This action might not be possible to undo. Are you sure you want to continue?
Topics 1. Structural Basis of Proteins 2. Proteins: Functional Design 3. Purifying Technique of Proteins 4. Exploring Proteins: Immunological Assay 5. Protein Conformation: Substantial Methods 6. Membrane Proteins
Structural Basis of Proteins:
Proteins are the important biomacromolecules present in every living cell and play a significant role in its survival. These organic compounds are found versatile in our body in the form of hair, cartilage, muscles, tendons, ligaments and skin. Proteins are considered as polypeptides. Each protein is a linear chain of amino acids linked together by peptide bonds.
Amino acids are the fundamental units of proteins. Naturally existing amino acids are of 20 in number. These 20 amino acids arranged together in different manner construct the proteins. All these amino acids possess the same common structure and differ only in their sidechain groups. The nature of individual amino acids like shape, size, charge and reactivity depends on these amino, carboxyl and R functional groups attached to them.
A typical protein contains approximately 200–300 amino acids. Some proteins like peptides are very small and some like titin present in skeletal muscle are very long having 26,926 amino acids in a single chain.
Amino acids are linked to each other by the formation of the single chemical association called ‘peptide bond’. The peptide bond is resulted from the dehydration reaction between the amino groups of one amino acid with the carboxyl group of the adjacent one.
Formation of a peptide bond:
It is basically an amide bond which is formed as a result of the linkage between the C – terminal of one amino acid with the N – terminal of another. This is an endergonic reaction and requires the hydrolysis of a high energy phosphate bond. This repeated reaction of peptide bond formation forms the back bone from which the various side-chain groups project. The linear protein chain is thus formed with a free amino group at one end and a free carboxyl group at the other end.
In conventional terms, a protein chain is represented with its C–terminal amino acid on the right and the N –terminal amino acid on the left.
Structure of Proteins:
Proteins are heteropolymers formed by the polymerization of amino acids. In organic chemistry, a two dimensional view of the molecule is applied while depicting the structure of the molecule. In basic biology, the structure of proteins is depicted at four hierarchical levels. (i) Primary structure (ii) Secondary structure (iii) Tertiary structure (iv) Quaternary structure
The sequence of amino acids or the linear arrangement of amino acids is called as the primary structure of proteins. Primary structure gives us the positional information of amino acids in a protein. It lets us know which amino acid starts the chain, which is the second and so on.
In short, a protein is imagined as a line, where the left end is represented by the first amino acid and the right end is represented by the last amino acid. The first amino acid is termed as the N-terminal amino acid and the last amino acid is termed as the Cterminal amino acid.
A protein does not extend through out as a rigid linear stick. The thread is folded in the form of a helix which resembles a revolving stair case’s course. Only some portions of the protein are arranged in the form of a helix popularly known as ‘localized organization’.
Other regions of the protein thread are folded into forms like an alpha helix which is a rod like spiral structure and a beta sheet, a planar structure formed by the combination of two or more beta strands. This arrangement is referred as the secondary structure.
The third level of hierarchy is the tertiary structure. The long chain protein molecule can also be folded like a hollow woolen ball giving rise to this peculiar structure which gives a three dimensional view to a protein. Tertiary structure is absolutely necessary for the many biological activities of proteins. For monomeric proteins which consist of a single polypeptide chain, tertiary structure is the highest level of organization.
Some proteins are an assembly of more than one polypeptide or subunits commonly known as ‘multimeric proteins’. The architecture of these proteins is such that these individually folded polypeptides or subunits are arranged one upon another in the form of a cube or a plate. This arrangement which consists of two or more subunits is termed as the quaternary structure of proteins.
Adult hemoglobin consists of 4 subunits. Two of these are identical to each other. Hence two subunits of alpha type and two subunits of beta type together constitute the human hemoglobin (Hb). Proteins can themselves be associated into larger assemblies which give rise to macromolecular assemblies. The protein coat of a virus is a best example of such macromolecular assemblies.
Proteins: Functional design
Proteins form the physical basis of life and each and every function inside the living cell relies upon proteins. Enzymes which take part in the catalysis of all biochemical reactions contain proteins. The well known example of an enzyme is the zymase which brings about the hydrolysis of starch. Heterotrophic organisms consume proteins as their major nutrient.
The extra cellular matrixes in which the cells are embedded are largely made of proteins like collagen. Contractile proteins determine the locomotion and movement of cells and organisms. Transportation of materials inside the body fluids is carried out by proteins. For the proteins to perform this variety of functions, they need to bind themselves to a wide range of molecules. This binding of molecules is not so general and is done with a high degree of specificity. Proteins as enzymes are termed as the ‘biological catalysts’. During such catalysis reactions, the enzymes bind to their target molecules or the substrates.
‘Affinity’ and ‘Specificity’ of Protein:
Interaction of a protein with its ligands can be determined by two important properties ‘Affinity’ and ‘Specificity’. The strength with which the protein and the ligand bind with each other is termed as ‘affinity’. Specificity indicates the exclusiveness of a protein’s ability to bind to a particular molecule when it is present along with the other molecules. The effectiveness of both the properties depends upon the ligand-binding site on the protein. It resembles a mold which fits exactly into its companion molecule. If the shape of the ligand-binding site is exactly complementary to the ligand molecule, then highly specific interactions with increased affinity will occur. The function of almost all the proteins depends on their ability to bind the ligand molecules. Ligand-binding sites on proteins and the corresponding ligands are chemically and morphologically complementary to each other. The affinity of a protein for a particular ligand refers to the strength of binding, its specificity, and also the restriction of binding to one or a few preferred ligands. The binding of an antibody with an antigen is a best example for such highly specific interactions. Proteins hold together, protect, and provide structure to the body of a multicelled organism. They catalyze, regulate, and protect the chemistry of body cells in the form of enzymes, hormones, antibodies, and globulins. They effect the transport of oxygen and other significant substances within an organism in the form of hemoglobin, myoglobin and various lipoproteins.
Purifying Technique of Proteins
Basis of Purification:
In order to identify the working mechanism of a particular protein, it is essential to know about the sequence of its amino acids and their nature. Once the purification of protein is accomplished, it is simple to determine the sequence of amino acids. After the application of appropriate purifying techniques, a sample is yielded which contains only one protein that is the protein of interest. The proteins need to be released out of the cell and can be separated into different densities using the method of differential centrifugation. Proteins can be distinguished from one another on the basis of their size, solubility, charges contained and the binding capacity. The followings are the popular methods of proteins purification.
Chromatography is used to separate the organic compounds based on the charge they possess, their size, shape, and solubilities. A chromatography commonly consists of a mobile phase and a stationary phase. The solvent and the molecules to be separated form the mobile phase and a stationary phase includes either a paper or a column called ‘resin’ through which the mobile phase travels. Depending upon their chemical nature, molecules travel through the stationary phase at different rates.
Ion –exchange chromatography:
Ion Exchange Chromatography relies on charge-charge interactions between the proteins in a selected sample and the charges immobilized on the resin taken. Ion exchange chromatography can be subdivided into anion exchange chromatography, in which the negatively charged ions bind to positively charged ions whereas vise versa in cation exchange chromatography. In living things, two third of the dry matter contains protein. Hence, efforts are being made to purify a protein of interest from other proteins in a cell. In order to separate any of the macromolecules the knowledge about the chemistry of proteins like molecular weight, its charge, and its shape is required.
Gel filtration chromatography:
Gel filtration chromatography is based on the principle of gravity. It allows separating proteins depending on their size. In this type of chromatography, microscopic glass beads with tinier holes are packed to form a column. A solvent sample is passed through the beads. Molecules that are smaller than the holes in the beads get suspended up in the beads. Hence, only the smaller molecules move through the column comparatively slowly than larger molecules. Based on their size alone, fractions containing different protein molecules can be easily collected.
In affinity chromatography, a molecule which is often an antibody that will bind to the protein of interest to be purified is attached to the glass beads. A mixture of proteins is added to the column. The protein of interest will bind to the antibody and everything else passes through the column. A buffer containing high concentrations of salt or acid is usually used to get the protein elute from the column.
High – Pressure Liquid Chromatography:
Liquid chromatography (LC) is a specific technique in which the mobile phase is a liquid. In this type, the stationary phase consists of a finely powdered solid adsorbent packed into a thin metal column. The mobile phase consists of an eluting solvent forced through the column by a highpressure pump. The mixture of molecules to be analyzed is injected into the column. A detector monitors this process. Many different LC packing and eluting solvents can be sued according to the requirements of desired resolution.
Dialysis is simple separation technique which involves a semipermeable membrane like cellulose. The molecules which are having dimensions more than the size of the pores retain inside the membrane whereas smaller ones emerge out. It is not considered as an effective method of separating proteins.
Salting out Technique:
In a given definite concentration of salt, the ability to precipitate differs from one protein to another. This method is employed to fractionate proteins. It is also found useful in concentrating the dilute solutions of proteins separated from other purification techniques.
Exploring proteins: Immunological assay
ELISA stands for Enzyme-Linked Immunosorbent Assay. In this technique, specific antibodies are employed as analyzing agents to quantify the amount of proteins or any other antigens. It is also known as enzyme immunoassay or EIA. The molecule is detected by antibodies that have been made against it, means for which it is the antigen. Both polyclonal and monoclonal antibodies are often used in this technique, but the results produced by monoclonal antibodies are more accurate. Here, an enzyme which can react with a colorless substrate to produce a colored product is linked covalently to a precise antibody. This antibody is capable of recognizing a particular target antigen. If presence of antigen is detected, then the enzyme – antibody complex will bind and the enzyme part serves as the catalyst for the reaction enabling the formation of a colored product. Thus, if we get a colored product, the presence of antigen can be confirmed. Two types of ELISA are most popular. Indirect ELISA and sandwich ELISA allow for the detection of antibodies, while sandwich ELISA can be used both for quantifying and detecting the antigens. The indirect ELISA is used as a test for the HIV infection.
Western blotting is an immunoassay technique which helps us to detect the very minute quantities of protein of interest in a particular or body fluids. A mixture of protein sample is loaded on a SDS-polyacrylamide gel and is subjected to electrophoresis. The process of electroblotting transfers the determined protein on the surface of the gel into a polymer sheet. If an antibody which is specific for the protein of interest is added to the sheet, the antigen –antibody complex will be formed accordingly. It can be easily detected by the addition of a second antibody which is specific to the first one. Like ELISA, an enzyme in the second antibody which produces a colored product.
Western Blotting technique is constructive in the cloning of genes. This is the basic test for detecting infection of Hepatitis C, where it is employed for detecting the presence of virus’ core protein.
Examination of proteins is made more powerful with the help of fluorescent markers. In this technique, the cells are coated with fluorescent –labeled antibodies or other fluorescent proteins, and are subjected to examination under fluorescent microscope. With the help of fluorescence microscopy, it is revealed that the receptors are located in the cytoplasm, if their respective hormones are not present. Once, if the steroids are added, they are translocated into the nucleus where they bind with DNA. Electron microscopy also can give a finer spatial resolution, using the antibodies tagged with electron-dense markers. For example, gold clusters can be conjugated with the antibodies for their high visibility under the electron microscope.
Protein conformation: Substantial methods
The different conformations of proteins increase their stability substantially. If we take a desirable protein as native (N) where N denotes the globular, native state of the protein, it can be now well defined as a result of numerous structural determinations by substantial methods like NMR and X-ray diffraction studies.
Nuclear Magnetic Resonance:
Spectroscopy is defined as the study of the interaction of electromagnetic radiation with matter. Nuclear magnetic resonance spectroscopy is the use of the NMR phenomenon to study physical, biological and chemical properties of matter. NMR spectroscopy is often used to study chemical structure using simple one-dimensional techniques. Nuclear magnetic resonance can be abbreviated as NMR. NMR spectroscopy discloses lot of information about hoe proteins fold, recognize and interact with the other molecules. The catalyzing reactions of the proteins and their intermediates are also made clear through this efficient method. This is a phenomenon occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. Some nuclei experience this phenomenon, and others do not, dependent upon whether they possess a property called spin. NMR reveals the structure and dynamics of proteins in solution. X-Ray Crystallography: X-ray crystallography is used for the determination of protein structure. The finest visualization of the available protein structure is obtained from X-ray crystallography only. The precise three dimensional structures of the atoms in a protein molecule is exposed with this technique. The reason behind this best resolution is the wavelength of the X-rays is about the same length of the covalent bond between the atoms.
Purified protein crystals are formed by the salting out technique. If the protein crystal is mounted is a specific orientation, with respect to the beam of X-rays, and is rotated in such a way that the beam strikes the crystal from various directions, an X-ray photograph is obtained on the film. The intensities of the spots are measured and reconstructed as an image of the protein. The three dimensional view of thousands of proteins is known as detail up to atomic level.
Cells form the basic unit of life and the universal feature of all cells is an outer selectively permeable membrane called the plasma membrane. Also, almost all eukaryotic cells contain complicated systems of internal membranes which give rise to various membrane-enclosed compartments within the cell. Structurally, cell membranes are composed of lipids and proteins.
The Plasma Membrane:
The plasma membrane serves as the boundary between the interior of the cell and the extra cellular fluid that bathes all cells. The lipids in the plasma membrane are chiefly phospholipids which are amphiphilic with the hydrocarbon tail of the molecule being hydrophobic, while its polar head hydrophilic. Cholesterol and phosphatidyl ethanolamine are the common phospholipids present in the plasma membrane. Plasma membrane has watery surfaces on both the sides, inside and outside of the cell and hence its phospholipids form a phospholipid bilayer with the hydrophobic tails facing each other. Although all membrane proteins are located at the membrane, they are both structurally and functionally diverse. Every biological membrane has the same basic phospholipid bilayer structure associated with a set of membrane proteins that enables the membrane to carry out its individual activities. Some proteins are bound only to the surface of the membrane, whereas others have one region masked within the membrane and domains on one or both sides of it. Generally protein domains on the extracellular membrane surface carry out cell-cell signaling processes. Domains within the membrane, specifically which form channels and pores, help to transport molecules across the membrane. Domains lying along the cytosolic face of the membrane deal with a variety of functions. They anchor the cytoskeletal proteins to the membrane and trigger the intracellular signaling pathways. Membrane proteins can be classified into two broad categories—integral or intrinsic and peripheral or extrinsic depending upon the nature of the membrane-protein interactions. Most of the biomembranes contain both types of membrane proteins.
Integral Membrane Proteins:
Integral membrane proteins, also called intrinsic proteins, have one or more parts that are embedded in the phospholipid bilayer. Many of the proteins associated with the plasma membrane are tightly bound to it. Integral proteins contain residues with hydrophobic side chains that interact with fatty acyl groups of the membrane phospholipids. This enables the anchoring the protein strongly into the membrane. Most integral proteins span the entire phospholipid bilayer. These transmembrane proteins contain one or more membrane-spanning domains. Domains are of four to several hundred residues long, which extend into the aqueous medium on either side of the bilayer. Two types of membrane-spanning domains are found in transmembrane proteins: one or more α helices or, multiple β strands.
Proteins containing seven membrane-spanning α helices form a major class that includes bacteriorhodopsin and many cell-surface receptors. In few cases, some are anchored to one of the membrane leaflets by covalently bound fatty acids which are embedded in the membrane. Some transmembrane proteins that span the bilayer several times form a hydrophilic channel through which certain ions and molecules can enter or leave away from the cell and, and the surface is hydrophobic, permitting interaction with the interior of the lipid bilayer. For example, all G-protein-coupled receptors like receptors of peptide hormones, each span the plasma membrane seven times. In all these cases, the portion within the lipid bilayer consists primarily of hydrophobic amino acids. The amino acids are often arranged in an alpha helix so that the polar C=O and -NH groups at the peptide bonds can interact with each other rather than interacting with their hydrophobic surroundings. Those portions of the polypeptide that project out from the phospholipid bilayer are liable to have more hydrophilic amino acids. Furthermore, those that project into the aqueous surroundings of the cell are usually glycoproteins, with many hydrophilic sugar residues that are attached to the part of the polypeptide exposed at the cell surface.
Peripheral Membrane Proteins:
Peripheral membrane proteins, or extrinsic proteins are more loosely associated with the membrane. They are usually attached noncovalently to the protruding portions of integral membrane proteins and do not interact with the hydrophobic core of the phospholipid bilayer. Cytoskeletal proteins spectrin and actin in erythrocytes are examples of peripheral proteins confined to the cytosolic face of the plasma membrane. The enzyme protein kinase C which shuttles between the cytosol and the cytosolic face of the plasma membrane, having a significant role in signal transduction is also a peripheral protein. Other peripheral proteins like extracellular matrix proteins are localized to the outer surface of the plasma membrane. Membrane proteins are often restricted in their movements. Since the lipid bilayer is indeed a film of oil, there is possibility of the structures immersed in it would be relatively free. Except for some membrane proteins, the mobility of other proteins is limited. Actin microfilaments are best examples of proteins present at the interior face of the plasma membrane that are tethered to cytoskeletal elements. Some proteins on the exterior of the membrane are anchored to components of the extracellular matrix like collagen. Integral membrane proteins like epithelial cells cannot pass through the stiff junctions found between certain kinds of cells.
Points to Remember:
• • • • • • • • • • • Proteins are heteropolymers formed by the polymerization of amino acids in which each individual protein is linked together by means of peptide bonds. Peptide bond is resulted from the dehydration reaction between the amino groups of one amino acid with the carboxyl group of the adjacent one. All the amino acids possess the same common structure differs only in their sidechain groups. Structure of proteins is depicted at four hierarchical levels as primary structure, secondary structure, tertiary structure and quaternary structure. The strength with which the protein and the ligand bind with each other is termed as ‘affinity’. ‘Specificity’ indicates the exclusiveness of a protein’s ability to bind to a particular molecule when it is present along with the other molecules. Working mechanism of a particular protein can be identified by knowing about the sequence of its amino acids and their nature. Chromatography is used to separate the organic compounds based on the charge they possess, size, shape, and their solubilities. Ion Exchange Chromatography relies on charge-charge interactions between the proteins in a selected sample and the charges immobilized on the resin taken. Gel filtration chromatography is based on the principle of gravity. It allows separating proteins depending on their size. In affinity chromatography, a molecule which is often an antibody that will bind to the protein of interest to be purified is attached to the glass beads and a mixture of proteins is added to the column. The protein of interest will bind to the antibody and everything else passes through the column. • Liquid chromatography (LC) is a specific technique in which the mobile phase is a liquid and the stationary phase consists of a finely powdered solid adsorbent packed into a thin metal column. • ELISA stands for Enzyme-Linked Immunosorbent Assay which is also known as enzyme immunoassay or EIA.
Two types of ELISA are in use: Indirect ELISA and sandwich ELISA allows for the detection of antibodies, while sandwich ELISA can be used both for quantifying and detecting the antigens.
Indirect ELISA is used as a test for the HIV infection. Western Blotting technique is an immunoassay technique which helps us to detect the very minute quantities of protein of interest in a particular or body fluids. It is the basic test for detecting infection of Hepatitis C, where it is employed for detecting the presence of virus’ core protein.
In fluorescent spectroscopy, the cells are coated with fluorescent –labeled antibodies or other fluorescent proteins, and are subjected to examination under fluorescent microscope.
• • •
Spectroscopy is defined as the study of the interaction of electromagnetic radiation with matter. Nuclear magnetic resonance spectroscopy is the use of the NMR phenomenon to study physical, biological and chemical properties of matter. X-ray crystallography is used for the determination of precise three dimensional structures of proteins. The finest visualization of the available protein structure is obtained from X-ray crystallography only.
• • • •
Cell membranes are structurally composed of lipids and proteins. Cholesterol and phosphatidyl ethanolamine are the common phospholipids present in the plasma membrane. Membrane proteins can be classified into integral or intrinsic and peripheral or extrinsic depending upon the nature of the membrane-protein interactions. Cytoskeletal proteins spectrin and actin in erythrocytes are examples of peripheral proteins confined to the cytosolic face of the plasma membrane.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.