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Transcript
25. Methods of protein study. Physic- chemical properties of proteins useful in their separation:- salting-out, denaturation, charge, isoelectric point, utilization of specificity of proteases. Principles of protein separation: - Electrophoresis, chromatography, and sequencing. Salting-out:Salting out is a method of separating proteins based on the principle that proteins are less soluble at high salt concentrations. The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein. This process is also used to concentrate dilute solutions of proteins. Hydrophobic and hydrophilic aminoacids are present in protein molecules. After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acid interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules. Protein can be dissolved in water, if the protein surface is hydrophilic. When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out. Solvation:-A sodium ion solvated by water molecules Denaturation:Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process. Denaturation disrupts the normal alphahelix and beta sheets in a protein and uncoils it into a random shape. Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. Which may be disrupted. Therefore, a variety of reagents and conditions can cause denaturation. The most common observation in the denaturation process is the precipitation or coagulation of the protein. Charge:-Protein The charge on the protein affects its behavior in ion exchange chromatography. Proteins contain many ionizable groups on the side chains of their amino acids as well as their amino - and carboxyl - termini. These include basic groups on the side chains of lysine, arginine and histidine and acidic groups on the side chains or glutamate, aspartate, cysteine and tyrosine. The pH of the solution, the pK of the side chain and the side chain’s environment influence the charge on each side chain. The relationship between pH, pK and charge for individual amino acids can be described by the HendersonHasselbalch equation In general terms, as the pH of a solution increases, deprotonation of the acidic and basic groups on proteins occur, so that carboxyl groups are converted to carboxylate anions (R-COOH to R-COO-) and ammonium groups are converted to amino groups (R-NH3+ to R-NH2). In proteins the isoelectric point (pI) is defined as the pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge. The pI varies for different proteins. The charge on proteins arises from some of the amino acid side chains, as well as the carboxy- and amino-termini, some prosthetic groups and bound ions. This application is designed to calculate charge based only on the side chains and carboxy - and aminotermini. The charge on amino acid side chains depends on the pH of the solution and the pKA of the side chains. It is also affected by the localized environment around a side chain. We assume the following pKA values for ionizable groups on the protein and that the side chains will have these pKA values regardless of their environment within the protein. We also assume that the separation is based on the total charge on the protein, not the mass-to-charge ratios. Therefore, a protein with a charge of +15 will bind more tightly to a cation exchange stationary phase than a protein with a charge of +10, regardless of size. Group pKA Acids Carboxy-terminus 3.1 Aspartate 4.4 Glutamate 4.4 Cysteine 8.5 Tyrosine 10.0 Bases Amino-terminus 8.0 Lysine 10.0 Arginine 12.0 Histidine 6.5 In short:-when the pH is less than the pKA of a group, the protonated form of the group predominates. This leaves the acidic side chains with a charge approaching 0 and the basic side chains with a charge approaching a limiting value of +1. Conversely, when the pH is greater than the pKA of a group, the deprotonated form predominates, giving acidic side chains a charge approaching -1 and basic side chains a charge approaching 0. The charge on the protein is the sum of the charges on the individual amino acid side chains. However, the charge on individual amino acid side chains can vary when they are near a group of non-polar or highly charged side chains. Isoelectric point:The isoelectric point (IEP), is the pH at which a particular molecule or surface carries no net electrical charge. Amphoteric molecules which are also known as zwitterions contain both +ve and -ve charges depending on the functional groups present in the molecule. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). The isoeletric point is pH value at which the molecule carries no electrical charge or the negative and positive charges are equal. Surfaces naturally charge to form a double layer. In the common case when the surface charge-determining ions are H+/OH-, the net surface charge is affected by the pH of the liquid in which the solid is submerged. Again, the isoeletric point is the pH value of the solution at which the surfaces carries no net charge. The isoeletric point value can affect the solubility of a molecule at a given pH. Such molecules have minimum solubility in water or salt solutions at the pH which corresponds to their isoeletric point and often precipitate out of solution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids which make up proteins may be positive, negative, neutral or polar in nature, and together give a protein its overall charge. At a pH below their isoelectric point, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can thus be separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a technique called isoelectric focusing, which uses a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis. (also read from practical notes”eletrophresis page 6”) Principles of protein separation Chromatography:- It is a separation method based on the different migration of solutes through a system of two diverse phases, one of which is mobile and the other stationary. Chromatographic methods can be classified according to: A. Mobile phase arrangement • Liquid chromatography (LC) - mobile phase is a liquid • Gas chromatography (GC) - mobile phase is a gas. B. Stationary phase arrangement • Column chromatography – stationary phase is placed in a column • Planar techniques: Paper chromatography (PC) – stationary phase is a special paper, either as such or modified with other compounds. Thin-layer chromatography (TLC) – stationary phase is spread on a solid flat support (e.g., glass plate or aluminum foil) C. The process, which prevails in separation(usually several physical and chemical processes take place in separation but one of them prevails) • Partition chromatography – separation is based on different solubility of sample components in a stationary phase (a liquid) and in a mobile phase (a liquid or a gas). • Adsorption chromatography – separation is based on different abilities of components to adsorb on the surface of stationary phase (a solid). • Ion-exchange chromatography – separation is based on exchange of the ionic sample with the ionic group of the stationary phase and is governed by electrostatic interaction. • Size exclusion chromatography (gel chromatography) – components are separated according to the size and shape of their molecules as well as the size and shape of the pores of the stationary phase (size-exclusion effect). The large molecules elute at the beginning, and the small molecules at the end. • Affinity chromatography – separation is based on molecular recognition. Only those components, which are complementary to stationary phase, are adsorbed by their affinity.Affinity interactions are very strong. (In detail - check practical lab note topic “chromatography”) Electrophoresis:- (read practical notes topic “Electrophoresis”) Sequencing:Its major aim is to determine the primary sequence or primary structure of an unbranched biopolymer. Proteins and peptides, and DNA & RNA all are the examples of biopolymer which is a class of polymers produced by living organisms, in which monomeric units are sugars, amino acids and nucleotides. Pyrosequencing:Pyrosequencing, which was originally developed by Mostafa Ronaghi, has been commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases in a 7-hour run with a single machine. In the array-based method, single-stranded DNA is annealed to beads and amplified via EmPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. Sanger sequencing (chain termination method) Developer -Frederick Sanger The key principle of the Sanger method was the of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators. use In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer. An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability. This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used 'universal' primers. Dye-terminator sequencing:Dye-terminator sequencing utilizes labeling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labeledprimer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labeled with fluorescent dyes, each of which with different wavelengths of fluorescence and emission. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labeled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis. This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects. Maxam-Gilbert sequencing:The method requires radioactive labeling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, and C+ T). Thus a series of labeled fragments is generated, from the radio labeled end to the first 'cut' site in each molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radio labeled DNA fragment, from which the sequence may be inferred. Also sometimes known as 'chemical sequencing', this method originated in the study of DNA-protein interactions (foot printing), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.