Proteins PDF

Proteins 1  What is a Protein A protein is a long chain of amino acids – most proteins 250 – 500 aa’s – a few (e.g. titin) can be 10,000+ Connected by single covalent bonds – “peptide” bond—proteins aka “polypeptides” – flexible, rotational 20 “biologically i...

Proteins 1  What is a Protein A protein is a long chain of amino acids – most proteins 250 – 500 aa’s – a few (e.g. titin) can be 10,000+ Connected by single covalent bonds – “peptide” bond—proteins aka “polypeptides” – flexible, rotational 20 “biologically important” amino acids – aka proteinogenic – there are a few other amino acids beyond 20 e.g., hydroxyproline, selenocysteine, pyrrolysine – chemical properties of each are different 2 Over 500 Different Amino Acids 3  Acquiring a Unique Structure Protein 3D structure is determined by chemical interactions between: – pairs of side chains – side chains and peptide bond – two or more peptide bonds – side chains and water Protein (or any molecule) continues to fold, unfold, twist and turn as it is synthesized Final conformation = lowest possible energy state for that molecule 4  Interactions Electrostatic forces – hydrogen bonds – van der Waals attractions – ionic bonds—interior only Hydrophobic interactions Covalent disulfide bonds (between cys side chains) All interactions constrained by van der Waals dimension 5  Levels of Structure Primary – amino acid sequence—covalent (peptide) bonds Secondary – hydrogen bond between peptide bonds Tertiary – higher order folding patterns – primarily weak noncovalent – sometimes ionic and/or covalent Quaternary – two or more proteins complexed together – primarily weak noncovalent – very occasionally ionic and/or covalent 6  Secondary Structures Maintained by H-bond between N-Hδ+ of one peptide bond – and C=Oδ- of another Two structural patterns—α helix and β sheet 7  Tertiary Structure 8  Quaternary Structure Two or more (often many more) separate proteins combined into a multi-subunit structure Individual proteins—subunits Assembled structure—protein complex Quaternary structure – held together primarily by weak noncovalent interactions – very occasionally some ionic and/or covalent 9  Protein Quaternary Structure 10  Other Quaternary Structures Proteins also form complexes with other types of molecules – protein + RNA (ribonucleoproteins) ribosomes, spliceosome – protein + lipids membranes Also maintained primarily by weak noncovalent interactions 11  Domains A specific feature found in different proteins – associated with a specific function or binding capacity – e.g. DNA binding domain – ATP binding domain – many different domains have been discovered 12  Many Possible Proteins 20 amino acids (plus a few others) 10390 possible arrangements of a protein 300 amino acids long Far, far fewer actual proteins exist In order for a protein to be selected for: – final structure must be much lower energy than any alternative structure – it must do something – it must do the same thing every single time – the thing it does must be beneficial...and more beneficial than the metabolic cost of making it 13  Protein Function 14  Protein Function Surface shape and surface chemistry are key to protein interactions and functions Proteins function by binding to other molecules – other proteins, DNA, RNA, etc. – aka “ligands” or “substrates” Binding involves many weak, noncovalent interactions—high specificity between interacting molecules 15  “Complementary” Molecules complementary Van der Waals dimensions complementary distribution of electro-chemical properties numerous weak, non-covalent interactions form 16  Protein Function Location on the protein where the binding interaction occurs is the binding site Often within a cavity on protein surface – or on surface of ligand or substrate 17  Protein Binding Binding with other molecules alters the protein’s chemical environment – change in chemistry leads to change in shape Shape change = conformational change Conformational change may result in – creation of new binding site – destruction of original binding site – gain, loss, or change of function 18  Many Proteins Are Enzymes Enzymes are biological catalysts Bind one or more reactants (substrates) Mediate reactions that convert substrate(s) into product(s) E + S → ES → EP → E + P 19  Enzyme Catalyzed Reactions Proceed Extremely Fast Reactants held in proper orientation Precisely positioned atoms on enzyme: – alter electron distributions of atoms involved in covalent bonds Very importantly : – enzymes bind to and stabilize reaction intermediates 20  Acid – Base Catalysis Acids and bases improve ability of water to hydrolyze many types of bonds e.g. peptide bond: δ+ – acid donates H+ to carbonyl O of peptide bond δ- Electrons move away from C – C acquires partial charge δ+ C now susceptible to attack by water’s electronegative O 21  Acid – Base Catalysis Base attracts H+ of water Electrons move closer to δ-- the already electronegative O Water becomes a better attacking molecule 22  Acid – Base Catalysis In aqueous solution acid- base catalysis either/or Acids and bases will react preferentially with δ+ each other δ-- Enzymes utilize both – acidic side chain in one region – basic side chain elsewhere – prevent them from reacting with each other 23  Lysozyme Lysozyme cuts polysaccharide component of bacterial cell walls Lysozyme catalyzes hydrolysis of glycosidic bond between adjacent sugars – energetically favorable reaction Although favorable – high activation energy – bond geometry must be distorted for reaction to proceed Distortion would eventually result from rare, high energy random collision 24  Lysozyme Lysozyme binds polysaccharide in elongated groove – numerous noncovalent bonds Binding causes sugar monomer to bend – sugar conformation is strained – however, arrangement is lowest energy level possible for sugar:protein complex 25  Lysozyme Acidic Glu on enzyme donates H+ to O on sugar #2 – O pulls electrons from C on sugar #1, C becomes δ+ (-) charged Asp on enzyme attacks δ+ C on sugar #1 δ+ #2 – temporary covalent bond #1 forms – H is displaced 26 26  Lysozyme Covalent bond between sugar monomers is now broken O on Asp is now temporarily covalently bonded to C of sugar #1 #1 #2 H from Glu is bound to O of sugar #2 27  Lysozyme (-) Glu pulls H+ off water molecule – original Glu chemistry restored Resulting OH- – attacks the δ+ C 28 28  Lysozyme Attack of OH- from water displaces Asp – Asp back to original chemistry Enzyme changes back to original conformation Cut sugars released 29  Non-Protein Parts of Enzymes Many enzymes have small molecules or metal atoms/ions bound to them – may be permanent covalent attachment – may be temporarily bound by noncovalent interactions Assist with catalytic function – usually in or very near active sites Inorganic = cofactor Organic = coenzyme – many “vitamins” are actually coenzymes 30  Molecular “Tunnels” Physical pathways that connect different active sites Prevent intermediate products from encountering the cytosol – keeps highly reactive intermediates isolated – prevents diffusion out of cell Often seen in reactions that produce ammonia as an intermediate – NH3 would readily diffuse out of the cell e.g. carbamoyl phosphate synthetase 31  Molecular Tunnel 32  Control of Protein Function 33  Regulating Protein Function Protein function controlled at many levels – rate of synthesis (gene expression, chap 7) – rate of degradation (chap 6) – segregation (compartmentalization, 12 & 14) Direct control of protein activity – most important and common control mechanism – especially important and elaborate in the control of protein enzymes 34  Protein Regulation Based on the ability to change a protein’s conformation – change in conformation = change in function Three main regulatory mechanisms – regulatory ligand binding – phosphorylation – GTP-GDP switching – a 4th, mechanical, will be discussed later 35  Regulatory Ligand Binding Many proteins have two or more binding sites 1) main binding site(s) for ligand(s) or substrate(s) 2) regulatory binding site(s) Regulatory ligand binds – protein chemistry changes – protein shape changes – main binding site changes Many proteins have more than one regulatory binding site – and more than one main binding site 36  Types of Ligands A ligand is typically a small molecule or ion – small proteins – small organic or inorganic molecules – ions (e.g. Ca2+) Ligands may be of extracellular origin – extra-organismal or intra-organismal Ligand may be of intracellular origin Ligand-protein interactions essentially form the basis by which cells “understand” what’s going on around them – and how to respond 37  Ligand Mediated Feedback Regulation Common mechanism to control enzyme function – occasionally used to control other protein types Final or intermediate product of an enzyme catalyzed biochemical reaction serves two functions: – 1) contribute to the overall outcome of the reaction – 2) function as a controlling ligand for one or more of the enzymes involved in the reaction 38  Negative Feedback Regulation 39  Positive Feedback Regulation Accumulation of substrate activates enzymes – or enzymatic pathways e.g.. accumulation of ADP stimulates activity of enzymes involved in sugar-oxidation pathway Result = conversion of ADP + P → ATP 40  Protein Phosphorylation Phosphorylation – Covalent addition of P to certain aa side chains Phosphorylation = conformational change Phosphate itself may also constitute a structural feature recognized by other molecules 41  Protein Phosphorylation Protein kinases phosphorylate Protein phosphatases dephosphorylate Phosphate always derived from ATP – P added only to Ser, Thr, or Tyr Hundreds (thousands?) of kinases – each specific to some target enzyme Phosphorylation may activate or inactivate depending on protein – phosphorylation = chemical change = conformational change= functional change 42  GTP-Controlled Proteins Some proteins have a GTP binding site (GTPases) – GTP binds to GTP binding site on protein – protein is now “active” At some point the GTP is hydrolyzed to GDP – this leads to conformational change – the protein is now “in-active” Some GTPases catalyze the hydrolysis of GTP themselves – others require a separate enzyme 43  GTPases At some point the GDP is forced to dissociate This allows a new GTP to immediately re-bind to the GTP binding site – protein is reactivated 44  Control of GTPase Activity Regulatory proteins determine whether bound molecule is GTP or GDP GTPase-activating protein (GAP) – catalyzes hydrolysis of GTP → GDP – leads to deactivation – sometimes a domain on the GTPase itself Guanine nucleotide exchange factor (GEF) – induces release of GDP – induces GTP binding to vacated GDP site – leads to activation 45  Four Main Regulatory Mechanisms – regulatory ligand binding add ligand, change chemistry change chemistry, change conformation change conformation, change function – phosphorylation add phosphate, change chemistry change chemistry, change conformation change conformation, change function – GTP-GDP switching GTP bound = one chemistry = one conformation = one function GDP bound = different chemistry = different conformation = different function – mechanical 46 change conformation, change function  Some Other Protein Functions 47  Transport Pumps Proteins bound to membranes – conformational changes drive molecules across membranes Conformational changes often coupled to ATP-binding followed by ATP hydrolysis 48  Synthetases Convert energy stored in an electrochemical gradient – into energy stored in a high energy chemical bond ATP Synthetase – energy in form of proton gradient – protons flow through synthase – synthase spins – mechanical energy used to synthesize ATP from ADP + Pi 49 Transport Pumps and Synthases use stored energy to move materials use movement of materials to store energy 50  Motor Proteins (eucaryotic only) Muscle contraction Crawling and swimming Chromosomal segregation Organelle movement Move enzymes along DNA strand during replication 51  Protein Machines Most cell processes carried out by large protein complexes (10+ subunits) Ordered series of conformational changes in each subunit contributes to coordinated process of entire complex These protein machines and protein switches control elaborate and complex cellular processes 52