MBC 201 General Biochemistry I PDF

**MBC 201 General Biochemistry I** **Primary, secondary, tertiary and quaternary structures of proteins** **Methods of isolation, purification and identification of Proteins** **Basic principles of tests for proteins and amino acids** **PROTEINS** ***Introduction*** *Proteins are macromolecule...

**MBC 201 General Biochemistry I** **Primary, secondary, tertiary and quaternary structures of proteins** **Methods of isolation, purification and identification of Proteins** **Basic principles of tests for proteins and amino acids** **PROTEINS** ***Introduction*** *Proteins are macromolecules formed by amino acids. A total of 20 different amino acids exist in proteins and hundreds to thousands of these amino acids are attached to each other by the peptide linkage in long chains to form a protein. Amino acids can be released from proteins by hydrolysis. (Hydrolysis is the cleavage of a covalent bond by addition of water in adequate conditions.) A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short chains of amino acids, containing less than 20--30 residues, are rarely considered to be proteins and are commonly called peptides, or sometimes oligopeptides* *The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids. Shortly after or during synthesis, the amino acid residues in a protein can be chemically modified in a process known as post-translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors.* Structure of Proteins --------------------- *There are four levels of protein structures and these are distinguished from each other by the degree of complexity in the polypeptide chain. A single protein molecule may contain one or more of the protein structure types which are: primary, secondary, tertiary, and quaternary structure.* ### Primary structure: *This is the amino acid sequence. A protein is a polyamide.* *The primary structure describes the unique order in which amino acids are linked together to form a protein.* *Primary structure of protein* ### Secondary structure: *Regularly repeating local structures stabilized by hydrogen bonds. It refers to the coiling or folding of a polypeptide chain that gives the protein its 3-D shape.* *There are two types of secondary structures observed in proteins.* ***Alpha (α) helix structure**.* *This structure resembles a coiled spring and is secured by hydrogen bonding in the polypeptide chain.* ***Beta (β) pleated sheet**.* *This structure appears to be folded or pleated and is held together by hydrogen bonding between polypeptide units of the folded chain that lie adjacent to one another.*  ***Secondary structure of protein*** ### Tertiary structure: *This is the overall shape of a single protein molecule. It is formed by the spatial relationship of the secondary structures to one another. Tertiary Structure refers to the comprehensive 3-D structure of the polypeptide chain of a protein. There are several types of bonds and forces that hold a protein in its tertiary structure. * ***Tertiary structure of proteins*** ### Bonds and Forces Found in Tertiary Protein Structures *Hydrophobic interactions * *Hydrophobic interactions greatly contribute to the folding and shaping of a protein. The \"R\" group of the amino acid is either hydrophobic or hydrophilic. The amino acids with hydrophilic \"R\" groups will seek contact with their aqueous environment, while amino acids with hydrophobic \"R\" groups will seek to avoid water and position themselves towards the center of the protein. ​* *Hydrogen bonding* *Hydrogen bonding in the polypeptide chain and between amino acid \"R\" groups helps to stabilize protein structure by holding the protein in the shape established by the hydrophobic interactions. Due to protein folding, ionic bonding can occur between the positively and negatively charged \"R\" groups that come in close contact with one another.* *Covalent bonding* *Folding can also result in covalent bonding between the \"R\" groups of cysteine amino acids. This type of bonding forms what is called a disulfide bridge.* *Van der Waals forces* *Interactions called van der Waals forces also assist in the stabilization of protein structure. These interactions pertain to the attractive and repulsive forces that occur between molecules that become polarized.* *The term \"tertiary structure\" as often used is synonymous with the term fold. The tertiary structure is what controls the basic function of the protein.* ### Quaternary structure: *This refers to the structure of a protein macromolecule formed by interactions between multiple polypeptide chains. Each polypeptide chain is referred to as a subunit. Proteins with quaternary structure may consist of more than one of the same type of protein subunit. They may also be composed of different subunits.*  ***Quaternary structure of protein*** *Hemoglobin is an example of a protein with quaternary structure. Hemoglobin, found in the blood, is an iron-containing protein that binds oxygen molecules. It contains four subunits: two alpha subunits and two beta subunits which function as a single protein complex. Another example are protein existing as a multienzyme complex. Proteins are not entirely rigid molecules.* *In addition to these levels of structure, proteins may shift between several related structures while they perform their functions. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as \"conformations\", and transitions between them are called conformational changes. Such changes are often induced by the binding of a substrate molecule to an enzyme\'s active site, or the physical region of the protein that participates in chemical catalysis. In solution, proteins also undergo variation in structure through thermal vibration and the collision with other molecules non-proteogenic amino acids.* **SIMPLE METHODS OF THE ISOLATION, SEPARATION, PURIFICATIONAND IDENTIFICATION OF PROTEINS** **Sample Preparation** Before samples can be analysed using advanced scientific equipment and instruments, they must be properly treated and prepared. This [**preliminary step**](https://www.labmate-online.com/news/laboratory-products/3/breaking-news/sample-handling-vs-preparation-what-is-the-difference/55541) is an important stage of the overall analysis process as it helps to prevent contamination, improve accuracy and minimise the risk of results distortion. Almost always, sample preparation starts with extraction. This involves isolating a representative piece of material from a larger source. To begin any sort of purification it is important that an assay be available to identify where the protein of interest is after the fractionation. Assays come in many different forms and depends in a large part on the type of protein to be purified (i.e. is it an enzyme?). Commonly used assay technologies are: **Crude Extracts** To being any sort of purification procedure you need to obtain the material from which you plan to isolate the protein of interest. Historically the abundance and ease of isolation dictated which proteins were first studied (e.g. hemoglobin). Also many proteins are common to a large number of species (e.g. metabolic enzymes) so they could be isolated in large abundance from other sources, such as yeast. Once the material containing the protein of interest has been obtained, it is necessary to generate a crude extract \-- for proteins from muscle that would mean grinding it up, for an intercellular protein that would mean breaking the cells open, etc. This is always done in the presence of a buffer and inhibitors. This is necessary to avoid denaturing the protein of interest due to change in pH and from being cleaved by enzymes that will be released in the process. **ISOLATION, SEPARATION, PURIFICATION AND CHARACTERIZATION OF PROTEINS** Protein purification methods make it possible to study and understand proteins in detail. Processes for the separation and purification of proteins exploit differences in physico-chemical properties of biological molecules, including molecular size, charge, solubility, hydrophilic or hydrophobic nature (charge) and biological affinity (binding specificity). These properties of a protein are derived from the AA properties composing the protein. For example the molecular weight (MW) of a protein is just the summation of the masses of the individual AAs composing the protein. MW is usually expressed in daltons (Da) or kilodaltons (kDa). Important methods used in biochemical purification include precipitation, crystallization, centrifugation, chromatography, electrophoresis, etc. **Precipitation** In bioprocessing, precipitation of biological material is usually achieved by the use of solvents, salts or increased temperature. Most of these precipitations produce a non-crystalline product and may constitute aggregates of several molecular species or contain significant amounts of occluded solvent or absorbed salts. Precipitates are therefore impure compared to crystals which, are produced by temperature decreases. Precipitation of proteins by \'salting out\' can serve to both purify and concentrate the particular protein fraction. This process depends on a number of factors: pH, temperature, protein concentration and the salt used. At constant ionic strength, the solubility of a protein is least at its isoelectric point and increases on either side of this. Neutral salts have a pronounced effect on the solubility of globular proteins. Protein solubility is increased with increasing low salt concentrations, a phenomenon known as salting-in. As the ionic strength is further increased, the solubility of a protein begins to decrease, eventually precipitating or salting-out the protein. Salts of divalent ions such as magnesium chloride and ammonium sulphate are far more effective than salts of monovalent ions such as sodium chloride, potassium chloride and ammonium chloride. Ammonium sulphate is the salt most commonly used for protein precipitation because of its high solubility (enabling very high ionic strengths to be attained), its lack of toxicity to most proteins, its cheapness, and, in some cases, its protein-stabilizing effect. A contributing factor to salting out of proteins from aqueous solutions is that the high salt concentration may remove water of hydration from the protein molecules, thus reducing their solubility. **Centrifugation** Generally the first step after forming a crude extract is a simple filtration or centrifugation to remove the large material. Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge. This process is used to separate two immiscible liquids with more-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Centrifugation alters the effective gravitational force on to tube/bottle so as to more rapidly and completely cause the precipitate (\"pellet\") to gather on the bottom of the tube. The remaining solution is properly called the \"supernatant\". The supernatant liquid is quickly decanted from the tube/bottle without disturbing the precipitate. differential\_centrifugation.jpg Differential centrifugation, as shown in the figure, is multiple rounds of centrifugation at increased speeds and time allows for different cellular fractions to be separated.     **Dialysis** Dialysis is a procedure for exchanging the solvent around a protein. In general the protein solution is placed inside a semi-permeable membrane (dialysis bag) which is suspended in a larger volume of buffered solution. The key to this procedure working is that the membrane has to be permeable to water and ions, but not to the protein of interest. Thus buffers & salts exchange until an equilibrium is established between the inside & outside of the membrane.   **Column Chromatography** Column chromatography is one of the most powerful fractionation methods. It can separate components of mixtures based upon:   Commonalities between all three types of chromatography methods is that they all use a resin (solid phase) with special chemical properties held in a glass cylinder (called a \"column\"). A buffered solution (mobile phase) percolates through the column and is collected in tubes (\"fractions\") upon exiting the column. A protein mixture is applied in the mobile phase & percolates through the column as an expanding band. Different proteins migrate differently depending on their properties and those of the resin. Below we will examine the different forms and examine their particular properties.   **Gel Filtration/Size Exclusion** gel\_filtration1.png In gel filtration, or as it is sometimes referred to as size exclusion, chromatography the resin are porous. Some molecules (blue here) can enter the resin and as the lines try to indicate it is not a straight path through; thus it takes longer for small molecules to traverse the column than large molecules which travel around the outside of the resin. This is highlighted in the figure below where big molecules (blue) come off first and smaller molecules (red) later.   **Ion Exchange** Ion exchange chromatography is broken in to two types - anion & cation exchangers. There are many different types of moieties that are used from weakly to very strongly charged thus allowing a huge range of molecules the ability to interact. ion\_exchange\_stepwise.gif Unlike gel filtration chromatography, here proteins directly interact with the resin. So generally the column is equilibrated in a buffer solution to establish a constant pH in the column, then the protein mixture is loaded where all or some of the proteins interact with the resin depending upon their own charge. Buffer is continued to be applied until all proteins not interacting with the resin have been washed off. At that point usually a gradient of increasing salt concentration (disrupts ionic and hydrogen binding) in the buffer is applied to column allowing the most weakly interacting proteins to release first followed by the more strongly and finally the most strongly interacting. This can also be accomplished by changing the pH of the buffer being applied to the column. **Anion Exchanger** Anion exchanger means that it removes anions from protein mixture so that means the resin must be decorated with positively charged moieties. Before elution begins all positively and uncharged proteins will fall through the column. When you start eluting, first you will knock off the weakly negative proteins (e.g. -1 charge), followed by those with a stronger negative charge (-2), and finally the most negatively charged proteins (-3).  **Cation Exchange** It is exactly oppose with a cation exchanger \-- here cations are removed from the protein solution so the resin must be negatively charged. Again before elution begins all negatively and uncharged proteins will fall through the column. When you start eluting, first you will knock off the weakly positive proteins (e.g. +1 charge), followed by those with a stronger positive charge (+2), and finally the most positively charged proteins (+3).   **Affinity** Affinity chromatography requires that you know something specific about your protein \-- that it has a specific tag engineered into the sequence, that it binds NAD+, you know the ligand it binds or that you have a specific monoclonal antibody that interacts with your protein. For this type of chromatography your resin is decorated with the antibody, NAD+ or a divalent metal (most popular engineered tag is the 6xHis tag \-- 6 His residues at either the N- or C-termini of the protein). Again the column resin is pre-equilibrated in the appropriate buffer before the protein sample is loaded. It is expected that only the protein of interest will interact. Once all non-interacting proteins have been removed from the column then your protein can be eluted by changing the pH, adding salt, adding a metal chelator, or a high concentration of the ligand.    **Electrophoresis** Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a uniform electric field. Thus it separates components of a mixture based on their size and/or charge. Many important biological molecules such as proteins, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) exist in solution as cations (+) or anions (-). Under the influence of an electric field, these molecules migrate at a rate that depends on their net charge, size and shape, the field strength, and the nature of the medium in which the molecules are moving. Electrophoresis in biology uses porous gels as the media. The sample mixture is loaded into a gel, the electric field is applied, and the molecules migrate through the gel **matrix**. Thus, separation is based on both the molecular sieve effect and on the electrophoretic mobility of the molecules. This method determines the size of biomolecules. It is used to separate proteins, for identification, sequencing, or further manipulation visuallization.gif   Visualization of proteins on paper or in a gel is an important step in any electrophoresis. Proteins may be visualized using silver stain or Coomassie Brilliant Blue dye (right image). In some cases the gels are transferred to a solid support (nitrocellulose) and then probed with specific antibodies (Western Blot).   **Paper electrophoresis** Generally used to separate AAs or peptides of differing charge. As shown in the figure, AAs and peptides will separate based on their charge with the most highly charged species moving the furthest.    **PAGE (PolyAcrylamide Gel Electrophoresis) \-- Native Gel** gel\_electrophoresis.jpg It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent). The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel. The term \"gel\" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Agarose is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules and macromolecular complexes.   \"Electrophoresis\" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass but also their charge and shape which varies widely for proteins. Electrophoretic mobility of small molecules is greater than the mobility of large molecules with the same charge density thus allowing separation. To separate proteins or DNA generally the pH of the buffer and protein mixture is high (\~9) so that the proteins carry a net-negative charge. However, because size, charge and shape all play a role in how a molecule will behave in a native gel most scientists use a SDS-PAGE gel which is predictable.     **CHROMATOGRAPHY** Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separated from each other while moving with the aid of a mobile phase. The factors that affect this separation process include molecular characteristics related to adsorption, partition, and affinity or differences in molecular weights. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster. Based on this approach three components form the basis of the chromatography technique: **Stationary phase:** This phase is always composed of a "solid" phase or "a layer of a liquid adsorbed on the surface a solid support". **Mobile phase:** This phase is always composed of "liquid" or a "gaseous component." **Separated molecules:** The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component that affects the separation of molecules from each other.. Stationary phase in chromatography is a solid phase or a liquid phase coated on the surface of a solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids, and solid material. Liquid chromatography is used especially for thermal unstable, and non-volatile samples. **Types of chromatography** Paper chromatography Column chromatography Ion-exchange chromatography Gel-filtration (molecular sieve) chromatography Affinity chromatography **Paper chromatography** In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method a thick filter paper comprised the support, and water drops settled in its pores made up the stationary "liquid phase." Mobile phase consists of an appropriate fluid placed in a developing tank. Paper chromatography is a "liquid-liquid" chromatography. https://cdn1.byjus.com/wp-content/uploads/2022/02/word-image104.png **R~F~ Value: =** Distance moved by solute divided by distance moved by solvent. The R~f~ (retardation/retention factor) value is the ratio of the solute's distance travelled to the solvent's distance travelled. The R~f~ value is a physical constant for organic molecules that can be used to verify a molecule's identity. Depending on the nature of the analytes and the stationary phases, a chromatogram must first be generated with an appropriate solvent (mobile phase). After drying the chromatogram, the locations (migration values) of the analytes and the solvent front are measured. R~f~ values are always less than 1. An R~f~ value of 1 or too close to it means that the spot and the solvent front travel close together and are therefore unreliable. **Addition to Column Chromatography** As the length of the column increases, the resolution of two types of proteins with different net charges generally improved. However, the rate at which the protein solution can flow through the column usually decreases with column length. And as the length of time spent on the column increases, the resolution can decline as a result of diffusional spreading within each protein band. As the protein-containing solution exits a column, successive portions (fractions) of this effluent are collected in test tubes. Each fraction can be tested for the presence of the protein of interest as well as other properties, such as ionic strength or total protein concentration.