Protein Structure & Function (Cellular & Molecular Biology MD105) F2024 PDF

Cellular & Molecular Biology MD105 Dr C. Michaeloudes Protein structure and function Dr C. Michaeloudes Cellular & Molecular Biology MD105  Lecture Objectives To understand: The basic structure of amino acids and the formation of polypeptide chains...

Cellular & Molecular Biology MD105 Dr C. Michaeloudes Protein structure and function Dr C. Michaeloudes Cellular & Molecular Biology MD105  Lecture Objectives To understand: The basic structure of amino acids and the formation of polypeptide chains The basic process of protein folding and the levels of protein structure organization The importance of the 3D conformation in protein activity and the different roles of proteins in the cell The role of enzymes in the cell and how enzyme activity is regulated The role of protein misfolding in causing disease REMINDER – Chemical bonds  Chemical bonds Chemical bonds hold atoms together to form molecules Chemical bond = the attractive forces between two or more atoms leading to the formation of molecules Chemical bonds in biological molecules: 1. Covalent bonds 2. Weak non-covalent bonds Electrons and bonding Atomic nucleus consists of protons (positively charged) and neutrons (neutral) Ø Positive charge Electrons are continuously moving around the nucleus in specific regions (orbits) – shells § 3 shells (some atoms have 4) § Each shell can take a maximum number of electrons Atoms whose outer shells are completely filled are very stable (e.g helium, argon) Atoms with incomplete outer shells tend to interact with other atoms to fill their outer shells Ø Transfer electrons or share electrons Covalent bonds Covalent bonding involves sharing electrons between two or more atoms § Complete the outer shell of each atom Shared electrons hold the nuclei together by opposing their mutual repulsion Strongest bonds in the cell § Broken by specific chemical reactions catalyzed by enzymes Disulfide bonds § Covalent bonds between thiol (-SH) groups of two amino acids  Weak non-covalent interactions Electrostatic attractions § Electron transferred from one atom to the other creating a negative and a positive ion § Electrical attraction between the negative and positive ions  Weak non-covalent interactions Hydrogen bonds § Hydrogen and oxygen are held together by a polar covalent bond § one side of the molecule is slightly more positive or negative than the other side § electrical attraction between the more positively-charged H atom and the more negatively-charged O § Can form between molecules other than water Image from Essential Cell Biology, 5th Edition Weak non-covalent interactions Van der Waals attraction § Transient attractions that form when any two atoms approach each other § Non-specific § Form due to fluctuations in the electron distribution in each atom § Transient electrostatic attraction  Hydrophobic forces Not a considered as bonds Aqueous environment (containing water) - non-polar molecules move away from the hydrogen bonded water molecules Drive hydrophobic molecules to aggregate together away from water Bring molecules together to promoting bonding  Amino acid structure and formation of polypeptide chains  Amino acids Amino Carboxyl group group Side chain Small organic molecules, which possess a carboxyl group (-COOH) and an amino group (-NH2) attached to a central α-carbon The α-carbon carries a specific side chain that distinguishes one amino acid from the other § 20 different amino acids Side chains give amino acids unique chemical properties Amino acids  Hydrophilic side chains Water molecule Lysine (positive charge) Asparagine (polar) Hydrophilic = molecules that easily dissolve in water -“Water- loving” § Form hydrogen bonds with water Polar or charged (negative/positive) molecules Polar = molecule with uneven distribution of electrons § one side of the molecule is more positive or negative than the other side  Hydrophobic side chains Ethane Isoleucine (hydrocarbon chain) Tryptophan (ring structure) Hydrophobic = molecules that do not dissolve in water – “Water- fearing” § Do not form hydrogen bonds with water Non-polar/uncharged side chains Non-polar = hydrocarbon chains and ring structures  Proteins are made of amino acids Peptide bond A protein is made up of a long chain of amino acids § Polypeptide chains Amino acids are held together by covalent bonds called peptide bonds The carbon atom of the carboxyl group of one amino acid reacts with the nitrogen atom from the amino group of a second amino acid leading to removal of water (condensation) Protein synthesis  Polypeptide chains have a specific amino acid sequence Polypeptide backbone Amino Carboxy terminus terminus Peptide bond Side chains Amino acid sequence Each polypeptide chain has an amino (-N) terminus (-NH2 group) and a carboxy (-C) terminus (-COOH group) Side chains project from the polypeptide backbone and are involved in chemical bonding required for folding into active proteins For each protein, the amino acids are present in a unique order called the amino acid sequence  Protein folding Polypeptide chain Functional protein Polypeptide chains are folded into their 3D shapes in order to form a functional protein The final folded shape of a protein is called its functional conformation § changes in conformation lead to changes in protein function  Protein folding Polypeptide chain Polypeptide chain folded into a 3D structure – proteins Polypeptide backbone is flexible – some peptide bonds can rotate freely allowing folding of the chain Protein This allows the formation of bonds between atoms in the polypeptide backbone and the amino acid side chains Polypeptide chain can fold into different shapes  Where does protein folding occur? Protein folding in the endoplasmic reticulum Protein folding also occurs in other compartments i.e mitochondria, cytoplasm, nucleus  Chaperone proteins Newly synthesized Protein folding in living cells is polypeptide chain assisted by special proteins called chaperone proteins Bind newly-synthesized polypeptide Chaperone chains and make the folding process proteins more efficient by: § By providing energy obtained from ATP hydrolysis § By preventing polypeptide chains from forming aggregates Incorrectly folded Correctly folded protein protein (becoming tangled) with other chains  Levels of protein structure organization Amino acids Amino acid sequence – the order 1. Primary structure of amino acids in the polypeptide chain β-sheet Hydrogen bonding between atoms β-sheet α-helix α-helix 2. Secondary structure of the polypeptide backbone causes some sections of the chain to fold into repeating patterns Polypeptide chains undergo 3D 3. Tertiary structure folding due to covalent and non- covalent bonds between amino acid side chains 4. Quaternary structure Two or more polypeptide chains combine to form a complex Levels of protein structure organization Secondary structure Secondary structure Secondary structure = folding of regions of the polypeptide chain into specific patterns by forming hydrogen bonds between atoms in the backbone Two types of secondary structures: 1. α-helix 2. β-sheet Most proteins have a mix of the two patterns The α-helix A single polypeptide chain twists around itself to form a right-handed helix The α-helix structure is stabilized by hydrogen bonds A hydrogen bond is formed every 4th amino acid, linking the carbonyl group (C=O) of one peptide bond and an amino group (N-H) on another  Proteins containing α-helices α-helices are found in proteins Two or more α-helices can wrap located in cell membranes around each other to form § E.g transport proteins and structures called coiled-coils receptors § Found in elongated proteins like α-keratin (hair, skin, nails) and myosin (muscle contraction) β-sheet Segments of polypeptide chains lying side by side are held together by hydrogen bonds § Between carbonyl and amino groups of peptide bonds Very strong structure  β-sheets are found in immunoglobulins Immunoglobulins are made up of 4 polypeptide chains, which are folded into repeated domains containing 2 stacked layers of β- sheets Tertiary structure Tertiary structure is determined by bonding between side chains Tertiary structure = folding into the complete 3D conformation Electrostatic Hydrogen attractions bond The tertiary structure of Disulfide proteins is stabilised by bond bonding between amino acid side groups Hydrophobic § Electrostatic attractions interactions § Hydrogen bonds § Disulfide bonds  Hydrophobic forces also determine tertiary structure Unfolded polypeptide In aqueous environment (water) hydrophobic side chains cluster together in the interior of the Hydrophobic Hydrophilic Polypeptide protein, away from the side chains side chains backbone water Hydrophilic side chains are found near the Hydrophilic side Hydrophobic outside of the folded chains are on the side chains are clustered protein, where they form outside in the interior hydrogen bonds with of the protein water molecules Folded protein Quaternary structure  Quaternary structure Some proteins contain more than one polypeptide chains (subunits) associated together Subunits are held together by the same types of bonds that hold together the tertiary structure § Electrostatic interactions § Hydrogen bonds § Hydrophobic interactions The tertiary structure is required for the function of the protein  Haemoglobin quaternary structure β β Example: Haemoglobin consists of 4 polypeptide chains – tetramer § 2 α subunits and 2 β subunits Each polypeptide chain holds a haem molecule where oxygen is bound α α Protein function Which jobs do proteins do in the cell? Functional Proteins Structural Proteins  Protein domains AMP-binding Proteins are divided into functional units (40- domain 350 aa), which are conserved and fold into a 3D conformation independently of the rest of the chain § E.g α-helices and β-sheets Each domain has a specific function § E.g DNA-binding, calcium-binding Some proteins have more than one domains linked by a region of polypeptide chain Example: bacterial catabolite activator protein (CAP) has 2 domains DNA-binding domain § Binds to the signaling molecule cAMP § Binds to DNA Enzyme function  Enzymes Catalyze nearly all the chemical reactions in the cell § Accelerate the speed of chemical reactions Bind to one or more ligands (substrates) and convert them into chemically modified products § This happens again and again without the enzyme changing Usually involves making or breaking a covalent bond in the substrate molecule Grouped into functional classes based on the chemical reactions they catalyze Each enzyme type catalyzes one specific reaction § E.g hexokinase adds a phosphate group to D-glucose  Enzymes Substrate binds to binding site (active site) forming enzyme-substrate complex Covalent bonds are made or broken forming an enzyme-product complex Product is released and enzyme binds to additional substrate molecules  Active site Ligand-binding site is created by protein folding and the amino acid side chains Provides a complementary shape to its ligand and keeps the ligand in place via weak non-covalent bonding § Hydrogen bonding, electrostatic attractions Active site  Enzyme Cofactors Some enzymes need to be bound to non-protein molecules called cofactors to function normally Co-factors can be: § Inorganic ions such as Fe2+ and Mg2+ - minerals E.g DNA polymerase requires Mg2+ as a co-factor § Organic molecules mostly derived from dietary vitamins E.g Vitamin C is a coenzyme for enzymes involved in collagen synthesis Attach permanently (covalent bonds) or temporarily (electrostatic attractions or hydrogen bonding) to the enzyme Cofactors are required for the enzyme to catalyze the reaction Enzyme activity  Regulatory molecules Enzymes can be regulated by other molecules that either increase or reduce their activity. Molecules that increase the activity of an enzyme are called activators Molecules that decrease the activity of an enzyme are called inhibitors § Competitive and non-competitive inhibitors Competitive inhibition Inhibitor molecule is structurally similar to the substrate It binds to the active site and prevent the substrate from binding (competition) The rate of reaction slows down if the substrate concentration is low If the substrate concentration is high then the substrate occupies most of the active sites so the inhibitor cannot inhibit the rate of reaction Non-competitive inhibition Inhibitor molecule binds to a site away from the active site- allosteric site Changes the 3D structure of the protein The active site of the enzyme changes conformation so the substrate can bind but cannot activate the enzyme Regardless of how much substrate there is the rate of reaction is slow Competitive and non-competitive inhibition Protein misfolding and disease  Misfolded proteins can cause disease Proteins that are incorrectly folded (misfolded) are taken to the lysosomes for degradation using the process of autophagy In some pathological cases misfolded proteins are not degraded § Mutations that change the amino acid sequence § Defective lysosome function § Abnormal chaperone protein function Misfolding of α-helix into β-sheets § Misfolded proteins form filaments made of continuous stacks of β- sheets Accumulation of these filaments leads to the formation of called amyloid fibrils Accumulation of these insoluble fibrils lead to the formation of amyloid plagues that can damage cells and tissues  Misfolding and neurodegenerative diseases β-sheet stacks Amyloid fibrils Amyloid plagues on neurons (filaments) Accumulation of plagues on neurons causes them to die leading to impaired neurotransmission Amyloid plagues are characteristic of neurodegenerative diseases: § Alzheimer’s disease § Huntington’s disease § Bovine spongiform encephalopathy (mad cow disease) § Creutzfeldt–Jakob disease  Sickle cell anaemia Normal gene Single nucleotide mutation Pro Glu Glu Pro Val Glu Glutamate - hydrophilic Valine - hydrophobic Abnormal protein Normal protein conformation – conformation Subunits form long rigid protein fibers Die prematurely- Anaemia Rigid/sticky – blood Normal red blood cells Sickle cells vessel obstruction