L2 Structure and Function of Protein Lecture Notes PDF

STRUCTURE AND FUNCTION OF PROTEIN Lecture 2 Pn. Mariati Abdul Rahman Dept. of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, UKM. Objectives Students should be able to describe: 1. Physical and chemical properties of protein and thei...

STRUCTURE AND FUNCTION OF PROTEIN Lecture 2 Pn. Mariati Abdul Rahman Dept. of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, UKM. Objectives Students should be able to describe: 1. Physical and chemical properties of protein and their function. 2. Structural conformation and organization of protein. 3. Principles of protein separations. References: 1. Basic medical biochemistry, Marks and Marks. 2. Biochemistry. Lippincott’s Illustrated Reviews. Champe and Harvey.  Tertiery structure of protein Primary structure determines its tertiary structure. The shape of a 3-D conformation of a globular protein involves interactions between amino acid residues that may be located far from each other. Globular proteins in aqueous solution is compact with high density atoms in the core of the Also includes α-helices and β-sheets. molecules. Covalent (sharing a pair of electrons) Hydrophobic side chains buried i.e. disulfide bonds and non-covalent in the interior, while hydrophilic (hydrogen & ionic) bonds are involved. groups on the surface.  Non-covalent interactions between the side chains of aa residues Important to stabilize the tertiary structure. 1. Electrostatic interactions- Interaction between & among cations and anions. 2. Hydrogen bonds - A chemical bond in which a hydrogen atom of one molecule is attracted to an electronegative atom, especially a nitrogen, oxygen, or fluorine atom 3. Hydrophobic interaction -hydrophobic amino acids (such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan and methionine) clustered together within the protein. Tertiery structure of protein Domain - fundamental functional & 3-D structural units of polypeptide (with specific function i.e binding site of small molecule. Generally polypeptide with ≥ 200 amino acids will have ≥2 domains. Core domain is built from supersecondary structure ⚫ Therefore each domain has (motifs). characteristics of a small, Folding of peptide chain within compact globular proteins that domain usually occurs is structurally independent of independently of folding in other other domains. domains.  Proteins can be denatured with organic molecules e.g urea (disrupts hydrogen bonding patterns) & β- mercaptoethanol (disulphide bonds) or by heating. Under certain conditions, denatured proteins can refold into their native conformation, regaining original function. Many simple single-subunit proteins such as nucleases that are denatured, can refold spontaneously into native conformation if they are brought back carefully.  In the cell, not all proteins can fold back into their native conformation. As proteins fold & refold while searching for its lowest energy state, it passes thro many high-energy conformations that slow the process (kinetic barriers). These kinetic barriers can be overcome by heat-shock proteins, which use energy provided by ATP hydrolysis to assist in the folding.  Named HSPs because their synthesis in bacteria is increased when temperature increased suddenly. In human cells – different families of proteins with different activities. HSP70 bind to nascent (recently formed) polypeptide chain as their synthesis is being completed to prevent the incomplete chains from folding prematurely. The multisubunit barrel-shaped hsp60 family of proteins is called chaperonins. Unfolded protein fits into the barrels cavity that excludes water and serves as templates for folding  Tertiery structure of protein Chaperonins – a group of proteins that are required for proper folding of many proteins. Also known as polypeptide chain binding (PCBs) – acts as catalysts by increasing the rate of folding at the final stages of folding process. structure of protein 3D structure of a protein composed of multiple subunits. Theses subunits are held together by the same non-covalent interactions (hydrophobic interactions, hydrogen bonds, ionic bonds) involved in tertiary structure. In hemoglobin, subunits are similar. All four contains heme and bind oxygen. structure of protein 4 polypeptides 2 α-chains 2 β-chains 4 heme groups 4 subunits held together by non-covalent interactions tetramer with 2 identical dimers Dimers (αβ)1 and (αβ)2 2 polypeptide within the dimer is held by hydrophobic interactions Pyruvate dehydrogenase complex – has several different types of subunits. Diff color represents diff subunits.  Protein separation ⚫ Proteins are diverse ⚫ Differ in size, shape, charge, hydrophobicity and affinity towards other molecules. ⚫ principle of separation. ⚫ To isolate and characterize. ⚫ Chromatography – technique to isolate and purify all types of biomolecules. ⚫ The samples (solutes) are allowed to interact with mobile and stationary phase. ⚫ Mobile phase – gas /liquid moves the sample through the column. ⚫ Column contains stationary Molecules with weak affinity – spend phase – has ability to bind more time with mobile phase, rapidly solutes. removed from column. 3 kinds of chromatography: 1. Ion-exchange chromatograp hy 3 kinds of chromatography: 2. Gel filtration chromatograp hy 3 kinds of chromatography: 3. Affinity chromatograp hy SDS Gel electrophoresis  Isoelectric focusing Proteins have many charged groups on their surface. Each functional group has pKa – half of the members of the group are protonated (addition of H+). Fully protonated Low pH – net positive charge Some intermediate pH pKa – net charge – zero (0) Has equal number of positive & negative charges. pH = pI (isoelectric Fully de point). protonated High pH – net negative charge When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge. Antibody technique Western blot 1. Proteins are separated by SDS 4. Primary antibody detected by Gel electrophoresis. secondary ab. 2. Proteins in gel are transferred 5. Secondary a/b is already to a membrane labelled with enzyme (alkaline (Nitrocellulose/PVDF). phosphatase). 3. Selected proteins are detected 6. Substrate is attached to by antibody (primary). enzyme – give color. Posttranslational modification (PTM) Protein synthesis – translation Changes (chemical modification) after synthesis – PTM. Common PTM – 1. Phosphorylation – addition of phosphate group. Frequently used by the cells to alter activity of enzymes. Fatty acids, ADP-ribose, methyl, acetyl &CHO groups may be added to proteins via PTM by specific enzymes. 2. Glycosylation – addition of sugar moiety on serine, threonine or asparagine. For secreted proteins or to the cell surface. Can occur spontaneously. Occurs at the Golgi app. Posttranslational modification (PTM) 3. Enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. ⚫ For instance, the peptide hormone insulin is cut twice, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. + C peptide