Blocking (Blocking is achieved by placing the membrane in a dilute solution of protein with a minute amount of detergent)
- 30. Procedure…. Detection(A modified antibody is used which is linked to reporter enzyme , when exposed to substrate it produces color) Analysis (Size approximation are taken)
- 31. Advantages It is specific test for detection of HIV as compare to other tests. It tells us how much protein has accumulated inside the cell. It gives the information about the size of protein. It can detect one protein in a mixture of proteins.
- 32. Disadvantages If a protein degraded , western blotting detect it well. This test takes longer than other existing tests. It might also be more costly.
- 33. Applications It can identify the nature of the protein it can be applicated as a tool of quantitative analysis of the micro molecule antigen in cooperation with Immunoprecipitation.
- 34. Applications… The western blot is extensively used in biochemistry for the qualitative detection of single proteins protein-modifications (such as post-translational modifications) are also determined.
- 35. Medical diagnostic applications • The virus is enveloped with different proteins • The detection of presence of these proteins is useful in the detection of presence of virus. • Some forms of Lyme disease testing employ western blotting.
- 37. SDS-PAGE SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its molecules have a net negative charge within a wide pH range.
- 38. SDS-PAGE A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field.
- 39. 2D Gel Electrophoresis 2D gel electrophoresis (2DE) is a key technique for purifying individual proteins from complex samples based on their Isoelectric points and molecular weights.
- 40. Isoelectric Focusing Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their Isoelectric point (pI.
- 41. Isoelectric Focusing Type of zone electrophoresis Electrophoresis is the separation of charged molecules, particles and ions in a liquid medium under the influence of an electric field. Is ideal for separation of amphoteric compounds. Separation is achieved by applying a potential difference across the gel that contains a pH gradient.
- 42. Required for Isoelectric focusing Sample Ampholytes Buffer Voltage Supporting media Gel