219103 lecture 8
- 1. HBC1011 Biochemistry I Trimester I, 2018/2019 Lecture 8 – Protein structure and function Ng Chong Han, PhD MNAR1010, 06-2523751 chng@mmu.edu.my
- 2. Overview • Tertiary structure – Supersecondary structure/motif – Protein domain – Type of interactions • Quaternary structure • Amino acid and protein function • Protein folding 2
- 3. Tertiary structure • Protein tertiary structure - a protein's geometric shape. • The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. • This final shape is determined by a variety of bonding interactions between the "side chains" on the amino acids. • These bonding interactions may be stronger than the hydrogen bonds between amide groups holding the helical structure. • As a result, bonding interactions between "side chains" may cause a number of folds, bends, and loops in the protein chain. Different fragments of the same chain may become bonded together. 3
- 4. Supersecondary structures/motifs • Combinations of different secondary structure. • Sometimes motifs are associated with a particular function, although structurally similar motifs may have different functions in different proteins. 4 The helix-turn-helix motif, a supersecondary structural element found in many DNA binding proteins.
- 5. Protein domain • A protein domain - may consist of combination of motifs, a conserved part of a given protein sequence and structure that can evolve, function, and exist independently. • Each domain forms a compact 3D structure and often can be independently stable and folded. Many proteins consist of several structural domains. One domain may appear in a variety of different proteins. 5
- 6. Protein classes - 1 • Globular protein : spherical proteins that are somewhat water-soluble (they actually form colloids in water), eg. hemoglobin 6
- 7. Protein classes - 2 • Fibrous protein: forms long protein filaments, which are shaped like rods or wires, structural proteins or storage proteins that are typically inert and water-insoluble, occurs as an aggregate due to hydrophobic side chains that protrude from the molecule, eg collagen, keratin 7
- 8. Protein classes - 3 • Membrane protein: interact with biological membrane, may exist as both water-soluble and lipid bilayer states, are relatively flexible and are expressed at relatively low levels, eg G protein- coupled receptor 8
- 9. Tertiary structure Water-soluble proteins fold into compact structures within nonpolar cores • In an aqueous environment, protein folding is driven by the strong tendency of hydrophobic residue to be excluded from water. • A system is more thermodynamically stable when hydrophobic groups are clustered rather than extended into the aqueous surroundings. • The polypeptide chain therefore folds so that its hydrophobic side chains are buried and its polar, charged chains are on the surface. 9
- 11. Tertiary structure: Types of interactions • Four types of bonding interactions between "side chains” are involved in protein folding: • Covalent bond – Disulfide bonds • Non covalent bond – Hydrophobic interactions – Electrostatic interaction – Hydrogen bonding – Salt bridges/ionic bond 11
- 12. Tertiary structure: Disulfide bonds • Covalent bonds are the strongest chemical bonds contributing to protein structure. Covalent bonds arise when two atoms share electrons. • Disulfide bonds - Formed by oxidation of the thiol groups on cysteine. Different protein chains or loops within a single chain can be held together by the strong covalent disulfide bonds 12 • Important for protein folding and stability, usually extracellular protein. • Since most cellular compartments are reducing environments, in general, disulfide bonds are unstable in the cytosol, unless a sulfhydryl oxidase is present cysteine oxidation
- 13. Tertiary structure: Ionic interaction • Ionic bonds are formed as amino acids bearing opposite electrical charges are juxtaposed in the hydrophobic core of proteins. • Some amino acids (such as aspartic acid and glutamic acid) contain an extra -COOH group. Some amino acids (such as lysine) contain an extra -NH2 group. 13 • Ionic bonding in the interior is rare because most charged amino acids lie on the protein surface. • These bonds are easier to break than covalent bonds because there is very little sharing of electrons between the two ions.
- 14. Tertiary structure: Hydrogen bonding • Hydrogen bonds between side groups - not between groups actually in the backbone of the chain, likes those in 2nd structure. • Lots of amino acids contain groups in the side chains which have a hydrogen atom attached to either an oxygen or a nitrogen atom. For example, the amino acid serine contains an -OH group in the side chain. 14 However, a -COOH group and an- NH2 group would form a zwitterion and produce stronger ionic bonding instead of hydrogen bonds.
- 15. Tertiary structure: Hydrophobic interaction • The hydrophobic effect is the desire for non-polar molecules to aggregate in aqueous solutions in order to separate from water. • Hydrophobic bonding forms an interior, hydrophobic protein core, where most hydrophobic side chains can closely associate and are shielded from interactions with solvent H2O. 15
- 17. Quaternary structure • Quaternary structure - the combination of two or more polypeptides chains, to form a complete unit. Each polypeptide chain in such a protein is called a subunit. • The interactions between the chains are not different from those in tertiary structure, but are distinguished only by being interchain rather than intrachain. 17 Simplest form: dimer with two identical subunits Common form: consists of more than two different subunits
- 18. Quaternary structure • Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. • Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-", for example, a heterotetramer, such as the two alpha and two beta chains of hemoglobin. 18
- 19. The α2β2 Tetramer of Human Hemoglobin.The structure of the two identical α subunits is similar to but not identical with that of the two identical β subunits. The molecule contains four heme groups (black with the iron atom shown in purple).
- 20. The amino acid sequence of a protein determines its 3-D structure • The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. • The experiment: To destroy the 3D structure of the protein using reducing agents and to determine the conditions required for the structure and protein activity 20
- 21. Amino Acid Sequence of Bovine Ribonuclease. The four disulfide bonds are shown in color.
- 22. Reducing agents 22 These reducing agents denature the proteins, disrupting the protein structure by reducing the disulphide bonds (-S-S-). The proteins loses the enzymatic activities after the structure is disrupted.
- 23. Reduction and Denaturation of Ribonuclease, the protein activity is destroyed. The denatured ribonuclease, freed of urea and β-mercaptoethanol by dialysis, slowly regained enzymatic activity.
- 24. Reestablishing Correct Disulfide Pairing. Native ribonuclease can be reformed from scrambled ribonuclease in the presence of a trace of β-mercaptoethanol.
- 25. Sequence specifies conformation • The sulfhydryl groups of the denatured enzyme became oxidized by air, and the enzyme spontaneously refolded into a catalytically active form. • All the measured physical and chemical properties of the refolded enzyme were virtually identical with those of the native enzyme. • Information needed to specify the catalytically active structure of ribonuclease is contained in its amino acid sequence. 25 Mutation in amino acid residues may affect the protein functions.
- 26. Protein folding • Protein folding is the process by which a protein structure assumes its functional shape or conformation. • Each protein exists as an unfolded polypeptide when translated from a sequence of mRNA to a linear chain of amino acids. • This polypeptide lacks any stable 3D structure. Amino acids interact with each other to produce a well-defined 3D structure, the folded protein, known as the native state. 26
- 27. Protein folding is a highly cooperative process • Protein can be denatured by heat or chemical denaturants to disrupt the weak bonds stabilizing tertiary structure. • Under the cooperative transition, most proteins show a sharp transition from the folded to unfolded form on treatment with increasing concentrations of denaturants, such as β-mercaptoethanol. 27
- 28. Components of a Partially Denatured Protein Solution. In a half- unfolded protein solution, half the molecules are fully folded and half are fully unfolded. Structures that are partly intact and partly disrupted are not thermodynamically stable and exist only transiently. Cooperative folding ensures that partly folded structures that might interfere with processes within cells do not accumulate.
- 29. Protein misfolding 1. Protein folding is a complex, trial-and-error process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell. 2. However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. 29 Deposits of these misfolded proteins are associated with a number of diseases, eg. amyloid disease, prion disease.
- 30. Protein misfolding disease 1. In Alzheimer disease, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid protein assemblies consisting of α- pleated sheets. 2. In prion disease, the infective agent is an altered version of a normal prion protein that acts as a “template” for converting normal protein to the pathogenic conformation 30
- 31. Protein functions • Each type of protein with its distinctive sequence of amino acids residues, has a characteristic shape, size & biological function. • Classified according to their biological roles. 31 Function Examples Structural muscle, skin, hair Signalling insulin, growth hormone Catalyst Enzymes Immunity antibodies Regulation DNA-binding proteins Poison Toxins in snake, spiders Transport Hemoglobin
- 33. Summary 1. Proteins are built from a repertoire of 20 amino acids. 2. Primary structure: Amino acids are linked by peptides bonds to form polypeptide chains. 3. Secondary structure: Polypeptide chains can fold into regular structures such as the alpha helix, the beta sheet, and turns and loops. 4. Tertiary structure: Water-soluble proteins fold into compact structures with nonpolar cores. 5. Quaternary structure: Polypeptide chains can assemble into multisubunit structures. 6. The amino acid sequence of a protein determine its 3D structure. 33
- 34. Study questions 1. What are three main protein classes based on their structure? 2. What are the similarity and difference in chemical bonding between secondary and tertiary structures? 3. Why are side chains important for protein tertiary and quaternary structures? 4. What are the covalent and non-covalent bond in tertiary structure? Identify them in peptide structure. 5. What amino acid is required for disulfide bond formation? 6. What is the difference in hydrogen bonding between secondary and tertiary structures? 7. What is the main difference between tertiary and quaternary structures? 34