BC 351 Lecture 4 Protein Function Ligand Binding Terms Ligand Association constant Dissociation constant Fraction bound Conformational Change Modulator Heterotropic modulator Homotropic modulator Allosteic protein Cooperative binding Negative modulation Positive modulation Principles 1 Protein function ligand binding a Specific examples will be myoglobin and hemoglobin 2 Protein dynamics conformational changes a Specific example will be hemoglobin 3 Affects of protein dynamics allosteric proteins and cooperative binding a Specific example will be hemoglobin I Introduction to protein function pgs 153 154 II Ligand binding pgs 155 157 a Definition of a ligand i b Examples of ligands i A single atom 1 Calmodulin a Ligand Ca2 ii A diatomic molecule 1 Myoglobin and hemoglobin a Ligand O2 iii A small organic molecule 1 Avidin a Ligand Biotin iv DNA 1 TATA Binding Protein a Ligand TATA box DNA element v Protein 1 Myosin a Ligand actin filament LN04 1 c Ligand binding equilibrium i Ligand binding by its definition is a reversible process meaning that equilibrium will exist b w free and bound forms of the ligand This equilibrium will be dependent upon the protein s affinity for its ligand ii The association constant Ka P free L PL Ka PL Pfree L iii The dissociation constant Kd 1 Definition of the dissociation constant a Kd Pfree L PL 2 The Kd is an expression or a measure of the proteins affinity for its ligand a A large Kd means the protein has a weak affinity for its ligand b A small Kd means the protein has a strong affinity for its ligand LN04 2 iv Binding curves 1 Definition of fraction bound a PL P free PL L 2 Binding curves are basically titrations a Ligand is being increased incrementally b This change in ligand affects the equilibrium that was previously established b w protein bound ligand and free ligand i This then changes the value of the fraction bound LN04 3 3 How to interpret the binding curve in regards to protein affinity L a One can demonstrate using the equation for and Kd that the Kd can also be defined as i The concentration of ligand where the binding sites are occupied or 0 5 b Therefore the smaller the Kd the smaller the L required to reach half maximal binding capacity III Myoglobin Hemoglobin pgs 158 159 a Our specific examples i In the next section of notes we will be looking at the proteins myoglobin hemoglobin to illustrate the principles of 1 Ligand binding affinity binding curves LN04 4 2 How form affects function and physiology a We are going to primarily focus on myoglobin oxygen binding curves b Structural overview i Myoglobin 1 Monomer no quaternary structure a 153 amino acids b 8 helices ii Hemoglobin 1 Tetramer quaternary structure a 2 subunits 141 residues and 2 subunits 146 residues b 8 helices each iii Comparison 1 Tertiary and Secondary Structure very similar 2 Primary Structure very different a Amino acids are identical at only 27 positions LN04 5 c Structural nomenclature i Helices names 1 A B C etc ii Undefined regions names 1 AB BC CD etc iii Residue names 1 A10 CD2 E7 etc F C G H CD E D B AB A A10 RESIDUE 12 LN04 6 d Specific structural elements i Oxygen binding site 1 Heme is a protoporphyrin ring an Fe atom 2 Myoglobin s distal and proximal His a Distal His His E7 His 64 b Proximal His His F8 His 93 3 Hemoglobin s distal and proximal His a Distal His His58 or His63 b Proximal His His87 or His92 LN04 7 e How form affects function and physiology i Why bind heme in these proteins myoglobin hemoglobin 1 Some clarifications a Concentration becomes partial pressures pO2 and P50 is the Kd Just to confuse you 2 Unbound or free heme vs protein bound heme a Free heme and CO vs O2 binding i Affinity of the heme is far greater 20000x for CO than it is for O2 pCO or pO2 b Protein bound heme i CO vs O2 binding in protein bound heme 1 Affinity of the heme drops drastically for CO when it is covalently bound to the protein LN04 8 ii Why does the affinity for CO drops so drastically when the heme is found in the protein 1 This is due to a steric clash b w the distal His and the CO IV Conformational changes in proteins pgs 158 162 a Definition of a Conformational Change i ii In general 1 Protein structure is far from static 2 Changes in structure relate to the function LN04 9 b Hemoglobin Specific example i Hemoglobin vs myoglobin in terms of function and O2 binding capacity 1 Hemoglobin acts to carry oxygen from the lungs to the tissues a As it is exposed to different environments lungs vs tissues it experiences different pO2 i Hemoglobin s structure is designed and optimized to bind maximal amounts of oxygen at pO2 in the lungs and at the same time release significant amounts of oxygen at pO2 in the tissues Not in book LN04 10 2 Myoglobin would be a poor oxygen carrier but is an excellent oxygen bank a Why would it be a poor oxygen carrier i Because at pO2 found in the tissues 91 of the myoglobin oxygen bind sites will be saturated with O2 b Why is it a great oxygen bank i For the same reason stated above When oxygen levels drop below normal conditions then it will start to release the oxygen it was able to store ii This is especially important in marine mammals such as the blue whale LN04 11 ii But why the difference 1 In other words what are the physical chemical structural features that affect hemoglobin s O2 binding capacity as compared to myoglobin iii Hemoglobin s structure accommodates two structural forms or conformations 1 One of these conformations is a The T state whose affinity for O2 is quite low 2 The other conformation is a The R states whose affinity for O2 is quite high LN04 12 3 The sigmoid curve demonstrated by a hemoglobin oxygenbinding experiment represents a A protein that can change its conformation b w a high and low affinity state L LN04 13 iv But how does this conformational change occur 1 The first event a We are going to start with a T state hemoglobin with no oxygen bound b Upon oxygen binding the heme group undergoes a structural change of its own i Pucker Planer Puckered Planer 2 The second event a The proximal His is taken along for the ride due to the fact that it is covalently bound to the Fe atom of the heme group b This also results in the movement of the entire Fhelix LN04 14 3 The third event a To understand the 3rd event we must look at the atomic structural details of the subunit interfaces 2 1 and 1 2 in the T state i Salt bridge between His 146 HC3 and Asp 94 FG1 subunit only and His 146 the carboxyl terminal of